Substrate diversity of the cAMP-dependent protein kinase: regulation based upon multiple binding interactions

Substrate diversity of the CAMP-dependent regulation based upon multiple binding Donal School

of Medicine,

A. Walsh, University

David

protein kinase: interactions

B. Glass and Ryan D. Mitchell

of California, Davis, California, and Emory Medicine, Atlanta, Georgia, USA

University

School

of

The proposition is forwarded that the CAMP-dependent protein kinase is one of quite a small class of enzymes wherein differential modes of binding of its multiple substrates make an important contribution to the end physiological response. It is postulated that a variety of different substrate affinities may have evolved in order to regulate the order of substrate phosphorylation. The recent elucidation of the protein’s three-dimensional structure provides the opening to test this as a new concept of cellular regulation.

Current

Opinion

in Cell

Biology 1992, 4:241-251

Introduction

(R2CL). These forms clearly differ in several attributes, including their sensitivity to CAMP, tissue-specific expression, autophosphorylation, and targeting to distinct intracellular sites directed by protein-specific binding or possible signal sequences. These differences may be sufficient to account for the physiological importance of the multiple forms, although intuitively a more comprehensive set of differences might be expected and may later be found. One noted absence in the list of distinctive attributes is any recognized difference in substrate specificity of the multiple forms of the protein kinase catalytic subunit, this despite the wide range of protein substrates so far identified. Of the three forms of catalytic subunit, Ca, Cp and Cy, both the a and p forms have been expressed as proteins from the respective cDNAs [ 41. The y isoform has so far only been evaluated at the level of the cDNA [5] and has yet to be characterized as a protein, but critical parts of its sequence are identical to Ca and Cp (see below). At the level of experimental evaluation now available, in particular considering the properties of the enzymes isolated from tissues with a known isoform mRNA selectivity, no differences in isoform substrate specificity are apparent. This will be expanded upon in this review. Currently, there is no evidence to suggest that there is any selectivity in the phosphorylation of different substrates by different isozyme forms. However, further investigation of this is warranted as a truly in-depth experimental analysis remains to be undertaken.

Last year’s contribution by McKnight [l] on the ‘Cyclic AMP second messenger systems’ provides an excellent review of the identity and multiplicit)l of the components of the CAMP second messenger system, particularly the subunits of the cAMP-dependent protein kinase. Because the review is still current, this year’s assessment will concentrate on just one aspect of the CAMP regulatory .systern, namely, the characteristics of the substrates for this enzyme. We present here a collection of thoughts, aided in particular by the elucidation of the tertiary structure of the catalytic subunit of the protein kinase by the Taylor and Sowadski laboratories [ 2**,3**], which suggest that an expanded insight of the interactions of the CAMPdependent protein kinase with its protein substrates is necessar)’ for a hIII understanding of what occurs physiologically. For most enzymes, once the substrate has been identified and the binding constants determined, understanding the reaction mechanism and the molecular interactions involved in catalysis can be of considerable interest to those inquisitive about physical biochemical principles. Rarely do such studies expand upon our knowledge of that enzyme’s physiological role. It is suggested that, for the CAMP-dependent protein kinase class of enzymes, further inquiry into reaction mechanisms and the details of substrate interactions with the active site will lead to important physiological insights. For the purposes of the thesis presented here, several issues reviewed by M&night [ 1) merit reemphasis. Unraveled initially by the use of molecular biology, there is now evidence for at least three forms of catalytic (C) subunit and four forms of regulatory (R) subunit of the CAMPdependent protein kinase, which together could produce at least twelve forms of protein kinase holoenzyme

C-catalytic;

PKI-protein @

Current

Proposition One of the first hallmarks identified for the CAMPdependent protein kinase was that it could catalyze the

Abbreviations kinase

Biology

Ltd

inhibitor; ISSN

R-regulatory.

0955-0674

241

242

Cell

regulation

-phosphon4ation . .

of many. .proteins. The first two protein substrates of well recognized physiological importance that were found were phosphorylase kinase and glycogen synthase. Since then, a broad range of proteins have been identified that are candidate substrates for cAMPdependent phosphorylation. Proving that they are so, using stringent criteria such as those of Beavo and Krebs [6], has not always been so readily accomplished but, nevertheless, there are clearly many proteins that are bona fide substrates for the &&P-dependent protein kinase. The bulk of known enzymes ( > 90% of those characterized) under normaI physiological conditions only catalyze a single unique reaction with unique substrates (i.e. A + B, or A i- B + C + D, etc.). For this class of erizymes, i.e. those that catalyze reactions of this simple type, a straightforryard reaction mechanism can be elivisioned wherein an identical *set of binding interactions, conformational changes and inter-atom interactions occur for each cycle of catalysis. For convenience, we will tern7 this class of enzymes as ‘unique substrate(s) enzymes’. The cAMP-dependent protein kinase is different from the above ‘unique substrate(s) enzymes’ in that it has multiple substrates and can catalyze multiple reactions (i.e., Prot, + ATP 4 ProL-P -I- ADP, or Protb + ATP + ProtbP + ADP, etc.). Additionally, in some cases, the protein kinase can cat&ze multiple reactions with the same substrate (i.e., Prot, + nATP + Pro%-P, + nADP). Having one enzyme catalyze multiple reactions is by no means unique. The gastrointestinal proteases, for exanmmpie, catallqe the hydrolysis of many proteins. However, it is suggested that a vev specific difference exists between a gastrointestinal protease such as trypsin and the class of multi-substrate regulatory enzymes of which the cAMP-dependent protein kinase was the first discovered example. With trypsin, chymotrypsin and other GI proteases, their substrate is no longer destined to perform its pre-ordained catalytic (or other) function. Such hydrolytic rrdctions are for the most part irreversible, leading to the destruction of the substrate. The specitic function of the substrate of such hydrolytic enzymes becomes irrelevant the moment it has become a substrate for a multi-substrate enzyme. The order of hydrolysis of proteins by chymotrypsin and trypsin is not known to play any physiological function. For convenience, we have termed this simple type of enzyme ‘substrateindependent multi-substrate enzymes’. The catalytic mechanism of this group of multi-substrate enzymes appears to be just about as straight forward as for the ‘unique substrate(s) enzymes’. The mode of recognition of the different substrates by the enzyme is normally quite simple, and the same set of binding interactions, conformational changes and inter-atom interactions occur, independent of the protein (or other) substrate being degraded. How many multi-substrate enzymes fit into this &SS of ‘substrate-independent multi-substrate enzymes is difficult to evaluate. A broad range of hydrolases exist, some with quite broad specificity, whose modes of interaction with multiple substrates are probably identical and the order of hydrolysis of different substrates of no ph)‘-

