Regulation of fatty acid synthesis via phosphorylation of acetyl-CoA carboxylase

Prog. Lipid Res. Vol. 28, pp. 117-146. 1989 Printed in Great Britain. All rights reserved

0163-7827/89/$0.00 + 0.50 @, 1989 Maxwell Pergamon Macmillan plc

REGULATION OF FATTY ACID SYNTHESIS PHOSPHORYLATION OF ACETYL-CoA CARBOXYLASE

VIA

D. GRAHAME HARDIE MRC Protein Phosphorylation Group, Biochemistry Department, The University, Dundee, DDI 4HN, Scotland, U.K. CONTENTS I. INTRODUCTION II. FATTYACID SYNTHESIS--PATHWAYSAND PRECURSORS III. ACETYL-CoACARBOXYLASE A. Structure and function B. Allosteric regulation C. Phosphorylation I. Protein kinases acting on purified acetyl-CoA carboxylase (a) Cyclic AMP-dependent protein kinase (b) Acetyl-CoA carboxylase kinase-2 (ACK2) (c) The AMP-activated protein kinase (d) The calmodulin-dependent multiprotein kinase (e) The Ca 2+- and phospholipid-dependent protein kinase (protein kinase C) (f) Casein kinases-I and -2 2. Protein phosphatases acting on acetyI-CoA carboxylase (a) Protein phosphatase-I (b) Protein phosphatase-2A (c) Protein phosphatase-2B (d) Protein phosphatase-2C (e) AcetyI-CoA carboxylase phosphatases 3. Phosphorylation of acetyI-CoA carboxylase in intact cells (a) Hepatocytes (b) Adipocytes (c) Mammary acinar cells IV. PHYSIOLOGICALROLESOF THE AMP-ACTIVATEDPROTEINKINASE A. Phosphorylation of proteins other than acetyI-CoA carboxylase B. How is the AMP-activated protein kinase regulated in vivo? I. Regulation by hormones 2. Regulation by AMP 3. Regulation by fatty acyI-CoA esters 4. Regulation by cholesterol metabolites V. CONCLUSIONS REFERENCES

117 118 119 119 120 121 121 121 123 123 126 127 128 129 129 129 129 130 130 130 130 133 138 139 140 141 141 141 142 142 142 143

I. I N T R O D U C T I O N

AcetyI-CoA carboxylase catalyzes the first step which is committed to fatty acid synthesis, i.e. the carboxylation of acetyl-CoA to malonyl-CoA. The available evidence suggests that the enzyme exerts a major controlling influence on the rate of fatty acid synthesis in liver, and is one of several enzymes regulating the overall flux from glucose to fatty acids in adipose tissue and lactating mammary gland. The review that follows will be a somewhat personal account of current ideas on the regulation of fatty acid synthesis by hormones and other extracellular factors in mammals. It will concentrate on the regulation of acetyI-CoA carboxylase itself, but where there is a good evidence that the pathway of fatty acid synthesis is regulated at additional sites, these will be discussed briefly. The conclusions presented are based largely on experiments carried out on the laboratory rat, but where there is evidence that mechanisms differ in other species, this will be mentioned. 117

D. G. Hardie

118

G L U C ~ //

GLUCOSE

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~' GLYCOGEN

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TRIACYLGLYCEROL

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FattyacyI-CoA

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--..... CHOLESTEROLESTER ~)~ BILEACIDS

!

FATTY ACID

CHOLESTEROL PEP f' t LACTATE i j,s AMINOACIDS malT(~CoA mev~ate ~pyruvite acetylcCoA =.acetoacetyt-CoA = HMG-CoA i

FiG. I. Major pathways of fatty acid and cholesterol synthesis in mammals. Potential sites of regulation, mentioned in the text, are (1) glucose transport across the plasma membrane, (2) pyruvate dehydrogenase, (3) acetyl-CoA carboxylase and (4) HMG-CoA reductase. II. F A T T Y A C I D S Y N T H E S I S - - P A T H W A Y S

AND PRECURSORS

The major pathways which provide acetyl-CoA for fatty acid synthesis in mammals are summarized in Fig. 1. The most important site of fatty acid synthesis in most mammals and birds is the liver, but in some mammals, including the rat, adipose tissue also contributes a large proportion of whole body fatty acid synthesis. 33 In the special case of the lactating mammal, the mammary gland is also an extremely active site for the pathway, particularly in those mammals which produce lipid-rich milk) °4 In adipose tissue and lactating mammary gland, glucose is the most important precursor for fatty acid synthesis. The uptake of glucose by mammary gland at peak lactation in the rat is of the order of 30 mmoles per day, which is roughly equivalent to the whole body glucose consumption of an adult male rat of the same weight. ~°4 In non-lactating rats, the conversion of glucose to fatty acids in the post-absorptive state is also high in adipose tissue. In mouse and rat liver, by contrast, it appears that neither glucose, nor glycogen, are utilized significantly as precursors for fatty acid synthesis. 26037'N9 This may seem surprising until it is pointed out that the liver is normally metabolizing in the net direction of gluconeogenesis rather than glycolysis. This is probably true even in the fed state, 9~ although there is evidence for a zonation of intact liver into periportal "gluconeogenetic" and perivenous "glycolytic" areas, t35 and experiments carried out with a mixed population of isolated hepatocytes should perhaps be interpreted with caution. The major precursor for hepatic fatty acid synthesis is almost certainly lactate. Although some of this metabolite may be derived from erythrocytes and anaerobic muscle metabolism, a more important source is likely to be metabolism of glucose in intestinal mucosa, where up to 40% of glucose taken up from the gut is converted to lactate before secretion into the bloodstream, at least in the rat) °7 In starved rats, the concentration of lactate in the portal vein is 1-2 raM, and this rises to 3--4 mM after acarbohydrate-rich meal. 64 When isolated hepatocytes are presented with lactate in this concentration range, they will use it preferentially as a carbon source for fatty acid synthesis and there is little or no flow of carbon from glucose into fatty acids. 26 Isolated hepatocytes will also utilize amino acids as precursors for fatty acid synthesis, 26 and it seems likely that on high protein diets amino acids may act as important precursors for the pathway. This may be particularly important for the ketogenic amino acids which are broken down to acetyl-CoA and cannot be used as precursors for gluconeogenesis.

Regulation of fatty acid synthesis

119

Whichever of the above precursors are used, they are metabolized to mitochondrial acetyl-CoA, whereas cytoplasmic acetyl-CoA is the substrate for acetyl-CoA carboxylase. Acetyl-CoA cannot cross the inner mitochondrial membrane, and must first be converted to citrate, which is exported via a protein carrier, and is then converted back to acetyl-CoA by the cytoplasmic enzyme ATP-citrate lyase. In ruminants and other herbivorous animals such as the rabbit, cytoplasmic acyl-CoA esters can be synthesized directly from acetate, propionate or butyrate, which are important products of metabolism by the gut bacteria. In these species, ATP-citrate lyase is usually absent, and cytoplasmic acyl-CoA synthetases appear to provide essentially all of the short chain acyl-CoA precursors for fatty acid synthesis. III. A C E T Y L - C o A C A R B O X Y L A S E

A. Structure and Function

Although ATP-citrate lyase or short chain acyl-CoA synthetases can be regarded as enzymes of fatty acid synthesis, they do also provide acetyl-CoA for other pathways (e.g. cholesterol synthesis). However, malonyI-CoA is not used in any pathway other than fatty acid synthesis, and, therefore, acetyl-CoA carboxylase catalyzes the first step committed to fatty acid synthesis. An abundance of evidence, reviewed in the remainder of the section, suggests that this enzyme is of major importance in overall regulation of the pathway. The extent to which acetyl-CoA carboxylase can be regarded as rate-limiting (or to put it more correctly, the extent to which the control strength approaches unity 74) will depend on the tissue under consideration and the precursor that is being utilized. This question will be discussed more fully when regulation of fatty acid synthesis in the three main lipogenic tissues is reviewed in later sections. Acetyl-CoA carboxylase is a biotin-containing enzyme which catalyzes two partial reactions: ATP + HCO; + Enz.biotin ~ Enz.biotin.CO~- + ADP + Pi

(i)

Enz.biotin.CO~- + acetyl-CoA ~ Enz.biotin + malonyl-CoA.

(ii)

In Escherichia coli, acetyl-CoA carboxylase can be resolved4~ into three protein components, i.e. a biotin carboxylase [catalyzing reaction (i)], a carboxyl transferase [catalyzing reaction (ii)] and a carboxyl carrier protein [a non-enzymic protein which contains the covalently bound biotin]. However, numerous different purification protocols have demonstrated that in fungi, higher plants, avian and mammalian liver, and mammalian adipose tissue and mammary gland, the enzyme is composed of a single type of subunit of molecular mass 200 to 250 kDa. 2.~s'126,t33.~37.147The partial reactions can still be measured independently using isotopic exhange reactions, 89and it seems very likely that eukaryotic acetyl-CoA carboxylases are multifunctional polypeptides containing two active sites which have been combined by gene fusion events. The recent cloning and sequencing of cDNAs coding for rat and chicken acetyl-CoA carboxylase have supported this view. s3'~31 There is weak homology between the amino-terminal region of both carboxylases and carbamoyl phosphate synthetase (Fig. 2). Since carbamoyl phosphate synthetase catalyzes at ATP-dependent carboxylation reaction analogous to reaction (i) above, the amino terminal regions may represent the biotin carboxylase domain. The ~iotin-binding region is the center of the molecule, corresponding presumably to a ~rboxyl carrier domain. There is a weak homology in the C-terminal region with the biotin carboxylase domain r

N~

biotin domain

11

carboxyl b'ansferue domain 1

l~otin I

J

l

l

C

FIG. 2. Model for the domain structure of aeetyl-CoA earboxylase, based on the location of the eovalently-bound biotin, and homologies with earbamoyl phosphate synthetase and propionylCoA earboxylase. The location .of the boundaries between domains are not yet well defined.

120

D.G. Hardie

//-subunit of propionyl-CoA carboxylase (Fig. 2). The latter enzyme contains distinct aand fl-subunits, with the ~-subunit containing the biotin and being able to catalyze the biotin carboxylase reaction, but not the overall reaction. 43 The ]/-subunit of propionylCoA carboxylase may,'therefore, be the carboxyl transferase subunit, and the C-terminal domain of acetyl-CoA carboxylase may represent the carboxyl transferase domain of that enzyme. It is well known that acetyI-CoA carboxylase can be readily cleaved by proteinases to yield two fragments of ,--120 kDa. 133'~43Although the sites of cleavage have not been precisely defined, they would appear to be between the putative carboxyl carrier and carboxyl transferase domains, and this cleavage may occur because there is an exposed "hinge region" between the domains. These hypotheses have been put together in the suggested domain structure shown in Fig. 2, which should be regarded as a working model only. It is interesting to note that the proposed evolutionary history of acetyl-CoA carboxylase from discrete monofunctional proteins to a fused multidomain protein is probably valid also for the two other major lipogenic enzymes, i.e. fatty acid synthase 9° and ATP-citrate lyase) 2 The discussion of quaternary structure which follows refers to the avian and mammalian enzymes, which appear to be very similar. The minimal molecular weight of native acetyl-CoA carboxylase suggests that the smallest form that occurs in vivo is a homodimer of the 200-250 kDa subunit. In the presence of the allosteric activator citrate (see below), the enzyme readily forms long linear polymers consisting of up to 32 subunits disposed in an extended helical array. TM These polymers have been observed by electron microscopy, viscometry and ultracentrifugation of the purified enzyme, and can also be detected in crude extracts by gel filtration or sucrose gradient velocity sedimentation. A view, which had at one time almost the status of dogma, is that the polymeric species is the only active species of the enzyme. However, recent light-scattering measurements have shown that activation by citrate precedes polymerization, 7'8 and there is no direct evidence that the polymers exist in vivo. Observations that a proportion of acetyl-CoA carboxylase permeates very slowly from digitonin-treated hepatocytes, which has been interpreted as evidence for the existence of the polymeric form in intact cells, 95 could equally well be explained by binding of the enzyme of intracellular membranes. Similarly, observations 6 that a proportion of acetyl-CoA carboxylase in liver extracts is insensitive to the biotin-binding protein, avidin, can be explained by a shielding of the biotin in the active conformation of the dimer, and there is no necessity to invoke the polymerized form to explain these data. In the opinion of this author, the existence of acetyi-CoA carboxylase polymers in vivo remains unproven, although there is no doubt that the active conformation of the enzyme readily polymerizes in vitro. B. Allosteric Regulation

In vertebrates, but not in fungi or plants, acetyl-CoA carboxylase is dramatically activated by citrate. In most vertebrates, but excluding ruminants and other herbivores, citrate is the immediate precursor of cytoplasmic acetyl-CoA (Fig. 1), so that citrate can be regarded as a "feed-forward activator". Purified avian acetyl-CoA carboxylase binds citrate with a dissociation constant around 2-3/~M, 41 although concentrations in the miilimolar range are required for activation of the enzyme. The likely explanation for this apparently anomaly is that free citrate 3- is the true activator, whereas in activation assays a large proportion of the citrate is complexed by Mg 2+, which must be added due to the fact that Mg.ATP 2- is one of the other substrates. 7.s Although citrate-independent enzyme activity can be measured both in crude extracts and in the purified enzyme, this may be due to traces of endogenous citrate which remain bound during enzyme preparation, and under most conditions the purified enzyme is essentially completely citrate-dependent. Citrate stimulates both partial reactions, 12s and appears to act by stabilizing the active conformation of the carboxylated enzyme. 49 The susceptibility of the citrate-activated enzyme to polymerization is discussed in the previous section.

Regulation of fatty acid synthesis

121

Long chain acyl-CoA esters are very potent inhibitors of acetyl-CoA carboxylase, and appear to oppose the activation by citrate. The most potent inhibitors, such as palmitoyiCoA and stearoyI-CoA, have K~ values as low as 1-5 riM,j°8 and inhibit very significantly when present at a concentration equimolar with that of the enzyme. ~09Since the fatty acids produced by the lipogenic pathway must be converted to acyl-CoA esters prior to further metabolism, long chain acyl-CoA esters can be regarded as classical feedback inhibitors. In intact cells, the bulk of the acyl-CoA esters are bound to proteins, ~' 97 and it is difficult to know how important this type of regulation is in vivo, although exogenous fatty acids do inhibit lipogenesis in perfused liver, 2° (see also Section IV.B.3). Two other metabolites have been reported to activate acetyl-CoA carboxylase in vitro, i.e. CoA and guanine nucleotides, t44.~5°Although these observations are interesting, their physiological significance remains unclear. CoA does not activate the enzyme in the presence of Mg 2÷, a finding which casts some doubt on the importance of this regulator in vivo. Guanine nucleotides promote the formation of polymeric acetyl-CoA carboxylase. However, activation by guanine nucleotides is only observed in crude extracts and the effect is lost on purification of the enzyme, so the mechanism of this effect remains obscure. The location of the binding sites for any of these allosteric effectors, or of citrate or fatty acyl-CoA, with respect to the primary structure of the protein is not known. C. Phosphorylation The first evidence that mammalian acetyI-CoA carboxylase could be regulated by reversible phosphorylation came from the detection of phosphate in the purified rat liver enzyme, 7° and from observations that crude preparations of rat liver acetyI-CoA carboxylase became inactivated in a time-dependent manner when incubated with Mg.ATP. 24 Although the interpretation of these preliminary experiments was initially challenged, this mechanism of regulation of enzyme activity is now firmly established. The purified enzyme has now been shown to be phosphorylated in vitro by at least seven distinct protein kinases, while there is direct evidence for regulation by phosphorylation in intact cells for each of the three major lipogenic cell types. These studies will now be reviewed, beginning with the studies on the phosphorylation of the purified enzyme by each class of protein kinase, followed by a review of our knowledge of the protein phosphatases which act in this system. In later sections, we dicuss the evidence for regulation of acetyI-CoA carboxylase by phosphorylation in vivo or in intact cells, and discuss the role of the regulation in the overall control of fatty acid synthesis in each of the three major lipogenic cells types (hepatocytes, adipocytes and mammary acinar cells). 1. Protein Kinases Acting on Purified Acetyl-CoA Carboxylase (a) Cyclic AMP-dependent protein kinase. The free catalytic subunit of cyclic AMP-dependent protein kinase was the first enzyme shown to phosphorylate highly purified acetyl-CoA carboxylase. ~ Phosphorylation was stoichiometric and was completely blocked by the heat-stable protein inhibitor of cyclic AMP-dependent protein kinase, although the initial experiments were complicated by the presence of a cyclic AMP-independent protein kinase which contaminated the acetyi-CoA carboxylase preparations. We originally reported that a single major site was phosphorylated, based on the finding of a single major phosphorylated peptide after total digestion with trypsin or chymotrypsin, with peptide analysis by reversed phase high performance liquid chromotography (HPLC) (Fig. 3) or thin layer isoelectric focussing. 18,47.62However, recent amino acid sequencing in this laboratory has shown that these tryptic and chymotryptic peptides are not derived from the same site, as we initially tacitly assumed, and are, in fact, quite different sequences. 98 The two sites of phosphorylation can now be identified by comparison with the complete sequence of the rat enzyme derived from cDNA cloning, as ser-77 and ser-1200 (Fig. 4). The sequences around these two sites are conserved in the sequence of the chicken enzyme, with the residues corresponding to the phosphorylation sites being

D . G . Hardie

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FIG. 3. Phosphopeptide mapping by HPLC of acetyl.CoA carboxylase phosphorylated by cyclic AMP-dependent protein kinase. The enzyme was labelled using [7-~=P]ATPand the kinase, digested with trypsin (T), chymotrypsin (C) or trypsin plus chymotrypsin (T + C), and radioactive peptides

were analyzed by reversed phase HPLC using an on-line radioactivity monitor. Reproduced from Munday e t al. ~

ser-80 and ser-1193. If reversed phase HPLC analysis is carried out after a double digestion with trypsin and chymotrypsin, then both sites of phosphorylation can be recovered in separate peptides (TC1 and TC2, respectively, Fig. 3) in a single run. 98 Phosphorylation by cyclic AMP-dependent protein kinase results in inactivation of acetyl-CoA carboxylase. Kinetic analysis shows that this inactivation results from a modest depression of V ~ couples with an increase in the K, for the allosteric activator, citrate. 's'~ The maximum effects of phosphorylation are, therefore, observed at low, subsaturating concentrations of citrate (0.1-1 raM) which corresponds well with the range of estimated physiological concentrations of citrate in hepatocyte cytosol3 2 Inactivation definitely results from the phosphorylation itself, since it is blocked by the protein inhibitor of cyclic AMP-dependent protein kinase/8 and is totally reversed by dephosphorylation using any of three different purified protein phosphatases (phosphatases-1, -2A or -2C). e Kim and coworkers have challenged these findings and have reported that they were unable to obtain direct phosphorylation of acetyl-CoA carboxylase purified from rat liver by cyclic AMP-dependent protein kinase. 8mThey suggested instead that the phosphorylation of acetyl-CoA carboxylase was catalyzed by a specific acetyl-CoA carboxylase kinase which was itself activated by phosphorylation by cyclic AMP-dependent protein kinase. While we cannot rule out the existence of such a specific kinase, both this and at least three

Regulation of fatty acid synthesis biotin J

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FIG. 4. Domain map of acctyl-CoA carboxylar¢ (cf. Fig. 2) showing locations of the phosphoryl-

ation sites. other laboratories 15'~'47'71'137have shown that acetyl-CoA carboxylase purified from rat or

rabbit mammary gland, rat liver or rat adipose tissue is phosphorylated and inactivated directly by cyclic AMP-dependent protein kinase. We have also shown that addition of the heat-stable protein inhibitor at any stage during the reaction instantly abolishes phosphorylation by cyclic AMP-dependent protein kinase.'°m This finding is not consistent with the phosphorylation cascade model of Kim and coworkers, although there is clear evidence for such a cascade involving the AMP-activated protein kinase (see below). It should also be added that the available evidence now suggests that direct phosphorylation of acetyl-CoA carboxylase by cyclic AMP-dependent protein kinase does not occur in intact cells (see Section III.C.3). (b) Acetyl-CoA carboxylase kinase-2 (ACK2). A cyclic AMP-independent protein kinase phosphorylating acetyl-CoA carboxylase, originally identified as a contaminant in preparations of the rabbit mammary enzyme.~ has been partially purified in this laboratory from lactating rat mammary gland, t°' Although not yet completely characterized, this activity is clearly distinct from the catalytic subunit of cyclic AMP-dependent protein kinase since it has a different apparent molecular weight on gel filtration, is more sensitive to inhibition by heparin, and is completely insensitive to the heat-stable protein inhibitor of cyclic AMP-dependent protein kinase.~ We initially reported that the sites of phosphorylation for cyclic AMP-dependent protein kinase and ACK2 were similar, based on HPLC analysis of chymotryptic phosphopeptides. Recent experiments using the double digestion (trypsin + chymotrypsin) technique have confirmed that ACK2 does indeed phosphorylate sites within peptides TC1 and TC2 (Fig. 5), although TC2 is phosphorylated much more rapidly than TC1, unlike cyclic AMP-dependent protein kinase which labels both peptides with similar kinetics. Amino acid sequencing of TC2 after phosphorylation by ACK2 confirmed that ser-1200 was the major site of phosphorylation by this kinase. 98 This is of interest because cyclic AMP-dependent protein kinase and ACK2 have very similar effects on acetyl-CoA carboxylase activity (predominantly an increase in the K, for citrate), indicating that this effect may be caused by phosphorylation at ser-1200. The physiological role of ACK2 remains unclear. Further work on the purification of this kinase is needed in order to ascertain its specificity for acetyl-CoA carboxylase, its tissue distribution and its regulation. (c) The AMP-activated protein kinase. Studies in our laboratory using rat hepatocytes, described in more detail in a later section, showed that acetyl-CoA carboxylase was present in a form of low specific activity that was highly phosphorylated (4--5 molecules phosphate per subunit), even in basal cells. 62At least some of the phosphate was clearly of regulatory significance, since treatment of the purified enzyme with protein phosphatase produced a dramatic activation, caused by both an increase in l/m~ and a decrease in K, for citrate. 62J25

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FroG.5. Phosphopeptide mapping by H P L C of acetyl-CoA carboxylas¢ phosphorylated by cyclic AMP-dependent protein kinas¢ (cAMP-PK), aeetyl-CoA earboxylase kinas¢-2 (ACK2) or The analysis was as in after digestion by trypsin plus chymotrypsin (but using a different elution gradient). Reproduced from Munday e t al. n