siological significance. However, there is currently ver)’ little information available to assess most of the situations in which this occurs. At our current level of knowledge, some multi-substrate enzymes clearly fit into the class of ‘substrate-independent multi-substrate enzymes and, therefore, can be placed in different categories to enrimes like the &IMP-dependent protein kinase, f%r the context that is presented here. The proposition is put forward that the CAMP-dependent protein kinase may represent a class of intracellular enzymes, probably rebtively small in number, that are distinct from the ‘unique substrate(s) enzymes’ that comprise the bulk of all enzymes, and also from the ‘substrate-independent multi-substrate enzymes’, as defined above. It is proposed that the cAMP-dependent protein kinase might have multiple diRerent (albeit sometimes onl). quite subtly) modes of interaction with different substrates, and that these differences are physiologitally relevant. It is proposed that diRerent substrates for the cAMPdependent protein kinase, even though they are bona lide physiological substrates, may not all be equally efYective as substrates (expressed zs K ,,,, kill, etc. 1, and these differences are rele\vant to cellular regulation. This variation in the abiliv of substrates to act as I~hosphonll-acceptors is a consequence of differences in the mode of substrate recognition and the resultant substrate-enzyme interactions. and induced conformational changes associated with catalysis. It is proposed that recognition between the substrates and the protein kinase may involve differential usage of the key recognition determinants. One important consequence of this unique property for this class of enzymes could be that it provides a mechanism to dictate the order of substrate I~hosphc))‘lation in a given cell type in response to a stimuli. If this proposition is correct then it ma). also apply to other regulator) proteins that act upon multiple substrates. Examp’les would be protein kinase C, Ca’+calmodulin-dependent protein kinase II, casein kineses I and II, receptor tyrosine kinases, and also the broadspecificity serineithreonine and tyrosine protein phosphatases. For the purposes of this review, this class of enzymes is termed ‘substrate-enjoilied multi-substrate enzymes (where ‘enjoined’ is defined as ‘to impose rules LIPOII'). A major difference between these enzymes and the multi-substrate hydrolases discussed above is that the substrate is not simply destroyed, but rather its activiv is regulated in a reversible manner. The substrate still plays its ordained role within the cell, and in most cases that is a regulatory one.

Background,

rationale

The

CAMP-dependent

role

of the

and key observations protein

kinase

Since its discovery the biological role of the CAMP dependent protein kinase has been well recognized. Many polypeptide, catccholamine and other hormones use the CAMP system ;LS a major means to elect the regulation of cell function. Receptor-activated adenyl cyclase causes cellular CAMP levels to increase, the cAMP-

Substrate

diver&v

dependent protein kinase becomes activated, and then a broad range of proteins are phosphorylated leading to alterations in their activity. The coordinated effect of this signal transduction pathway is, of course, the end physiological response to the hormone. Many of these steps, with the notable exception of the last, have been well studied and are well understood. Little is known in any cellular system of the steps and properties that allow a smooth coordination of the etfects of multiple substrate phosphorylatioli into the end cellular response. These protein l’hosl’horylations lead to a spectrum of enhanced or diminished catalytic activities, binding properties, etc. Is there some order of substrate phosphory lation imposed by the inherent properties of the components that is essential for an accurate and concerted end physiological response? Or is the mere change in catalytic (or other) activities of the phosphorylated protein suficient, and the remaining events a random consequence from that point on? The cAMP-dependent protein kinase and the other enzymes in its multi-substrate class are distinctive from essentiall~~all other intracellular enzymes, in that they have multiple substrates. (It would seem possible that this principle might appl!~ to IWII~ of the post-t~~islational modification enzymes that exist.) Once CAMP is elevated and the protein kinase activated, what determines which of its substrates are the first to be modified. This question would not apply with respect to most other enzymes in the cell, as most only catalyz one reaction. Once the protein kinase has been activated, is a specilic order of protein phosphorylation vital for a coordinated physiological response? If so, then can this order be modified, leading to a modulated end response? Can endogenous or esogenous signals modulate the order of phosphonllation so that the end physiological response will be altered, even if only quite subtly? Consider the simple process of hormonally regulated secretion. The end response is the consequence not only of what is secreted, but also of the rate and duration of secretion, and whether secretion occurs at a smooth constant level, in surges, or with some modulation between high and low rates. Depending upon the physiological circumstances, any of a wide \.ariety of responses might be optimal. As well as understanding which proteins are phosphorylated in response to a CAMP signal, which is what most studies have so far addressed, it is clearly also important to gain an insight into how the phosphorylation of different proteins is temporall~~ coordinated. For the most part, this entire are;L has been ignored. An example of the phosphor?,lation of multiple pro teins over time is shown in Fig. 1. The graph is derived from a combination of data from several studies carried out under closely parallel conditions. The studies involved the monitoring of perfused rat heart in re. sponse to catecholamines. \Xihat is clearly eLident under this one specific set of conditions is that even from this quite simple comparison there is a recognizable order of protein phosphorylation. The lirst identifiable protein phosphorylated is the p subunit of phosphorylase kinase. Next, clearly lagging behind phosphorylase kinase p subunit and coordinated with the contractile response, is the phosphoqUion of the TN1 subunit of troponin.

of the

c/IMP-deDendent

%

Maximum stimulated response

motein

kinase

Walsh, Glass, Mitchell

l-

loo’ 80. 60 40

20

Catecholamine

stimulation

(4

Fig. 1. Phosphorylation of cardiac proteins. The data have been combined from various studies of perfused heart stimulated by catecholamines under very closely identical conditions 125-281, and are presented as a percentage of total phosphorylation of the specific protein. 0, p subunit of phosphorylase kinase; 0, troponin I subunit; 0, 27 kD sarcolemma protein. Dotted line represents contractile response. (See text for further details.)

Markedly slower than both the inotropic response and the phosphorylation of TN1 is the CAMP-dependent phosphovlation of a protein of the sarcolemma. This simple experiment indicates quite straightforwardly, and not surprisingly, that there is a temporal order of protein phosphorylation in response to the cAMP signal. Not included in this comparison, but of interest, is the phosphorylation of the CYsubunit of phosphorylase kinase. It lags behind p subunit phosphoqllation and is clearly controlled by it. Under the precise conditions of the experiment of Fig. I, glycogen gmthase, a well established substrate of the cAMP-dependent protein kinase, is not phosphoqflated at all in response to epinephrine. Its epinephrine-dependent phospho+tion can be obsenred if the tissue is first treated with insulin. This is a clear example of the order of phosphorylation being regulated. The data of Fig. 1 are presented as a percentage of total phosphorylation of the specific protein. This format does not bring into focus any consideration of the proteins being present in different amounts. If two proteins are being phosphorylated, the one at lowest concentration might be fully phosphorylated first, even though it is being phosphorylated at a lower rate. There are multiple parameters that could and certainly do influence the order of phosphorylation of proteins within the cell, including cellular localization and targeting, the regulation of phosphorylation by the phosphorylation of other sites, and the regulation of phosphovlation by the binding of ligands. These are just three of many possibilities and clearly much remains to be learned. For the purposes of this reliew, however, we will concentrate on the single issue of whether proteins have ‘evolved’ to be better or worse substrates for the protein kinase, thereby dictating a preferred order of phosphorylation, and whether, as a consequence of this evolution, their modes of interaction with the protein kinase differ.

243

244

Cell

regulation

second domains n-ith residues from both haking critical roles in the mechanism of phosl’hotransfer. Knighton el al. [2-.3-l have provided an extensive discussion of the role of many of these amino acids, incorporating \\ithin their discussion extensive prior work. both of their o\vn and others, on the identification of crucial residues. There is excellent agreement bemeen the past chemical clcriKitization studies and nhat can non be \?sualized from the solved structure. mos NHz

kin

I &-V

1

Phospha

7 less.

Fig. 2. The

bi-lobular structure of the CAMP-dependent protein kinase containing bound protein kinase inhibitor PKl(S-24)amide. The upper lobule contains the ATP binding site, the lower the peptide docking sites. Outlined on the right and bound primarily to the lower lobule is PKI. The pseudosubstrate site of the carboxy1 half of the inhibitor peptide is at the cleft of the two lobules; the lower part of the peptide is an CL helix with PhelO within a hydrophobic cleft. Reprinted from [3*-l with permisslon. v

The

structure

of the

CAMP-dependent

protein

kinase

The elegant and arduous work of the Taylor and Sowaclski laboratories [2**,3**] has now provided us with the three-dimensional structure of the catalytic subunit of the cAMP-dependent protein kinase. Given their likel!. common evolutionary origin on the basis of their primav sequences [7], this structure should serve as the model for most if not all other protein kinases. The solved cq~,tal structure is for the binary Complex containing the protein kinase inhibitor (PKI). Elucidation of the ternaq’ complex with nucleotide is promised shortly [81. Key features of the structure are iHustrated in Figs 2 and 3. The protein is bi-lobular with the first lobule composed of residues -4S120, forming a core structure of anti-parallel p sheets, and containing the P-loop glycine-trio motif of the nucleotide-binding site. The second lobule, initiated by a 16.amino-acid OLhelix of the amino terminus, contains the remaining two-thirds of the molecule with a core of a helices. A small portion of the carboxyl terminus forms part of the first lobule. The recognition determinants for PKI are located almost completely on the second lobule. The site of catalysis is located at the cleft between the first and