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Neither cyclic AMP-dependent protein kinase nor ACK2 could account for this effect, since neither produced such a large change in Vm,~. It was, therefore, clear that there must be another acetyl-CoA carboxylase kinase present in rat liver. Several groups have described rat liver kinase activities that produce large decreases in the V~,x of acetyl-CoA carboxylase and could, therefore, potentially account for the low activity of the enzyme in liver cells. 5'7j'79'124Only the kinase activity described by Lent and Kim was characterized in any detail. 79 It was reported to copurify with acetyl-CoA carboxylase on polyethylene glycol precipitation and DEAE-cellulose but could be separated from it on Sepharose-2B. ~ The kinase was judged to be pure at this stage by SDS-polyacrylamide gel electrophoresis, with a subunit molecular weight of 170,000. However, no evidence was present that the 170 kDa polypeptide corresponded to the kinase activity, and the very low specific activity (,,, 10 nmol ACC inactivated/min/mg) makes the claim for purity somewhat doubtful. Kim's group subsequently reported that phosphorylation was stimulated by binding of Coenzyme A to acetyl-CoA carboxylase, and that the kinase was phosphorylated and activated by cyclic AMP-dependent protein kinase? °.81 We have recently purified a protein kinase from rat liver (originally called acetyI-CoA carboxylase kinase-3) which accounts for > 90% of the cyclic AMP-independent acetylCoA earboxylase kinase activity recovered after polyethylene glycol fractionation of a crude extract of the tissue. 2~3 The preparation is not yet homogeneous, although the specific activity of our final preparation (~500 nmoi/min/mg) is nearly 2 orders of

Regulation of fatty acid synthesis

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FIG. 6. [~2P]Phosphate content (circles) and activity (squares) of acetyl-CoA carboxylase during phosphorylation by AMP-PK, and dephosphorylation by protein phosphatasc-2A. Acetyl-CoA carboxylase activity was measured at l0 rnM citrate. Excess EDTA was added with the phosphatase to chelate Mg 2+ and prevent further phosphorylation. Reproduced from Carling et al. ~

magnitude greater than that reported by Lent and Kim of their preparation. The correlation between the phosphorylation of purified acetyl-CoA carboxylase and inactivation of the enzyme is shown in Fig. 6. Phosphorylation results in both an elevation of K= for citrate and a dramatic drop in Vm~.98 At physiological citrate concentration (0.5 ma), phosphorylated acetyI-CoA carboxylase is >95% inactivated compared with controls treated with Mg.ATP alone. This effect is completely reversed by dephosphorylation by protein phosphatase-2A. We have found two intriguing modes of regulation of the kinase in vitro. 23 It is activated 4- to 6-fold by Y-AMP, with a half-maximal effect at 14/~M, which is within the physiological range in rat liver. A wide range of other nucleotides and AMP analogues, including ADP and cyclic AMP, are ineffective, although the kinas¢ is inhibited by adenosine with a half-maximal effect at ~ 200 #M. Since we have discovered recently that the kinas¢ phosphorylates other substrates apart from acetyl-CoA carboxylase, we have now renamed it the AMP-activated protein kinase (AMP-PK). The second mode of regulation is phosphorylation. 23We found initially that the kinase was very unstable unless it was prepared in the presence of the protein phosphatase inhibitors, fluoride and pyrophosphate, and subsequently showed that the purified enzyme was inactivated by the purified catalytic subunits of protein phosphatase-! or -2A (see Section III.C.2 for nomenclature of phosphatases). If the kinase is partially purified in its inactive form by omitting the protein phosphatase inhibitors, it can be reactivated by addition of Mg-ATP (Fig. 7). This reactivation appears to be due to a distinct "kinase kinase" rather than to autophosphorylation by the kinase itself, because the reactivation does not occur after A

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126

D . G . Hardie

further purification of the inactive kinase by phosphocellulose chromatography. Intriguingly, the reactivation is markedly stimulated by nanomolar concentrations of palmitoyl-CoA. The important physiological implications of this finding are discussed in Section IV.B.3. Initial peptide mapping studies using the trypsin/chymotrypsin double digest suggested that AMP-PK phosphorylated sites within peptides (TC1 and TC2) identical to those phosphorylated by cyclic AMP-dependent protein kinase (Fig. 5). However, for the same degree of phosphorylation, inactivation by the kinase was much more dramatic than that by cyclic AMP-dependent protein kinase. The explanation for this apparent anomaly came from sequencing studies in which it was shown that AMP-PK phosphorylates exclusively the fourth residue within the TC1 peptide (corresponding to ser-79 in the rat enzyme, Fig. 4) whereas cyclic AMP-dependent protein kinase phosphorylates exclusively the second residue (ser-77). Both kinases also phosphorylate the TCI peptide, which contain a single phosphorylatable serine residue (ser-1200). 98 It is intriguing that such a subtle difference in the sites of phosphorylation should produce such a dramatic difference in the kinetic properties of acetyl-CoA carboxylase. The relationship between AMP-PK and the kinase activity described by Lent and K i m 79 is currently unclear. They have similar effects on acetyl-CoA carboxylase activity, and both are recovered in the 3-5% polyethylene glycol precipitate from a crude liver extract. AMP-PK fractionates in a similar manner to the kinase of Lent and Kim on ion exchange chromatography and gel filtration, and we have certainly not observed another acetyl-CoA carboxylase kinase which separated from AMP-PK beyond the first stage in the purification (polyethylene glycol precipitation). Although Lent and Kim do not appear to have examined the effects of AMP on their activity, there is evidence that both kinase activities are activated by phosphorylation. There are, however, some curious discrepancies. Lent and Kim reported that their kinase activity was activated by phosphorylation by cyclic AMP-dependent protein kinase, but we have failed to find any effect of the latter kinase on AMP-PK, even under conditions where the endogenous kinase kinase clearly activated the enzyme (Fig. 7). Lent and Kim also reported that phosphorylation of their kinase reduced the Km for acetyl-CoA carboxylase from 93 to 45 raM, without affecting Vm~However, the acetyl-CoA carboxylase kinase activity of AMP-PK is activated dramatically by phosphorylation when the substrate is at 2/ZM, which is close to a saturating concentration. Whatever the relationship between the kinase of Lent and Kim and AMP-PK, there is now very good evidence, discussed in Section III.C.3, that the latter is the most important protein kinase for physiological regulation of the activity of acetyl-CoA carboxylase. The kinase phosphorylates several other proteins in addition to acetyl-CoA carboxylase (Table 1), and its physiological role in fatty acid synthesis and other pathways is discussed in Section IV. (d) The calmodulin-dependent multiprotein kinase. This protein kinase (also known as calmodulin-dependent protein kinase II) was discovered independently in several different laboratories who were using various substrates for their kinase assays and different tissue sources for purification. It is now clear that these original reports were all describing the same activityJ° The enzyme does exist in at least three isozyme forms which are expressed to different degrees in different tissues and appear to be distinct gene products.~23 However, all of these isozymes have apparently identical substrate specificities. Substrates which are phosphorylated in vitro include enzymes (e.g. glycogen synthase, tyrosine hydroxylase, tryptophan hydroxylase), cytoskeletal proteins (e.g. tubulin, microtubule-associated protein 2) and proteins involved in neutrotransmitter release (synapsin-1). This broad specificity contrasts with the very restricted specificity of other Ca 2+- and calmodulindependent protein kinases such as mysosin light chain kinase and phosphorylase kinaseJ ° Numerous extracellular signals increase cytosolic Ca 2+ concentration, either by release from intracellular stores or by opening plasma membrane Ca ~+ channels. Although few of the substrates mentioned above have yet been firmly established to be physiological

Regulation of fatty acid synthesis

127

targets for protein kinase in vivo, the wide distribution and broad specificity of this enzyme suggest that it may be a general mediator of Ca 2+ effects in cells, somewhat analogous to the role of the cyclic AMP-dependent protein kinase in the actions of cyclic AMP. The calmodulin-dependent multiprotein kinase phosphorylates acetyl-CoA carboxylase purified from rat mammary gland in a Ca 2+- and calmodulin-dependent manner. 4~ Phosphorylation is stoichiometric and, at concentrations o f protein substrate o f 2/~ M, the initial rate is ~ 20% of the initial rate of phosphorylation of glycogen synthase by this kinase. We were unable to demonstrate any effect of this phosphorylation on either the K, of acetyi-CoA carboxylase for citrate or the Vm~. Thus, there is a marked difference between the effects on enzyme activity of the calmodulin-dependent multiprotein kinase and the other protein kinases mentioned above. This suggested that the former protein kinase must be phosphorylating novel site(s) on the enzyme, a suggestion that has now been confirmed by amino acid sequencing. 5~ The major site of phosphorylation was found in an 18 residue tryptic peptide (T4) quite distinct from those containing sites 1 and 2/3. 32p radioactivity was recovered in the seventh cycle of Edman degradation, giving the phosphorylation site sequence: -phe-ile-ile-gly-ser-val-ser(P)-glu-asp-asn-ser-glu-asp-glu-ile-ser-asn-leu. This phosphorylation site can now be identified with set-25 in the complete sequence of the rat enzyme; s3 somewhat surprisingly it does not fit with the sequence arg-X-ser(P) which has been suggested as the minimal sequence for recognition by the caimodulindependent multiprotein kinase, based on studies with synthetic peptidesJ ~2 Although phosphorylation of ser-25 has no effect on the activity of acetyi-CoA carboxylase under the conditions we have examined, we do have evidence that this site is phosphorylated in intact cells. This will be discussed in Section III.C.3.

(e) The Ca 2+- and phospholipid-dependent protein kinase (protein kinase C). This protein kinase was originally described in rat brain extracts as a protein kinase that was only active after treatment with proteases. In 1979, it was reported that the enzyme could be activated reversibly (i.e. without proteolysis) by Ca ~+ and a crude sonicated suspension o f m e m b r a n e phospholipids? 32 Subsequently, it was found that full activity could be achieved at physiological Ca 2+ concentrations using suspensions of pure phosphatidylserine containing trace amounts of diacylglycerol.75 The diacylglycerol, in which at least one of the fatty acids had to be unsaturated, both increased the maximal velocity and also greatly increased the sensitivity to Ca ~+. This was of great interest because diacylglycerol was already known to be produced transiently by a wide variety of extracellular signals which produce the "PI effect.''~2 This effect is a breakdown of plasma membrane phosphatidylinositol-4,5TABLE1. Initial Rate of Phosphorylation of Various Substrate Proteins by the AMP-activated Protein Kinase* Initial velocity (nmol/min/ml) Relative Concentration rate Substrate (~M) (mg/ml) + AMP - AMP (%) Acetyl-CoA carboxylas¢ 2.0 0.48 89 23 100 Glycogen synthase 2.0 0.18 64 17 73 Phosphorylase kinase 2.0 0.66 55 14 62 Hormone-sensitiye lipas¢ 1.0 0.08 35 10 40 Bovine casein -2.0 21 1.7 24 ATP-citrate lyase 2.0 0.23 4.8 1.5 5 6-Phosphofructo-2-kinase/ fructosc-2,6-bisphosphatase 2.0 0. l I 4.7 -5 Fructose-1,6-bisphosphatase 2.0 0.07 2.9 -3 Pyruvate kinase (L) 2.0 0.12 i.0 -2 6-Phosphofructo-l-kinase 2.0 0.16 <0,5 -<0.5 'The amount of kinase added was varied for different substrates, but under the conditions used (extent of phosphorylafion <0.I mol/subunit) the incorporation was linear with time and amount of kinase added. In the right hand column, rates are expressed as percentages of the rate with acetyl-CoA carboxylase as substrate. • .