‘CDC

7

Fig. 3. Ribbon structure of the CAMP-dependent protein klnase. The upper lobule has an anti-parallel P-sheet core, the lower lobule a primary u-helical core. The P-loop phosphate anchor IS part of the ATP-binding site. Residues in the catalytic loop participate in phosphotransferase catalysis. The positions of inserts from several other kinases are shown, Illustrating the likely generality oi this structure to other kmases. In this ribbon diagram the first helix at the amino terminal and the interaction of the carboxyl terminal with the first lobule have been omitted. Repnnted from l3**1 with permrssion.

The solved structure of the cAMI’-dependent protein ki nase makes a critical contribution to our knowledge. As the continuing studies provide further retinement there are several issues to be rcsol\~ed. In the cq5tal structure currently presented, the binding site of nucIcosi& was apparent but there was ambiguity as to the Iocation ot the p and y phosphates of ATP. The cAMI’-dependent protein kinase, like many protein kinases, exhibits a lou kzvel of ATPase activity, but nevertheless. with a turnover number of - 1 min - I, there would be a signiticant possibility that the ATP within the crystal was hydrolyLcd during the accumulation of the X-ray ditfraction clatd ( unless this was blocked suficiently by the presence of PKI [c)] ).

Substrate

diversitv

It is important to resolve whether the ternary structure proposed is for the ATP-enzyme or the ADP-enzyme complex. On the basis of circular dichroism data [lo], it has been suggested that there are several significant conformational changes that occur during the sequential binding of substrates. It is of specific interest to know which conformational state the solved structure represents. Extensive kinetic data [ 9.111 have indicated that optimum protein substrate or PKI binding occurs with the ATP-nzyme complex. It will be important to discover how closely the binary enzyme-PKI complex structure reflects that of the ternary enzyme-ATP-PKI complex. The hydroxyl group of the l~hosphor)ll-acceptor serine of substrates has been implicated to play a role in optimizing the conformation of the ternaT complex [ 1 I]. If this is true, it will be of interest to determine how closely the binding of PKI, and the conformational changes that it induces, retlccts that occurring with substrate proteins. All of these issues will probably be resolved as further refinement of the crystal structure occurs. The structure that is available already certainly provides very important first insights.

Amino acid recognition kinase

determinants and binding

for protein kinase inhibitor site attachments to the protein

PKI has been a major tool in the examination of the possible mode of interaction of protein kinase substrates with the protein kinase. The elucidation of a high-affinit) peptide [ PKI( &22 )amide] that retained 85% of the binding energy of the intact PKI has enabled a detailed e\ralu. ation of the recognition detemiinants essential for highaffinity binding to the protein kinase [ 12-1-r]. Further, a peptide based upon this sequence is the most efficacious substrate for the cAMI’-dependent protein kinase identitied so far, from the standpoint of both high aftinity and high rate of catalysis [ 131. PKI appears to have ‘evolved’ to optimize recognition determinants for the protein kiIKLW catal!dc site and indeed appc”rs to have done so far better than any protein substrate. or certainI!, any protein substrate so far identitied. This prompts the question of why the substrates of the protein kinase are not in fact ‘optimum’ as substrates. Optimum in this context is delined as minimum K,,. maximum V,,,;,,. Other important parameters provide further detinition of what may be op timum under physiological conditions (see later ). PKI( G22)amide is the shortest peptide based upon the PKI sequence that retains maximal inhibitory activit),. The key residues of PKI( 6-22 )amide that account for its high tinity for the protein kinase were determined by examining the effect of the substitution of each residue in a series of synthetic peptides. The conclusions from these studies were then reinforced by the identification of a second fomi of PKI in testis, termed PKItest [ 151. This form will be referred to as Type II PKI here, as its presence has now been demonstrated in several other tissues besides testis. Type II PKI has only ~10%homology overall with the well studied skeletal muscle PKI, and