128

D.G. Hardie

bisphosphate to produce inositol-l,4,5-triphosphate, now believed to be the second messenger responsible for the release of intracellular stores of Ca 2+, and diacylglycerol. Extracellular signals producing this response include hormones (e.g. adrenaline acting at at receptors, vasopressin acting at V, receptors), neurotransmitters (e.g. acetylcholine acting at at least one class of muscarinic receptor) and growth factors (e.g. platelet-derived growth factor). There are also now suggestions that other extracellular signals, which do not cause inositol trisphosphate production (e.g. insulin), may cause release of diacylglycerol from other derivatives of phosphatidylinositol. ~2° Interest in this protein kinase was further heightened when it was found that phorbol esters, which are pharmacological agents produced by certain plants and which were known to be tumor promoters, would substitute for diacylglycerol in the activation of protein kinase C. 25 There is now good evidence that protein kinase C is the only high affinity "receptor" for phorbol esters in cells. ~° As well as potentially explaining the tumor-promoting effects of these compounds, this observation meant that the phorbol esters could be used as experimental tools to activate protein kinase C specifically in intact cells. Protein kinase C phosphorylates acetyl-CoA carboxylase stoichiometrically, resulting in a modest depression of Vm,~.45 This inactivation is caused by the phosphorylation, since it is completely dependent on the presence of Ca 2+ and phospholipid and is reversed by protein phosphatase-2A. Amino acid sequencing shows that protein kinase C phosphorylates 2 sites, now identified as ser-77 and ser-95) 4's3 Since ser-77 is also phosphorylated by cyclic AMP-dependent protein kinase, 9s phosphorylation at this site may be responsible for the decrease in Vm,~ produced by protein kinase C. Despite the fact that protein kinase C does produce a modest effect on acetyl-CoA carboxylase activity, we do not believe that this kinase phosphorylates acetyl-CoA carboxylase under physiological conditions in the intact cell. We have not found any phosphorylation of ser-77 or ser-95 in intact cells, even after stimulation by phorbol esters, u These results are discussed in more detail in Section III.C.3(b). Also significant is the fact that ser-95 is the only one of the nine phosphorylation sites on the rat enzyme which is not conserved in the predicted sequence for the chicken enzyme (in the latter case, it is valine)) 3~ (f) Casein kinases-I and-2. Casein kinases-1 and -2 were initially described in extracts of rabbit reticulocytes, but have now been purified and characterized from a number of tissues, and appear to be very widely distributed) ° They are usually assayed using commercial casein as substrates, which has led to their naming as casein kinases. It should, however, be stressed that the protein kinase which phosphorylates casein physiologically, in lactating mammary gland, does not utilize commercial, mature casein as substrate unless it is first partially dephosphorylated, and is almost certainly a distinct protein from casein kinases-I and-2. 87 In experiments aimed at the isolation of cyclic AMP-independent acetyl-CoA carboxylase kinases, two such activities were separated from both rat liver extracts '36 and from extracts of lactating rat mammary gland, t°' On further examination, these two activities appeared to be identical with casein kinases-I and -2 by the criteria of subunit size, native molecular weight, chromatographic behavior on phosphocellulose, and sensitivity to inhibition by the glycosaminoglycan, heparin. Phosphorylation by either casein kinase-I or -2 does not affect the kinetic properties of acetyI-CoA carboxylase, so the physiological significance of these reactions, if any, is not clear. Casein kinase-2 phosphorylates a single site which has now been identified as ser-29, 5~'83almost adjacent to the calmodulin-dependent protein kinase site (Fig. 4). As will be discussed in a later section, ser-29 is phosphorylated in intact, isolated adipocytes, and phosphorylation at this site increases in response to insulin, s' Phosphorylation at ser-29 fits well with the "consensus" sequence that has been established for casein kinase-2 using studies of synthetic peptide substrates, i.e. a serine residue immediately followed on the C-terminal side by a cluster of acidic residues. 88

Regulation of fatty acid synthesis

129

At present, the site(s) phosphorylated by casein kinase-I on acetyl-CoA carboxylase have not been characterized in detail, although based on peptide mapping ~°1 they appear to be distinct from the sites shown in Fig. 4, and there is no evidence that they are phosphorylated in intact cells.

2. Protein Phosphatases Acting on Acetyl-CoA Carboxylase Extensive studies in the laboratories of Cohen and others have suggested that four protein phosphatases, i.e. phosphatases-1, -2A, -2B and -2C, account for all of the cytosolic protein phosphatase activity which is active against a wide range of phosphoserine/ phosphothreonine-containing substrates involved in glycogen metabolism, glycolysis/ gluconeogenesis, amino acid breakdown, and protein synthesis) °.67The properties of these four enzymes are summarized below: (a) Protein phosphatase-l. Protein phosphatase-1 has a catalytic subunit of 38 kDa ~39 which can exist as a free monomer, but is present in vivo as complexes with other polypeptides. In cytosol fractions, it is normally present as an inactive complex with a 23 kDa heat-stable protein called inhibitor-2. 4°'57'H7This inactive complex can be activated by phosphorylation of the inhibitor-2 subunit on a threonine residue, by a protein kinase termed glycogen synthase kinase-3, 5s'~4~although the physiological significance of this event remains uncertain. A substantial proportion of protein phosphatase-I is found in various particulate fractions, e.g. glycogen particles and microsomes. This association with particulate fractions is though to be mediated by binding of the catalytic subunit to distinct "targetting" subunits, which, in the case of glycogen particles, is a single polypeptide of 161 kDa (the G-subunit). 65 The free catalytic subunit is also regulated by another heat-stable protein called inhibitor-l, which inhibits the phosphatase at nanomolar concentrations, but only after inhibitor-I is phosphorylated on a threonine residue by cyclic AMP-dependent protein kinase. 3 For substrates for cyclic AMP-dependent protein kinase which are dephosphorylated exclusively by protein phosphatase-i, this would represent an attractive mechanism whereby cyclic AMP would simultaneously activate phosphorylation and inhibit dephosphorylation. It also represents a mechanism whereby substrates which are phosphorylated by protein kinases other than cyclic AMP-dependent protein kinase could still be regulated by cyclic AMP via protein phosphatase-l. The glycogen-bound form of protein phosphatase-I is also inhibited by inhibitor-l, although the inhibition is slow in onset. ~29 However, this may not matter because the glycogen-bound form dissociates to the free catalytic subunit in response to cyclic AMP elevation, due to phosphorylation of the G-subunit. 3° (b) Protein phosphatase-2A. In crude tissue extracts, protein phosphatase-2A is found to exist in three different forms, referred to as 2A0, 2A~ and 2A2. ~38All contain the same 35 kDa catalytic subunit (C) complexed to other subunits of 60 kDa (A), 55 kDa (B) and 54 kDa (B'). The free catalytic subunit (2A~) can be generated by various manipulations such as ethanol treatment, and is fully active. The native subunits structures of 2Ao, 2A~ and 2A 2 appear to be AB'C2, ABC2 and AC, respectively, and there is some evidence that 2A2 may be derived from 2A0 and/or 2A~ by dissociation or degradation of the B/B' subunits during extraction. None of the four forms of protein phosphatase-2A are inhibited by inhibitors-I and -2 in vitro, and the physiological regulation of protein phosphatase-2A, if any, remains unknown. However, the purified enzymes are dramatically activated in vitro by basic polypeptides such as historic HI, protamine and polylysine, and by Other naturally-occurring polycations such as spermine. H4 (c) Protein phosphatase-2B. Protein phosphatase-2B is a Ca~+-dependent protein phosphatase that contains a 61 kDa catalytic (A) subunit and a 15 kDa regulatory (B) J.P.L R 28/2--E

130

D.G. Hardie

subunit which is homologous with proteins of the Ca2+-binding family (e.g. calmodulin, troponin-C). 4"~7 Although the B-subunit alone does confer activation of the enzyme by physiological Ca z+ concentrations, an additional ten-fold activation is obtained by exogenous calmodulin in the presence of Ca 2+ ions. Protein phosphatase-2B is particularly abundant in brain and, when purified from this source, has been termed calcineurin,76The true physiological substrates for this protein phosphatase are not yet clear, but, compared with protein phosphatases-l, -2A and -2C, it appears to have a rather limited substrate specificity. (d) Protein phosphatase-2C. Protein phosphatase-2C has been purified from avian smooth muscle~H and, more recently, from rabbit skeletal muscle and liver. 59'92'96From the latter sources, the enzyme consists of two monomeric proteins of 42 and 44 kDa which are closely related but appear to be distinct gene products. Protein phosphatase-2C is completely dependent on Mg2+ ions for activity, and appears to have a relatively broad substrate specificity. (e) Acetyl-CoA carboxylase phosphatases. Using the criteria of Ingebritsen and Cohen, 67 we have examined the dephosphorylation of acetyl-CoA carboxylase (32P-labelled using cyclic AMP-dependent protein kinase) by protein phosphatase in crude cytosol fractions of rat liver. ~ Neither inhibitor-I nor inhibitor-2 had a significant effect on the dephosphorylation using this substrate. This suggested that protein phosphatase-I was not contributing to the dephosphorylation, although purified protein phosphatase-! can dephosphorylate the enzyme in vitro. 47 This result is not unexpected, since the protein phosphatase-I present in the cytosol fraction would be likely to be present as the inactive complex with inhibitor-2. Resolution of the acetyl-CoA carboxylase phosphatase activities in the crude cytosol fraction by ion exchange chromatography and gel filtration showed that they copurified with protein phosphatases-2A0, -2AI, -2Az and -2C. No evidence was obtained for any novel or specific acetyl-CoA carboxylase phosphatase. Although we have not examined the dephosphorylation of acetyl-CoA carboxylase phosphorylated by AMP-PK in such detail, purified protein phosphatases-2Ac, z3 -1 and -2C (unpublished results) will readily dephosphorylate this substrate in vitro. Krakower and Kim 77 have described a protein phosphatase in rat fat tissue which appears to associate with acetyl-CoA carboxylase as based on comigration with the polymerized form of carboxylase during sucrose gradient centrifugation. This activity was further purified by ethanol treatment and gel filtration. The activity had an apparent molecular weight of 26,000 on Sephadex G-75, dephosphorylated both acetyl-CoA carboxylase and phosphorylase, but was insensitive to inhibitor-2. These data suggest that it was probably a form of protein phosphataseo2A. Krakower and Kim claimed that this preparation was homogeneous, and had a molecular weight by SDS-polyacrylamide gel electrophoresis of 71,000. However, the low degree of purification and low specific activity compared with authentic phosphatase-2A, and the discrepancy with the native molecular weight by gel filtration, suggest that this claim was probably incorrect, and that the 71 kDa polypeptide was a contaminant in their preparation rather than the phosphatase itself. 3. Phosphorylation of Acetyl-CoA Carboxylase in Intact Cells

(a) Hepatocytes. In isolated rat hepatocytes, glucagon inhibits fatty acid synthesis (measured as incorporation of radioactivity from [3I-I]H20 into saponifiable lipid) by 50%, 62 whereas insulin stimulates the rate by 40%. 6° Since these hormones act on other pathways via protein phosphorylation, it seemed likely that they would modulate the phosphorylation state of acetyl-CoA carboxylase. Initial evidence that acetyl-CoA carboxylase is phosphorylated in vivo came from observations that enzyme purified from rat liver contained covalently bound phosphate. ~° Then Witters et al. ~45 reported that a prominent phosphoprotein of 240 kDa in the soluble fraction of 3eP-iabelled hepatocytes could be precipitated with antibody against acetyl-CoA carboxylase. Phosphorylation was

Regulation of fatty acid synthesis

131

evident in control cells incubated without hormones, but was also stimulated by glucagon. These studies were extended in our laboratory in collaboration with Witters, by purifying the enzyme to homogeneity on avidin-Sepharose columns in the presence of protein phosphatase inhibitors. 62 We were able to show that glucagon treatment increased the phosphate content of the enzyme from 4.5 to 5.2 tool per subunit, and that this increase was associated with a decrease in V,~ and an increase in the K, for citrate. At physiological citrate concentration (0.5 mM), the decrease in activity was about 50%, correlating rather well with the decrease in fatty acid synthesis observed under the same conditions. Analysis of 32P-labelled chymotryptic peptides by reversed phase HPLC showed that most of the increase due to glucagon was in a single peptide (peptide 1), which comigrated with the major chymotryptic phosphopeptide found after phosphorylation of the purified enzyme by cyclic AMP-dependent protein kinase. These results led to the tentative conclusion that inhibition of fatty acid synthesis by glucagon was the result of direct phosphorylation of acetyl-CoA carboxylase by cyclic AMP-dependent protein kinase.62 Pekala e t al. ~3 also found that the enzyme was highly phosphorylated in 32P-labelled monolayers of chicken hepatocytes, and estimated that the phosphate content was 10mol/subunit. However, the enzyme was isolated by immunoprecipitation, and the estimate of the amount of carboxylase protein recovered was based on the number of units of enzyme activity precipitated. Since the specific activity of the enzyme depends on its phosphorylation state, this method is prone to error. They also reported that dibutyryl cyclic AMP did not stimulate the phosphorylation. However, these agents were added at the same time as the [32P]phosphate, and steady-state of labelling was not achieved for several hours, by which time these agents may well have been degraded. In fact, inspection of their data reveals a stimulation of labelling by dibutyryl cyclic AMP in the early time points. Since carrying out our original studies with 32P-labelled hepatocytes, several observations accumulated which necessitated a re-examination of the involvement of cyclic AMP-dependent protein kinase. One conclusion that emerged was that the 4mol phosphate/subunit present in enzyme isolated from control cells was of regulatory significance, since treatment with purified protein phosphatase greatly activated the enzyme (Fig. 8). Since the activation involved a very large increase in Vm,~, phosphorylation by cyclic AMP-dependent protein kinase could not account for the low activity of the enzyme in the control cells. These observations triggered a search for the presence of cyclic AMP-independent acetyl-CoA carboxylase kinases in rat liver, a search which led to our discovery of AMP-PK. 2~3 The studies of the sites phosphorylated on acetyl-CoA carboxylase by this kinase and by cyclic AMP-dependent protein kinase9s then led to the realization that the phosphorylation of the enzyme in hepatocytes must be re-examined. The chymotryptic peptide 1 observed in the original study corresponded to the peptide C1 4

Control+phosphatas%

0CI

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F

Control gon

o

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'

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[citrate] (raM) FIG. 8. Activity (as a function of citrate concentration) of acctyi-CoA carboxylas¢ purified in the presence of protein phospbatas¢ inhibitors from isolated hepatocytes treated with or without glucagon (circles). Also shown are the activities after incubation of the purified enzyme with protein phosphatasc-2A (squares). Reproduced from Sire and Hardie. t'5

D. G. Hardie

132

[CONTROL 15[ TcTC2 10 S

,]'CONTROL :' ~" TI~ , ~ 6 0 ....

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20

40

60

40

80

120

Retention time (rain) FIG. 9. HPLC phosphopeptide maps of acetyi-CoA carboxylase phosphorylated in isolated 32P-labelledhepatocytes with or without glucagon (upper panels), or purified enzyme phosphorylated using [y-~2P]ATP and cyclic AMP-dependent protein kinase (cAMP-PK--lower panels). Digestion was with trypsin plus chymotrypsin (left panels) or trypsin alone (right panels) as in Fig. 3. Reproduced from Sim and Hardie.*~

(arg-met-ser-phe), containing serine-1200 (see Section III.C.l(a)). However, this serine is phosphorylated by both the AMP-activated and cyclic AMP-dependent protein kinases. To distinguish which kinase was responsible for the phosphorylation in the intact cells, it was necessary to analyze the tryptic peptide containing serines-77 and 79. Figure 9 shows HPLC profiles of acetyl-CoA carboxylase which had been purified from control or glucagon-treated hepatocytes and then digested with either trypsin (T) or trypsin plus chymotrypsin (TC). It is clear that glucagon stimulates the phosphorylation of peptide TI (containing ser-77/79) and also both TC1 and TC2 (containing ser-77/79 and ser-1200, respectively: TC2 is identical with chymotryptic peptide CI). Analysis of the location of the [32p]phosphate within T1 (Fig. 10) produced the surprising result that it was essentially all in ser-79 (phosphorylated by AMP-PK) and n o t in ser-77 (phosphorylated by cyclic AMP-dependent protein kinase). )2s Thus, it appears that AMP-PK is responsible not only for the low basal activity of acetyl-CoA carboxylase in rat hepatocytes, but also for the GLUCAGON

CONTROL 100 80 60 40

tide

20 Cycle No: Residue released:

1 S

2 S

3 M

4 S

5 G

6 L

S

2 S

3 M

4 S

5 G

6 L

FIG. 10. Analysis of the location of phosphoserine within peptide T1, purified from 32P-labelled hepatocytes which had been treated without hormone (control) or with glucagon. During the cleavage reaction in the gas phase sequencer, phosphoserine breaks down to free phosphate, which remains bound, along with any phosphopeptide remaining, to the ~ fiber filter. The analysis was, therefore, carried out by eluting radioactivity from sections of the filter after various cycles of degradation, and analyzing for content of phosphate/phosphopepfide by clectrophoresis at pH 1.9. Reproduced from Sim and Hardie.m

Regulation of fatty acid synthesis

133

further decrease in activity brought about by glucagon. Since glucagon is believed to exert most, if not all, of its actions in hepatocytes by increasing cyclic AMP, cyclic AMP-dependent protein kinase is probably involved, but not by directly phosphorylating acetyl-CoA carboxylase. This question is addressed further in Section III.C.3(b). Figure 9 shows that an additional tryptic peptide on the enzyme (T4) is phosphorylated in the hepatocytes, but does not change in response to glucagon. Phosphorylation within this peptide is increased in response to insulin, although this is not associated with a change in enzyme activity. Peptide T4 recovered from isolated hepatocytes comigrates with the peptide containing ser-23, ser-25 and ser-29, the latter being the predominant site for casein kinase-2. S~'6°Phosphorylation within this peptide has been studied in much more detail in isolated adipocytes 6° (see below). We also found that epidermal growth factor (EGF) had very similar effects to insulin both on the rate of fatty acid synthesis and on phosphorylation of peptide T4 in acetyI-CoA carboxylase. 6° This is of interest because it was subsequently shown that the receptors for insulin and EGF are related, ~4°both having hormone/growth factor-binding domains on the outside of the membrane (which show limited homology), a single transmembrane domain, and homologous protein (tyrosine) kinase domains on the inside of the membrane. The tyrosine kinase activities of the intracellular domains are activated by binding of hormone/growth factor to the extracellular domains. These results suggest that insulin and EGF activate acetyl-CoA carboxylase by a common mechanism, and it seems likely that the tyrosine kinase activity of the respective receptors will be involved in this mechanism. (b) Adipocytes. Phosphorylation of acetyl-CoA carboxylase in isolated rat adipocytes was first demonstrated by Brownsey et al., 19 who also showed that phosphorylation was stimulated by adrenaline. In collaboration with our laboratory, 18 it was subsequently shown that the adrenaline-induced increase was at least partly accounted for by phosphorylation of a tryptic peptide identical with that containing a site phosphorylated by cyclic AMP-dependent protein kinase. This peptide, originally analyzed by isoelectric focussing and termed the pI 7 peptide, can now be identified with T1, containing ser-77/79. In a later study carried out in collaboration with Clegg and Zammit, 6~ we reported that adrenaline and glucagon had similar effects on phosphorylation, increasing the phosphate content from 3.3 to 4.7 and 3.7 mol/subunit, respectively. This increased phosphorylation was at least partly accounted for by phosphorylation of chymotryptic peptide C1, now known to contain ser-1200, 9s and was associated with a large decrease in enzyme activity. As was the case with our hepatocyte studies, the finding that the AMP-activated and cyclic AMP-dependent protein kinase both phosphorylated ser-1200, but phosphorylated different residues within peptide T1, 9s necessitated a closer examination of the sites phosphorylated in isolated adipocytes. Figure ! 1 shows that adrenaline caused increased phosphorylation within peptide T1, and sequence analysis of TI (Fig. 12) confirmed that, as in hepatocytes, all of the phosphate was in ser-79 and not ser-77. 55 This demonstrates that the phosphorylation in response to adrenaline is catalyzed by AMP-PK and invalidated our previous tentative conclusions that the effects of adrenaline and glucagon were mediated directly by cyclic AMP-dependent protein kinase. '8 We considered several explanations for these unexpected findings: (1) Adrenaline may be acting through a mechanism not involving cyclic AMP, e.g. through binding to ct~ receptors. (2) Adrenaline may increase cyclic AMP, which is then broken down to Y-AMP, the latter activating AMP-PK. (3) Cyclic AMP-dependent protein kinase may phosphorylate and activate AMP-PK. (4) The effect of adrenaline may be mediated by inhibition of protein phosphatases, e.g. phosphorylation by cyclic AMP-dependent protein kinase of inhibitor1, which would then inhibit protein phosphatase-l. To examine the first hypothesis, we studied the effect of specific adrenergic agonists. Figure 13 shows that the ~-agonist isoproterenol produced an inactivation of acetyl-CoA carboxylase that was almost as large as that produced by adrenaline, and was stable during purification of the enzyme to homogeneity. The a-agonist phenylephrine had no effect (not

134

D . G . Hardie ISOPROTERENOL

CONTROL

100 30

80 20

60

40 A

I

10

/

/

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,dj

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100

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80

60 40

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50

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Retention Time (min)

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shown). These results indicate that in adipocytes adrenaline is acting on acetyl-CoA carboxylase entirely through/~-receptors, although it should be noted that Ly and Kim ss have reported phosphorylation and inactivation of acetyl-CoA carboxylase by =-agonists in rat hepatocytes. The second hypothesis was examined by using various pharmacological agents which activate cyclic AMP-dependent protein kinase independently of hormone receptors. Figure 14 shows that forskolin, IBMX or a combination of cyclic AMP analogues, the latter chosen for their ability to activate cyclic AMP-dependent protein kinase, all mimicked the effect of adrenaline. These studies strongly indicate that cyclic AMP-dependent protein kinase must be involved somewhere in the pathway upstream of AMP-PK. Since IBMX elevates cyclic AMP by inhibiting phosphodiesterase, the results with this agent rule out the possibility that the response is due to breakdown of cyclic AMP to Y-AMP. In view of the fact that Lent and Kim s~ have reported the existence of an acetyl-CoA carboxylase kinase in rat liver, which is apparently activated by cyclic AMP-dependent protein kinase [see Section III.C. 1(c)], the third hypothesis might appear to be particularly attractive. Unfortunately, we find that even very large amounts of cyclic AMP-dependent ADRENALINE

CONTROL

80 60 40 20 Cyde No: Residue released:

1 S

2 S

3 M

4 S

5 G

6 L

1 S

2 S

3 M

4 S

5 G

6 L

FIG. 12. Analysis of the location of phosphoserine within peptide TI, purified from 32P-labelled adipocytes which had been treated without hormone (control) or with adrenaline. See Fig. 10 for details of experimental methods.