of the

CAMP-deoendent

orotein

kinase

Walsh. Class. Mitchell

in the region of residues 6-22 is only 58% homologous and 47% identical. However, the protein is equipotent as an inhibitor of the protein kinase. Fig 4 provides an encapsulation of these two sets of results. From substitution studies, residues PhelO, Argl5, Argl8, Argl9 and Ile22 are clearly key recognition determinants for highaffinity binding. It is of note that these are five of the residues that have remained identical between the two forms or have a highly conservative substitution. Gly17 and hsn20 are both conserved in the two forms; they may either play a minor role in binding and/or their size and shape best permits other interactions. Shortening PKI(G22)amide from the amino terminus results in a marked loss of inhibitory affinity even though this region is not a consewed one, and residues 6, 7, 8, 9, 11, 12 and 13 can each be substituted with little effect on inhibitov potencJ7. However, this is well accounted for by the observation from solution structure studies that the amino-terminal one-third of the peptide is present as an o! helix. This amphiphilic helix establishes a hydrophobic environment for the binding of PhelO [ 14,161. The conclusions from these mrious studies and from the crystal structure of the PKI-catalytic subunit binary complex are reassuringly fully concordant. The X-ray crystallographic studies also identitied the docking site on the protein kinase for each of the recognition determinants of PKI. Thus at least for PKI we now have a well refined map of its mode of interaction with the catalytic site of the protein kinase. A comparison between the various cyclic nucleotidedependent protein kinases of these docking amino acids provides for some intriguing insights. Displayed in Fig. 5 are the sequences of the Ccr subunit containing in bold type the specitic amino acids identified by Knighton et LI/. [‘**I as binding sites for the PKI recognition determinants. For comparison, the companion sequences of the Co and Cy isofomls of mammalian cAh4P-dependent protein kinase are also shown. as well as the Cl, C2 and C, isoforms of yeast cAMP-dependent protein kinase and the Ia and Ip forms of mammalian cGMPdependent protein kinase. Comparison of the Ca, Cfi and Cy sequences indicates that, with the exception of an estremel), consenative tlip of Tyr235 and Phe239 in Cy, which together with Pro236 form the hydrophobic binding pocket for PhelO of PKI, the three are identical. LJnless the Tyr235-Phe239 flip would have produced a much more marked change than might be expected, it would appear highly likely that all three species of protein kinase cat$Tic subunit would bind PKI in an identical manner and with equal aftinity. Further, were the binding of a protein substrate to involve only the PKI recognition determinants, then it would probably be phosphotylated in an exactly equivalent manner b!r all three catalytic subunit isoforms. This data would suggest, therefore, that there is no difference in substrate specificity between the Ca, Cp and Cy isoforms of the protein kinase. Only if a substrate were bound to some part of the catalytic subunit other than the sites used by PKI could the three catlytic subunit isoforms display dilferent substrate sensitivities. This last concept is explored more fully below.

245

246

Cell

regulation

Fold increase in Ki (for sin le amino acid su%stitutionsl

I

6U>22

I

Fig. 4. Recognition determinants of protein kinase inhibitor (PKI). Two forms of PKI have been tdentliied, one from skeletal muscle, the second from testis; the amino acid sequence of both is shown in the upper portion of the figure illustrating a very low homology between the two even though they are equipotent 1151. The peptide PKl(6-22)amide has been isolated from PKl,k and retains 85% of its blnding energy to the protein kinase; its sequence is shown in the lower portion oi the figure in comparison with the same region oi PK&. Multiple studies have examined the effects of substitution of each of the residues 112-141; these results are bnetly summarized Illustrating the key importance of residues PhelO, Arg15, 18 and 19. and He22 as recognition determinants for high-affinity binding.

A comparison of the docking sequences of the mam malian CAMP-dependent protein kinase with comparable sequences in yeast &VP-dependent protein kinze and mammalian cGMP-dependent protein kinase, also yields some further information [ 170]. The docking site for PI(I PhelO (Tyr235-Pro23GPhe239) is totally absent in the cGMP-dependent protein kinase. Inhibition studies with PKI analogs have shown concordantly that PhelO is indeed not recognized by the cGMP-dependent protein kinase and in fact, when present, diminishes inhibition [17*,18]. In contrast to this, the PKI PhelO-binding site is present in yeast protein kinase, although the hydrophobicity of this site is compromised by the presence of Thr280, which is substituted for the corresponding proline residue in the mammalian enzyme.

hibitors of the yeast protein kinase. and even poorer inhibitors of the cG~lP-dep~nclelit enzyme [ 17*,18]. The docking site for PKI 11~1I (Leul98, Pro202, lxu205) is present in the !reast protein kinase and appears to be conse~~ti\~ely retained in the cGlMI’-clep~ndent kinase. Peptide substitution studies indicated that PKl Ile22 is a recognition determinant for the yeast enzyme but not for the cGMP-dependent enzyme [ 17.1. These apparently consen:lth,e changes in the cGMP-dependent protein kinase seem to have been sufficient to modi@ the h!~drophobic binding pocket for PKI Ile22.

PhelO does serve as a recognition determinant for the yeast kinase [17*] but does not contribute as much to the binding energy as it does when in a complex with the mammalian enzyme. Both the yeast cAMP-dependent protein kinase and the mammalian cGMP-dependent enzyme have retained the docking sites for both PKI Arg15 and PKI Arg19, but neither Asp329 nor Glu331, key residues for the docking of PKI ArglS, are present in either of these enzymes. It is probably these absences, together with the changes in the PKl PhelO docking site, that account for why PM-derived peptides are poor in-

Early work on the identilication of the protein kinase substrate determinants ( reviewed in [6,12,19*.20*] ) was based upon the peptide ‘Kemptide’, that had been derived from the cAMP-dependent protein kinase phosphorylation site in hepatic pyruvate kinase. The results suggested that the motif &g-Arg-X-Ser-Y contained most of what w;ts necessary to serve as a substrate for the c&UP-dependent protein kinase. Supporting this conclusion were the observations that Kemptide was just as good a substrate as the native protein from which it was derived (in fact, slightly better), that the arginines

The recognition CAMP-dependent

determinants of substrates protein kinase

of the

Substrate

diversity

could not be replaced by even lysine without serious detriment to substrate efficacy (2&100-fold decry&se in catalytic efficiency), and that moving the pair of arginines either one residue closer or one residue further from the phosphoryl-acceptor serine compromised both the rdte of phosphorylation and the binding of peptide to the enzyme ( - 150-riOO-fold change in K,,). A hydrophobic residue at Y appcqred favourable. Studies by the Zoller laboratory [ 21*,22*] with yeast cUlP-dependent protein kinase have provided quite separate support for the summation of docking determinants depicted in Fig. 5. By using a systematic mutagenic scanning strategy, charged amino acids were replaced by alanine and the effects tested by evaluating the resultant kinetics. This procedure identified for the binding of Kemptide (Leul-~g2-Arg3-Ala~Ser5-Ixl~1GGly7), that Glu171 of yeast Ct cAMP-dependent protein kinase was a docking determinant for Arg2 and that Glu21-1 and Glu274 were docking determinants for Argj. (Arg2 and Arg3 of Kemp tide are in positions equivalent to Argl8 and Argl9 of PKI.) Extending upon this, the PKI data now available suggest that the motif X-X-X-X-Phc-X-X-X-X-Arg-X-X-ArgArg-X-Ser-Ile would define the optimum substrate for the protein kinase. The data available appear to present a clear picture. Taken together, and with our now detailed knowledge of the docking sites on the catalytic subunit for the multiple interactions of PKI, they suggest that we now know the essential properties of a substrate for the cAh4P-dependent protein kinasc. This is not so, however. An examination of the phosphorylation site sequences that have been identified for a broad diversity of protein substrates suggests that our knowledge is far from complete (Table 1 ). Firstly, in none so far has there been identilied a phenylalanine (or other likely aromatic) in a comparable position to PKI PhelO. despite the clearly identified docking site for such a residue on the protein kinase. Secondly, instead of a precise alignment of arginines in strict topography to the acceptor serine, that the peptide studies would suggest as most important, a very diverse set of sequences of basic amino acids are observed. Lysine, suggested from the peptide studies to be quite unfavorable as a replacement for arginine, is present in a key position in the p subunit of phosphorylase kinase and in phenylalanine hydronlase. Both TN1 and hommon~-sensitive lipzse have a pair of arginines right nest to the l’hosl,ho-acceptor serine which, from peptide studies, would be suggested to decrease substrate efficacy 1~) r m 400.fold. Several substrates, but clearly not all, have an arginine present in a position comparable to PKI Argl5. A wide variety of amino acids are present as the residue immediately on the carboql side of the acceptor serine. In PKI, a hydrophobic residue is highly desirable at this location and inhibitor el%cacy is reduced 5&l 50.fold by the substitution of the isoleucine by glycine. For the protein kinasc substrates, this site is most frequently a hydrophobic residue, although a wide diversity of amino acids are present. The aspafate at this position in TN1 and the glutamate present in the protein phosphatase glycogen-binding subunit might certainly be expected to evoke quite disparate interactions from those

of the

CAMP-dependent

protein

kinase

Walsh, Class, Mitchell

of a hydrophobic residue. (The examples selected for the purposes of this discussion are from some of the most well founded substrates for the cAMP-dependent protein kinase. >

Conclusions

and extension

of the proposition

From the heterogeneity of substrate sequences that are now known, and from our current detailed knowledge of the binding site on the protein kinase for PKI, it is without doubt that the substrates for the CAMP-dependent protein kinases have not evolved to be ‘optimum substrates’. Optimum, that is, when considered in terms of highest affinity of binding and highest rate of catalysis. Why might this be so? Firstly, it must be recognized that those kinetic parameters alone do not define ‘physiologically’ optimum. If a substrate is present in the cell at relatively high concentration there would be little benefit for it to have evolved an extremely high affinity for the protein kinase. If that protein is already present in the cell at a concentration that is close to saturating the protein kinase, increasing the affinity of that protein for the kinase will not increase its rate of phosphotylation. (For a protein present in high concentration in the cell, if an increase in substrate affinity was also reflected by an increase in aflinity of the phosphtrprotein product, this could impede the rate of its phosphorylation; under these circumstances it would be more beneficial to be a ‘poor’ substrate and this could be the selection pressure.) This circumstance will apply to some proteins but is unlikely to be the situation for all of them. Why then is there such a heterogeneity of sequences around the phosp~ior)~latic,n site? There are two possible situations for the binding of protein substrates. The first would be the limiting circumstance where the only docking elements on the protein kinase for protein substrates that exist are those now identified for PKL An expanded possibility would be that, in addition to the binding sites with which PKI interacts, other sites exist with which some of the substrates interact but PKI does not. It is helpful to consider the more limiting condition lirst, and this is not too unreasonable an assumption to make at our current level of knowledge as, to date, PKI-derived peptides are the best substrates that have been identified i?z lift-o. Llnder this limiting condition, simply from an examination of the phosphonllatioli sequences, it is apparent that some substrates would not interact at all with some of the docking-site residues in the enzyme, and that at some sites, interactions would occur but the geometry would not be favourdble for these to be maximal. Even for a substrate protein containing the sequence Phe-X-X-X-X-Arg-X-X-Arg-Arg-X-Ser-Ile on its surface, this phosphorylation site structure is likely to be more constrained than the corresponding sequence in the small PKI peptide because of the three-dimensional structure of the protein substrate. Under these circumstances it is very clear that many of the protein substrates of the cAMP-dependent protein kinase have not evolved