135

Regulation of fatty acid synthesis

301CRUDE EXTRACT !__~E~t1,5 2"01"PURIFIEDT/~'~~--J-~ENZYME 25 Control 20 ~ p r o terenol 1015~5 TAdrenalin~"e 0

0

5 I

10 I

15 I

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r-

,

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[citrate] (raM)

,

10

,

15

[citrate] (mM)

FIG. 13. Activity (as a function of citrate concentration) of acetyl-CoA carboxylase in crude extracts (left panel) or after purification to homogeneity (right panel) from isolated adipocytes which had been treated with or without adrenaline or isoproterenol.

protein kinase do not activate the AMP-activated protein kinase under conditions where there is a large activation by endogenous "kinase kinase" (Fig. 7). However, given that the relationship of AMP-PK and the kinase described by Lent and Kim remains to be clarified, we cannot completely rule out the third hypothesis. At the moment, we favor the fourth hypothesis to explain the effects of adrenaline, i.e. that it inhibits protein phosphatase-I through phosphorylation of inhibitor-I by cyclic AMP-dependent protein kinase. Phosphatase-I in cell-free assays can dephosphorylate not only acetyl-CoA carboxylase phosphorylated by AMP-PK, but also the phosphorylated form of AMP-PK itself (unpublished results). Simultaneous inhibition of both of these phosphorylation events would be expected to cause a dramatic inactivation of acetyl-CoA carboxylase. A form of inhibitor-1 is present in rat adipocytes, and is known to be phosphorylated in response to isoproterenol, j°6 We have also found recently that protein phosphatase-1 accounts for most of the protein phosphatase activity in cell free extracts of adipocytes, using as substrate either phosphorylase a or acetyl-CoA carboxylase phosphorylated by AMP-PK (T. A. J. Haystead, unpublished). Unfortunately, this hypothesis cannot account for the effects of glucagon in rat hepatocytes, since these cells do not appear to contain inhibitor-l.~S In addition, phosphatase-2A appears to account for most of the acetyl-CoA carboxylase phosphatase activity in the soluble fraction of rat liver, ss although a shortcoming of these studies is that they were carried out using enzymes which had been phosphorylated using the cyclic AMP-dependent, rather than the AMP-activated, protein kinase. Possibly glucagon inhibits protein phosphatase-2A in rat hepatocytes via a novel mechanism, although there is no evidence for this at present. Insulin stimulates the incorporation of radioactivity from glucose into fatty acids in rat adipocytes by at least 25-fold. s' This dramatic effect is likely to be due to stimulation by

30 CRUDEEXTRACT a _ 25

ENZYME ~' 2.0 PURIFIED Control /

~

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10

0.5 I

I

5

10 I

[citrate] (raM)

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10 I

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15 I

[citrate] (raM)

FIG. 14. Activity (as a function of citrate concentration) of acetyi-CoA carboxylase in crude extracts (left panel) or after purification to homogeneity (right panel) from isolated adipocytes which had been treated with or without forskolin, isobutylmethylxanthine (IBMX) or a mixture of cyclic AMP analogues (N6-butyryl plus 8-thiomethyl-cyclic AMP).

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insulin of glucose transport and pyruvate dehydrogenase as well as acetyl-CoA carboxylase. However, there is also a I 0-fold increase in incorporation of radioactivity from acetate into lipidY Since acetate is taken up into cells by simple diffusion and is then converted directly into acetyl-CoA by cytoplasmic aeyl-CoA synthetase, this pathway is independent of glucose uptake and pyruvate dehydrogenase, and the large effect of insulin is likely to be mainly due to activation of acetyl-CoA carboxylase. An increase in acetyl-CoA carboxylase activity can indeed be measured in cell-free extracts after treatment of the cells with insulin. 44 This effect results from an increase in V,~x with no change in the Ka for citrate, and is much smaller (1.5- to 2-fold) than the effect on incorporation of radioactivity from acetate into lipid (9- to 10-fold). 52This suggests that, whatever is responsible for the effect of insulin on the pathway, it is only preserved to a small extent as a change in acetyl-CoA carboxylase activity in a cell-free extract. Since the well characterized effects of phosphorylation on acetyl-CoA carboxylase are to decrease enzyme activity, one would expect, if anything, that insulin would cause a d e p h o s p h o r y l a t i o n of the enzyme. However, Brownsey and Denton t6 reported the unexpected finding that insulin increased phosphorylation of acetyl-CoA carboxylase, albeit within a tryptic peptide (the "I site" peptide) distinct from those phosphorylated by cyclic AMP-dependent protein kinase. These observations were confirmed by Witters et al. ~ in adipocytes, and by our laboratory in hepatocytes6° and adipocytesY In adipocytes, EGF activates acetyI-CoA carboxylase in an identical manner to insulin, although it has a smaller effect on incorporation of radioactivity from glucose into lipid, possibly because it does not activate glucose transport to the same extent as insulin. 52 In any event, these results suggest that insulin and EGF activate acetyl-CoA carboxylase by a common mechanism, and it seems likely that the tyrosine kinase activity of the respective receptors will be involved in this mechanism. About the time of the original report that insulin increased the phosphorylation of acetyl-CoA carboxylase in adipocytes, Brownsey et al., ~5 reported that incubation of an adipocyte membrane fraction produced a time- and MgATP-dependent activation of partially purified adipocyte acetyl-CoA carboxylase. Using [~-32p]ATP, they also found phosphorylation of the enzyme, although they admitted that this preceded the activation. The same group subsequently found increased acetyl-CoA carboxylase activity in cell-free extracts of cells treated with insulin, and although there was no evidence that this was related to the membrane-bound kinase, they proposed a model in which insulin caused translocation from the membrane to the cytosol of a kinase which phosphorylated and activated acetyl-CoA carboxylase. 17 Although we have no reason to doubt that insulin increases a protein (serine) kinase which phosphorylates acetyl-CoA carboxylase, our data do not support a model in which this is responsible for activation of the enzyme. The evidence against this may be summarized as follows: (1) Brownsey and Denton ~6 originally reported that the effect of insulin on acetyI-CoA carboxylase activity survived partial purification of the enzyme, but both our laboratory 53and Witters et al. 146 have found that the effect is lost when the enzyme is purified to homogeneity on avidin-Sepharose. Although the yield on the affinity columns is <40% and it could be argued that we were selectively losing an insulin-activated forn~ of the enzyme, the effect on phosphorylation of the "I site" is still apparent after the affinity chromatographyY We have also found that the effect of insulin is lost when the cell free extracts are subjected to rapid gel filtration in high ionic strength, a result consistent with the idea that insulin is activating the enzyme via a low molecular weight dissociable factory This would also explain why the effect on enzyme activity in a cell-free extract is much smaller than the effect on incorporation of radioactivity from acetate into lipid. However, as yet, we have not succeeded in isolating this putative low molecular weight factor. (2) Incubation of cell-free extracts of hormone-treated adipocytes with large amounts of the catalytic subunit of protein phosphatase- 1 reversed the effects of adrenaline on acetyl-CoA carboxylase activity, but not those of insulin. However, in parallel experiments using extracts from [3~p]-labelled cells, it was clear that this treatment did cause almost complete dephosphorylation of the "I site"Y (3) We have recently identified the

Regulation of fatty acid synthesis

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major site phosphorylated in response to insulin as being within peptide T4 (Fig. 15), corresponding to residues 19 to 38 in the complete sequence. 5j Within this peptide, ser-23, ser-25 and ser-29 are all phosphorylated in control cells, but the effect of insulin seems to be on phosphorylation of set-29, based on findings that phosphorylation at this site causes a lower retention time for the peptide on reversed phase HPLC. Ser-29 is phosphorylated on the purified enzyme by casein kinase-2, 51 and phosphorylation by this kinase does not change the activity of purified enzyme.l°~ These findings are consistent with a recent report that casein kinase-2 is acutely activated when the adipocyte-like 3T3-LI mouse cell line is treated with insulin, j25aThey do not support the idea that the increased phosphorylation activates the enzyme. However, peptide T4, while similar to the insulin-sensitive peptide reported by Witters et al., appears to be different from the "I site" peptide as originally defined by Brownsey and Denton? We cannot rule out the possibility that phosphorylation within the "I site" peptide activates the enzyme, although the "I site" is a minor site which is only phosphorylated to a very small extent, even after insulin treatment. (4) The phorbol ester tetradecanoyl phorbol acetate (TPA), which activates protein kinase C, mimics the effect of insulin on phosphorylation of ser-29 in acetyl-CoA carboxylase (and of the "I site" peptide) when added to intact adipocytes. ~ However, it does not activate acetyl-CoA carboxylase or stimulate incorporation of radioactivity from acetate into lipid, even when in the same batch of cells insulin does stimulate these parameters. TPA does cause a modest stimulation of incorporation of radioactivity from glucose into lipid (probably because it activates glucose transport). It is worth noting that protein kinase C cannot be directly responsible for phosphorylation of acetyl-CoA carboxylase, because the two sites phosphorylated by this kinase on the purified enzyme (ser-77 and ser-95) are not phosphorylated in intact cells, even after phorbol ester treatment. (5) The toxin okadaic acid is a specific and potent inhibitor of protein phosphatases-I and -2A and causes dramatic increases in phosphorylation of many proteins, including acetyl-CoA carboxylase, when added to intact adipocytes. Although the toxin mimics the effect of insulin on glucose uptake (implying that this response involves increased phosphorylation), it completely blocks the effects of insulin on fatty acid synthesis. ~ Since we have shown that J.P.LR. 25,2--F