247

248

Cell

regulation

PKI-Phe-10 CAMP-PK, CAMP-PK, CAMP-PK,

Ca CR Cl

233 233 233

Ala

Gly

Tyr

Pro Pro

Pro Pro

Phe Phe Ala Phe Phe Ala

Asp Asp

CAMP-PK, CAMP-PK, CAMP-PK,

Cl C2 Cj

277 260 278

Ala Ala Ala

Gly Gly Gly

Tyr Tyr Tyr

Thr Thr Thr

Pro Pro Pro

Phe Tyr Phe Tyr Phe Tyr

Asp Asp Asn

Ser Thr Ser

552 567

Thr Thr

Gly

Ser Ser

Pro Pro

Pro Pro

Phe Ser Phe Ser

Gly

Gly

Pro Pro

cGMP-PK cGMP-PK

Ia II3

CAMP-PK, CAMP-PK, CAMP-PK,

Ca CD CI

124 124 124

CAMP-PK, CAMP-PK, CAMP-PK,

Cl C2 C3

168 151 169 441 456

cGMP-PK cGMP-PK

la ID

#pK1-w-

Gly

- - - - 327 - - - - 327 - - - - 327

Phe Asp Asp Tyr Phe Asp Asp Tyr Phe Asp Asp Tyr

Glu Glu Glu

Glu Glu Glu

Glu Glu Glu

Glu Glu Glu

- - - - 372 - - - - 355 - - - - 373

Phe Asp Lys Phe Asp Gin Phe Asp Aq

Tyr Tyr Tyr

Pro Pro Pm

Glu Glu Glu

Glu Glu Glu

Leu Leu

- - - - 648 - - - - 663

Phe Asp Ser Phe Asp Ser

Phe Pro Phe Pro

Glu Glu

Asp Asp

Ala Met Ala Met Ala

Ala Ala Val

Pro Pro .Pro

Gly

Gly

Gly Gly

Gly Gly

Glu Glu Glu

Met Phe Ser Met Phe Ser Met Phe Ser

_PKI-Arg-19 CAMP-PK, CAMP-PK, CAMP-PK,

Ca CD CI

167 167 167

Leu Lys Leu Lys Leu Lys

Pro Pro Pro

Glu Glu Glu

Asn Asn

CAMP-PK, CAMP-PK, CAMP-PK,

Cl C2 C3

211 194 212

Leu Leu Leu

Lys Lys Lys

Pro

484 499

Leu Leu

Lys Lys

cGMP-PK cGMP-PK

Ia IB

-

Leu Leu Leu

- - - - 227 - - - - 227 - - - - 227

Leu Ile Leu Ile Leu Ile

Tyr Tyr Tyr

Glu Glu Glu

Met

Asn

Leu Leu Leu

Asn

Ile

Pro

Glu Glu Glu

Asn Asn

Ile Ile

Leu Leu Leu

- - - - 271 - - - - 254 - - - - 272

Leu Ile Leu Ile Leu Ile

Tyr Tyr Tyr

Glu Glu Glu

Met Leu Ala Met Leu Ala Met Leu Ala

Pro Pro

Glu Glu

Asn

Leu Leu

Ile Ile

- - - - 546 - - - - 561

Leu Met Leu Met

Tyr Tyr

Glu Glu

Leu Leu Leu Leu

Pro

Asn

Thr Thr

PKI-Ile-22 CAMP-PK, CAMP-PK, CAMP-PK,

Ca CD CT

196 196 196

CAMP-PK, CAMP-PK, CAMP-PK,

Cl C2 C,

240 223 241

cGMP-PK cGMP-PK

Ia II3

515 530

Trp Trp Trp

Thr Thr Thr

Leu Leu Leu

Cys Gly Cys Gly Cys Gly

Thr Thr Thr

\\ Pro Pro Pro

Glu Glu Glu

Tyr Tyr Tyr

Leu Ala Leu Ala Leu Ala

Pro Pro Pro Pro Pro Pro

Trp Trp

Thr Thr

Phe Phe

Cys Gly Cys Gly

Thr Thr

Pro Pro

Glu Glu

Tyr Tyr

Val Val

Ala Ala

Pro Pro

I PKI-Arg-15

Fig. 5. The docking domains of mammalian CAMP-dependent protein klnase for the recognition determinants of protein klnase inhlbttor (PKI), and their conservation in other cyclic nucleotlde-dependent protein klnases. The key recognition cletermlnants of PKI (e.g. PKIArgl9) were determlned by the substitution studres Illustrated In Fig. 4. Shown here are the sequences of the Cr subunrt containing in bold type the specific amino acids Identified by Knighton e[ a/ [2**1. using X-ray crystallographic studies, as bindlng sites for the PKI recognition determinants. For comparsion, the equivalent sequences of the three forms of the yeast cAMP-dependent protein klnase, Cl, C, and C,, and the two forms of mammalian cCMP-dependent protein klnase, la and Ip, are shown. See text for further details. Adapted from 117.1.

to be as good a substrate as possible. If this is so, then it suggests that either there is very little e\4utionary pres-

sure for proteins to evol\~ as optimum subsmks. or that there is 3 good rc’;wn why evolving 3s optimum sub-

Substrate

Table

1. Phosphorylation

sequences

Muscle

glycogen

synthase:

site

la

Muscle

glycogen

synthase:

site

lb

site

2

Muscle

glycogen

Liver

synthase:

glycogen

synthase:

site

of substrates

of the

of the

CAMP-dependent

protein

kinase

(partial

Muscle

phosphorylase

kinase,

p subunit

Protein

phosphatase-I-glycogen

Protein

phosphatase

(P)-VYEPLKSINLPRPDNETLWD (P)-ESSEEVYVHTASSGGR

QFTVPLLEPHLDPEAAEQIRRRRP-T

6-phosphofructo-2-klnase!

listing).