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protein phosphatase-1 dephosphorylates the "I site", this is the opposite of what one would expect if phosphorylation at this site activated the enzyme, unless increased phosphorylation at AMP-PK sites could over-ride the effects of phosphorylation at the "I site". While it could be argued that none of the five types of evidence quoted above, taken in isolation, conclusively rule out the hypothesis that increased phosphorylation in response to insulin activates the enzyme, we believe that taken together they make this extremely unlikely. It is also clear that under conditions where insulin does rapidly activate the enzyme in adipocytes, it does not bring about a dephosphorylation. Recently Witters et al) 49 have reported that, in the insulin-sensitive Fao Reuber hepatoma cell line, insulin does bring about an activation of acetyl-CoA carboxylase which is stable during purification, and which is accompanied by dephosphorylation. We have made similar observations in mammary cells [see Section III.C.3(c)]. It is worth noting that in both of these cases the incubation time with insulin (30-45 min) was rather longer than in our experiments with adipocytes (15 min). In our opinion, the rapid effects of insulin on acetyl-CoA carboxylase activity in adipocytes, therefore, must be due to a change in concentration of a small molecular weight effector, at present unidentified. Saltiel et al. TM reported that a low-molecular weight activator of acetyl-CoA carboxylase was released when rat liver plasma membrane preparations were incubated with insulin. It was subsequently found that a similar factor, which activated a particulate phosphodiesterase from adipose tissue, could be released from the membranes using phosphatidylinositol-specific phospholipase C. '2°''2z The phospholipase-generated activator has been purified and shown to be a phospho-oligosaccharide in which a carbohydrate moiety, not yet fully characterized, is attached via glucosamine to inositol phosphate. It has been proposed that the activator is released from a glycosylated phosphatidylinositol precursor by an insulin-activated phospholipase. This model has attractive features, in that the two products of this reaction would be a water-soluble phospho-oligosaccharide which might be responsible for activation of acetyl-CoA carboxylase, plus a hydrophobic diacylglycerol which could activate protein kinase C, and explain how phorbol esters mimic the effects of insulin on acetyl-CoA carboxylase phosphorylation. However, there are several problems: (1) Phospho-oligosaccharide purified by Saltiel on the basis of phosphodiesterase activation did not activate acetyl-CoA carboxylase when added to purified enzyme or to extracts from control or insulin-treated adipocytes (T. A. J. Haystead and D. G. Hardie, unpublished) There could, of course, be a family of these compounds with different functions. (2) Although phorbol esters mimic some effects of insulin, the available evidence suggests that insulin does not activate protein kinase C) 3 (3) The phospho-oligosaccharide appears to be released on the outside of the cell where it does mimic some actions of insulin. There is no evidence that this very hydrophilic compound can enter cells. It currently appears more likely that the phospho-oligosaccharide represents an insulinomimetic local mediator, rather than an intracellular messenger.'48 The mechanism of action of insulin, therefore, remains as elusive as ever. (c) M a m m a r y acinar cells. Fatty acid synthesis in the mammary gland of lactating rats makes great demands on the supply of glucose: the glucose uptake by the mammary glands of a lactating female (most of which is used for fatty acid synthesis) is approximately equivalent to the whole body glucose consumption of a male rat of the same age and weight. 1°4 It is, therefore, not surprising that the pathway should be stringently regulated when required. Two situations are known to cause inhibition of the very high rate of synthesis of fatty acid in this tissue: (1) Starvation for 24hr, which results in >98% inhibition) ta This effect is due to a successive inhibition of several steps in the pathway, which include glucose transport, phosphofructokinase, pyruvate dehydrogenase and acetyl-CoA carboxylase. 1°4The inhibition is totally reversed by refeeding for 2 hr, an effect which is abolished in streptozotocin-diabetic animals, "8 suggesting that the inhibition is due to the low circulating insulin in starved animals. (2) Feeding a high-fat diet (20% fat) for several days. This causes a 50% reduction in fatty acid synthesis compared with

Regulation of fatty acid synthesis

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standard chow diet (2% fat), and, in this case, the inhibition appears to result entirely from reduction in acetyl-CoA carboxylase activity) .t°5 McNeiUie et aL 93 showed that acetyl-CoA carboxylase in rat mammary extracts was activated by divalent cations in a fluoride-sensitive manner, suggesting that the enzyme was phosphorylated in vivo and was activated by endogenous divalent cation-stimulated protein phosphatases present in the extract. They also demonstrated indirectly by acetyl-CoA carboxylase activity measurements that the phosphorylation state increased after 24hr starvation of the rats. 94 Munday and Hardie 1°2,t°3 have confirmed that the decrease in acetyl-CoA carboxylase activity due to both starvation and fat-feeding results from increased phosphorylation, although in the case of 24 hr starvation there is also a reduction in the amount of acetyl-CoA carboxylase protein. Increased phosphorylation was demonstrated by showing that the reduction in activity was stable to purification in the presence of protein phosphatase inhibitors, and could be reversed by incubating the purified enzymes with protein phosphatase-2A. The total phosphate content of enzyme purified from chow-fed rats increased from 3.3 + 0.21 to 4.46 + 0.42 mol/subunit after 24 hr of starvation and 4.33 -t- 0.43 mol/subunit after 7 days on a high-fat diet: phosphatase treatment reduced this to 2-3 mol/subunit in each case. Both the low specific activity and the high phosphate content of enzyme from 24 hr-starved rats was reversed by refeeding for 2 hr, but, as with the effect on overall fatty acid synthesis, this did not occur if the rats were made diabetic with streptozotocin. These data show that the effects of starvation or fat feeding on acetyl-CoA carboxylase in lactating mammary gland are due to increased phosphorylation, and, at least in the case of the starved animals, this may result from their low circulating insulin concentrations. 73 Mammary acinar cells lack glucagon receptors but do have fl-adrenergic receptors. 27 However, elevation of cyclic AMP using combinations of fl-agonists and/or phosphodiesterase inhibitors does not appear to lead to either phosphorylation of acetyl-CoA carboxylase or inhibition of fatty acid synthesis. 2a'29 The effects of starvation are not, therefore, likely to be mediated by rises in cyclic AMP. For both starvation and fat-feeding, the drop in specific activity of acetyI-CoA carboxylase is due to a large decrease in V=, as well as an increase in the Ka for citrate, and the only known kinase which can account for such a large decrease in V,~x is AMP-PK. AMP-activated protein kinase has been detected in mammary gland extracts (unpublished results), but phosphorylation site analysis will be necessary to prove that it is responsible for the increased phosphorylation. Fat feeding is not associated with a drop in circulating insulin concentration, and, in this case, it is tempting to propose that the effect is due to fatty acyl-CoA activation of the AMP-PK cascade (see Section IV.B.3). Certainly the mammary gland takes up more fatty acids when the animals are on a high-fat diet, and the fatty acid composition of the milk changes to reflect more closely that in the dietary fat, rather than the characteristic composition of fatty acids synthesized de novo. The effects of fat-feeding on fatty acid synthesis are at least partially preserved during isolation of mammary acinar cells by collagenase digestion) Although the effects of fat-feeding in vivo may, as discussed above, be due to changes in the plasma concentration of triglycerides rather than in insulin, treatment of isolated acinar cell preparations with insulin can reverse the effects of high fat feeding. 1°3 Insulin causes both an increase in the specific activity and a decrease in the phosphate content of acetyl-CoA carboxylase purified from the cells. Thus, in this case, as in the Fao hepatoma cells used by Witters et al., ~49 insulin appears to cause a dephosphorylation of acetyI-CoA carboxylase, probably at the sites phosphorylated by AMP-PK.

IV. PHYSIOLOGICAL ROLES OF THE AMP-ACTIVATED PROTEIN KINASE From the previous section, it should be clear that AMP-PK appears to be responsible for regulation of acetyl-CoA carboxylase under a variety of circumstances. We shall now discuss the possible physiological roles of this kinase.

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A. Phosphorylation of Proteins Other Than Acetyl-CoA Carboxyla~e

When we discovered that AMP-PK was regulated by AMP and by phosphorylation, n we noticed that these properties were very similar to those of a previously described activity termed HMG-CoA rcductase kinasc. This enzyme was known to phosphorylate and inactivate HMG-CoA reductase, the important rate-controlling enzyme of cholesterol synthesis which is an integral membrane protein of the smooth endoplasmic reticulum) 9 As long ago as 1975, Brown et al. '4 reported that HMG-CoA reductace in microsomal fractions from human fibroblasts or rat liver was inactivated by a cytosolic factor which appeared to require the presence of both ATP and ADP. The factor was later identified as a protein kinase which appeared to be activated allosterically by ADP. ¢ It was subsequently shown that AMP was a much more potent activator. ~ In independent studies, two groups had also identified an HMG-CoA reductase kinas¢ which was regulated by phosphorylation, being activated by a "kinase kinase" and inactivated by protein phosphatase. 9'69 When we commenced our studies, it was clear whether the HMG-CoA reductase kinase activities, which were activated by phosphorylation or by AMP, were the same enzyme. However, we were easily able to show that our highly purified AMP-activated protein kinase would inactivate HMG-CoA reductase. ~ We used as our source of enzyme either rat liver microsomes, or membranes from the HMG-CoA reductase-overproducing UT-I cell line. The latter membrane preparations had the advantage that there was much less contamination with adenylate kinase (which interconverts ADP, ATP and AMP), and, using these membranes, we were able to show that the acetyI-CoA carboxylase kinase and HMG-CoA reductase kinase activities of our purified kinase preparation had the same sensitivity to AMP and to protein phosphatase treatment. We have subsequently shown that the acetyl-CoA carboxylase kinase and HMG-CoA reductase kinase activites exactly copurify during a six-step, 1000-fold purification from rat liver (unpublished data). Another important enzyme of lipid metabolism is hormone-sensitive lipase, '3° which is responsible for breakdown both of triacylglycerols in adipose tissue and cholesteryl esters in steroidogenic tissues (adrenal cortex, testis and ovary). The lipase is phosphorylatcd at a single serine residue by cyclic AMP-dependent protein kinase ~ [now identified as serine-5636~], and this is responsible for activation of lipolysis or cholesteryl ester breakdown in response to hormones which elevate cyclic AMP (adrenaline and glucagon in adipose cells, ACTH in adrenal cortex, lutropin and choriogonadotropin in testis/ovary). In collaboration with the laboratory Steven Yeaman, we have found that AMP-PK phosphorylates hormone-sensitive lipase purified from bovine adipose tissue at a serine residue (ser-565) almost adjacent to that phosphorylated by cyclic AMP-dependent protein kinasc? S This occurs in a sequence remarkably similar to that around ser-77/79 in acetyl-CoA carboxylase (Fig. 16). When the peptide containing ser-563/565 from bovine hormone-sensitive lipase was analyzed by sequencing, unlabelled phosphate was found at set-565, showing that it is at least partially phosphorylated in vivo. Phosphorylation at set-565 by AMP-activated protein kinase does not activate hormone-sensitive lipase, but it blocks phosphorylation and activation by cyclic AMP-dependent protein kinase at set-563. This mutual exclusion works in the reverse direction, and has also been found for the two analogous sites in acetyI-CoA carboxylase (ser-77/79), suggesting that the presence J. (~r-1200)

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Regulation of fatty acid synthesis

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of a neighboring positive charge prevents phosphorylation by either kinase. 99 In the case of hormone-sensitive lipase, this mutual exclusion is of great interest because it implies that anything which activates AMP-PK (e.g. fatty acyl-CoA, which stimulates the kinase kinase reaction) would block lipolysis. AMP-PK would also, therefore, exert a feedback control on the release of fatty acids from intracellular stores, as well as their synthesis. Additional substrates which are phosphorylated in vitro by AMP-PK include casein, glycogen synthase and phosphorylase kinase (Table 1).22 The phosphorylation of all of these substrates is stimulated by AMP, showing that the reaction is catalyzed by AMP-PK and not a contaminating kinase. A variety of other proteins which are good substrates for cyclic AMP-dependent protein kinase (e.g. L-pyruvate kinase, 6-phosphofructo 2-kinase/fructose-2,6-bisphosphatase) are not phosphorylated at significant rates by AMP-PK. Glycogen synthetase is phosphorylated at serine-7 (Fig. 16), and phosphorylation at this site increases the dependence of the enzyme activity on glucose-6-phosphate. H5 Glycogen synthetase is known to be phosphorylated at ser-7 in vivo, "6 although there are a number of other protein kinases, including cyclic AMP-dependent protein kinase, which phosphorylate the same site in vitro. It is conceivable that glycogen synthesis could be decreased by phosphorylation of glycogen synthetase by AMP-PK in vivo, brought about by rises in AMP induced by anoxia. Phosphorylation of phosphorylase kinase occurred at several sites, one of which was identified as ser-1020. 22 Since none of the sites was phosphorylated stoichiometrically, a physiological role for this phosphorylation is less likely, although ser-1020 is at least partially phosphorylated in vivo. TM B. How is the AMP-activated Protein Kinase Regulated in vivo? 1. Regulation by Hormones

Phosphorylation of acetyl-CoA carboxylase at the AMP-PK sites in response to glucagon treatment of isolated hepatocytes, and in response to adrenaline treatment of adipocytes, has been directly demonstrated by amino acid sequencing (see Section III.C.3). Although phosphorylation of HMG-CoA reductase in intact cells has not yet been studied in the same way, it has been studied indirectly by means of enzyme activity measurements. This is done by preparing microsomes in the presence of protein phosphatase inhibitors to measure the so-called "expressed" HMG-CoA reductase activity, and also preparing them in the absence of phosphatase inhibitors and incubating to allow complete dephosphorylation by endogenous phosphatases ("total" activity). The ratio of expressed :total activity can be taken as an inverse measure of phosphorylation state. It seems likely that the AMP-PK is responsible for the observed changes in activity of HMG-CoA reductase, although it should be noted that protein kinase C, and a Ca2+/calmodulin-de pendent protein kinase, have also been shown to inactivate HMG-CoA reductase in vitro, l°'t~ Using the approach described above, it has been shown that glucagon promotes phosphorylation, and insulin promotes dephosphorylation, of HMG-CoA reductase in isolated rat hepatocytes, u Using a "cold-clamping" technique which prevents phosphorylation of HMG-CoA reductase post-mortem, 3~ Zammit and coworkers have observed similar changes in response to insulin and glucagon in vivo. 32 These results suggest that cyclic AMP-elevating hormones increase phosphorylation at AMP-PK sites on both acetyI-CoA carboxylase and HMG-CoA reductase. As discussed in Section III.C.3, the mechanism for this remains unclear, although it is most likely to be mediated by inhibition of protein phosphatase activity. Insulin appears to promote dephosphorylation of HMG-CoA reductase, and at least in mammary cells and Fao hepatoma cells (Section III.C.3), of acetyl-CoA carboxylase also. Once again the mechanism is unclear but it may involve activation of protein phosphatases. 2. Regulation by A M P

When hepatocytes are starved of oxygen, AMP rises dramatically due to the action of adenylate kinase, which ensures that any small drop in ATP levels is accompanied by much

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larger increases in AMP. t42 AMP-PK may, therefore, be a mechanism for switching off biosynthetic pathways (fatty acid and cholesterol synthesis) when ATP is limiting. Although anoxia in liver probably represents a pathological state rather than a normal occurrence in vivo, this is an important technical point because it explains the necessity for cold- or freeze-clamping 3t'72'1~ to preserve the phosphorylation state of enzymes regulated by AMP-PK.

3. Regulation by Fatty Acyl-CoA Esters We believe the observation that the reactivation of AMP-PK by the kinase kinase is stimulated by long chain acetyl-CoA esters, 23[Section I II.C. 1(c)] is a particularly important clue to its function. This suggests that the kinase cascade exerts a feedback control on fatty acid synthesis. It may at first be thought odd that such a system should be necessary, since purified acetyl-CoA carboxylase is subject to direct allosteric inhibition by fatty acyl-CoA esters (Section Ill.B). However, a problem with the allosteric mechanism is that, while the total concentration of fatty acyl-CoAs in the rat hepatocyte is in the range of 1-10/~M, the free concentration is probably orders of magnitude lower, due to binding to very abundant cytosolic fatty acid/acyl-CoA binding proteins. ~'97 The concentration of acetylCoA carboxylase varies dramatically with diet, but in rat liver can reach levels equivalent to ~ 1-2 /~M. Since this is probably much higher than the concentration of free fatty acyl-CoA esters, a kinase cascade may be necessary to amplify the feedback signal. The phosphorylation of hormone-sensitive lipase by AMP-PK also represents a feedback system. 35 Fatty acids would be converted in the adipose cell to the CoA esters which would activate the kinase cascade, causing phosphorylation of HSL at the AMP-PK site, thus blocking phosphorylation and activation of HSL by cyclic AMP-dependent protein kinase, and preventing release of further fatty acids. An additional question is why such a feedback system, activated by fatty acyl-CoAs, should also inhibit HMG-CoA reductase. A possible clue comes from observations that suggest that rat liver fatty acid and cholesterol synthesis are in competition for acetylC o m . 3s It is conceivable that, when availability of excess fatty acyl-CoA shuts down fatty acid synthesis, simultaneous inhibition of HMG-CoA reductase is necessary to prevent the flux of acetyl units merely being diverted through cholesterol synthesis. The coordinate regulation of acetyl-CoA carboxylase and HMG-CoA reductase would also balance the rates of synthesis of the two major components of VLDL.

3. Regulation by Cholesterol Metabolites Cholesterol feeding of rats in vivo, or treatment of isolated hepatocytes with LDL, oxygenated cholesterol, or the cholesterol precursor mevalonolactone, all lead to decreases in the expressed/total activity ratio of HMG-CoA reductase. 39These results are consistent with the idea that cholesterol, or a derived metabolite, can also exert a feedback control on HMG-CoA reductase via the AMP-PK system. It is not clear whether these effects are mediated by binding of cholesterol metabolite(s) to HMG-CoA reductase itself, or to component(s) of the phosphorylation cascade. If the latter was correct, then they could exert a feedback effect both on cholesterol synthesis, and on the release of cholesterol from stored cholesteryl esters, the latter via phosphorylation of hormone-sensitive lipase. V. C O N C L U S I O N S

Acetyl-CoA carboxylase catalyzes the first step committed to fatty acid synthesis, and an abundance of evidence shows that it has a major regulatory influence on the pathway, particularly in the liver. In adipocytes and lactating mammary gland, various steps involved in the provision of acetyl-CoA (e.g. glucose transport, pyruvate dehydrogenase) may play additional regulatory roles in the overall pathway of f a t t y acid synthesis. Although acetyl-CoA carboxylase can be regulated in vitro by allosteric effectors, the major

Regulation of fatty acid synthesis

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form of acute regulation in vivo appears to be protein phosphorylation. Purified acetyl-CoA carboxylase can be phosphorylated in v i t r o by seven different protein kinases at up to seven serine residues which have now been defined by amino acid and e D N A sequencing. At least five of these sites are phosphorylated in intact, isolated hepatocytes or adipocytes. O f these physiological sites, phosphorylation at serine-23, 25 and 29 does not appear to modulate enzyme activity. However, phosphorylation of ser-29 occurs in response to insulin and epidermal growth factor, and may be catalyzed by casein kinase-2. Phosphorylation at ser-79 and/or ser-1200 is responsible for the direct regulation of enzyme activity, and the AMP-activated protein kinase is the only kinase that can account for tl~is phosphorylation. The AMP-activated protein kinase, therefore, appears to be the kinase most important for regulation of acetyl-CoA carboxylase in vivo. The AMP-activated protein kinase also phosphorylates and regulates H M G - C o A reductase and hormone-sensitive lipase, and therefore appears to play a general role in regulation of lipid metabolism. As its name suggests, the kinase is activated by physiological concentrations of AMP. As well as providing a mechanism for switching off lipid biosynthesis when cells are compromised by anoxia, this mechanism also explains the necessity for freeze- or cold-clamping when attempting to estimate the in vivo activity of acetyl-CoA carboxylase or H M G - C o A reductase. The AMP-activated protein kinase is also the final component of a phosphorylation cascade, being activated by phosphorylation by a "kinase kinase", currently poorly characterized. Phosphorylation by the AMP-activated protein kinase cascade is increased by hormones which elevated cyclic A M P and, at least in certain cases, decreased by insulin, but the detailed mechanisms of these effects are not known. A potentially important finding is that the protein kinase cascade is activated by low concentrations of fatty acyI-CoA esters, so that it could act as a novel feedback mechanism operating on both synthesis of fatty acids and their release from triacylglycerols. Results from studies with H M G - C o A reductase suggest that cholesterol, or a related metabolite, could also have feedback input via this kinase system. (Received 9 December

1988)

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(1980). 7. BEATV,N. B. and LANE,M. D. J. Biol. Chem. 258, 13043-13050 (1983). 8. BEAT',',N. B. and LANE, M. D. J. Biol. Chem. 258, 13051-13055 (1983). 9. BEG, Z. H., STONIK,J. A. and BREWER,H. B. Proc. Natl. Acad. ScL U.S.A. 76, 4375--4379 (1979). 10. BEG,Z. H., STOSIK,J. A. and BREWER,H. B. J. Biol. Chem. 260, 1682-1687 (1985). I1. BEG,Z. H., STOSIK,J. A. and BREWER,H. B../. Biol. Chem. 262, 13228-13240 (1987). 12. BERRIDGE,M. J. and IRVIN'E,R. F. Nature 312, 315-321 (1984). 13. BLACKSHEAR,P. J., WITTERS, L. A., GIRARD,P. R., Kuo, J. F. and QUAMO,S. N. J. Biol. Chem. 260, 13304-13315 (1985). 14. BROWS,M. S., BRUNSCHEDE,G. Y. and GOLDSmlS,J. L. J. Biol. Chem. 250, 2502-2509 (1975). 15. BROWNSEY,R. W., BELSHAM,G. J. and DENTON,R. M. FEBS Lett. 124, 145-150 (1981). 16. BROWNSEY,R. W. and DENTON,R. M. Biochem. J. 202, 77-86 (1982). 17. BROWSSEY,R. W., EDGELL,N. J. HOPKIRK,T. J. and Dmcros, g. M. Biochem. J. 218, 733-743 (1984). 18. BROWSSEY,R. W. and HARDIE,D. G. FEBS Lett. 120, 67-70 (1980). 19. BROWNSEY,R. W., HUGHES,W. A. and DEi'crols,R. M. Biochem. Y. 184, 23-32 (1979). 20. BRUNENGRABER,H., BOUTRY,M. and LOWESSTEIS,J. M. Eur. J. Biochem. 82, 373-384 (1978). 21. CARLING0D. and HARDIE,D. G. Biochem. Soc. Trans. 14, 1076-1077 (1986). 22. C,,~tLtSt.3,D. and HARDIE,D. G. Biochem. Biophys. Acta in press (1989). 23. CARLING,D., ZAMMIT,V. A. and HARDIn,D. G. FEBS Lett. 223, 217-222 (1987). 24. CARLSON,C. A. and KIM,K. H. J. Biol. Chem. 248, 378-380 (1973). 25. CASTAGNA,M., TAKAI,Y., KARmUCm,K., SASO,K., KIKKAWA,U. and NISHIZUKA,Y. J. Biol. Chem. 257, 7847-7851 (1982). 26. CLARK,M. G., ROGNSTAD,R. and KATZ, J. J. Biol. Chem. 249, 2028-2036 (1974). 27. CLEC,G, R. A. and MULLANEY,I. Biochem. J. 2~1, 239-246 0985).

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