(P)-ISTESQPNGGHSLGADLMSPSF

KPGFSPQPSRRG-S

inhibitor-l

(P)-PATLVLTSDQSSPEVDEDRI

RLQKIWIPHSSSSSVLQRRRG-S

(P)-SIPQFTNSPTMVIMVGLPAR

2,6-bisphosphatase

Phosphofructo-I-kinase

DTSEHAHLEHISRKR-S

Fructose-1,6-bisphosphatase pyruvate

(PI-GEATV

FLEIYNKDKAKSRP-S

kinase

Rat adipocyte

(PI-LPLPQSRARESPVHSICD

NH,-EGPAGYLRRA-S

hormone

senslttve

lipase

(P,-LAQLTQELGTAFFQRQQLP

ACNRDTAPHGFWALTESMRR-S

(P)-VSEAALAQPEGLLGTDLKKLT

Acetyl

coenzyme

A carboxylase:

site

2

FMLPTSHPNRGNIPTLNRM-S

(P)-FASNLNHYGMTHVASVSDVL

Acetyl

coenzyme

A carboxylase:

site

1

LSDLGISALQDGLAFHMR-S

(P)-SMSGLHLVKQGRDRKKIDS

ATP

citrate

lyase

Liver

phenylalanine

Liver

tyrosine

Rat

brain

hydroxylase

CAMP

heart

element

type-2

troponin

gizzard

Canine Bovine

brain

brain

chain

(P)-YRKILNDLSSDAPG

ADSESEDEEDLDVPIPGRFDRRV-S

kinase

phospholamban protein

56

dopamine-regulated

phosphoprotein

(P)-VCAETYNPDEEEEDTDPRVI

(P)-MAMISGMSGRKAGSSPTSP

NH,-MDKVQYLTRSAIRRA-5

(PI-TIEMPQQARQNLQNLFINFC

AKRMKEAKEKRQEQIAKRRRL-S

(P)-SLRASTKSESSQK

VPAPPSQLDPRQVEMIRRRRP-T

(P)-AMLFRLSEHSSPEEEAS

MNYLRRRL-S 6 subunit

Acetylcholine

receptor:

y subunit

Pro-artrial

natriuretlc

Neurofilament

list contains

phosphorylated messenger

cell

with

cross

phosphorylatlon.

Even

when

With

muscle

sites

the

of phosphorylation were

extended

difficult

site

between

those

the

of an in wvo

glycogen

synthase,

following from

to a CAMP

In Interpretation talk

to establish

phosphorylation

in response

Difficulties

in interpretation. level

It remains

CAMP-dependent

intact

and

(P)-FGIMIKAGEYILKKPRSELMF (PI-CFGGRIDRIGAQSGGL

STERRAYSSYSAPVSSSLSVRR-S

ambiguities.

identified.

system,

(NF-L)

of ,n vitro

in the

been

(PI-SVGYISKAQEYFNIKSRSEL

LGMQLEPSEETPEKPQRRRS-S GPRSLRRS-S

subunit

some site

(P)-DSNFM

SRADESEQPDWQNDLKLRRS-S

peptide

70 kD

list the

IP)-DRAYATEPHAKSKKKISASR

ARRKWQKTGHAVRAIGRLS-S

synapsln receptor:

of the

(PI-LIEDARKEREAAAAAAAAAV

SVTDSQKRREILSRRP-S

NH,-ADESRDAAGEAKPAPAVRR-S light

Acetylcholine

highest

protein

I

ribosomal

actually

binding

CAMP-dependent

myosin

cardiac

Rat liver

This

(P)-DFGQETSYIEDNQ

kinase

Chlcken

above

(P)-FSESRADEVAP

AAVVLCNGVLSRKL-‘5 EQDAKQAEAVTSPRFIGRRQ-S

regulatory

subunit

protein

Rat

GSTSTPAPSRTA-S

hydroxylase

Regulatory Rabbit

Mitchell

(P)-VTSLGGLPQWEVEELPVDDLLL

MAEVSWKVLERRARTKRSG-S subunit

Glass,

(P)-VSSLPCLEDWEDFDLENSVLF

ERTGIMQLKSEIKQVEFRRL-S

binding

Walsh,

(P)-CTSSSCCSKRSNSVDTSSLSTP

NH,-MLRGRSL-S a subunit

kinase

(P)-VDTSSLSTPSEPLSSAPSLGEERN

NH,-PLSRTL-S

2

kinase,

Pig liver

protein

APQWPRRASCTSSSCCSKRSN-S

phosphorylase

fructose

CAMP-dependent

DEDEEAAKDRRNIRAPQWPRRA-S

Muscle

Liver

diversity

published

signal. arise

2nd

messenger

when

systems well

of whose full

and

cases

sequence

the

has

the

signal

a CAMP

is identified

the

of CAMP-dependent

a hormonal

the

bulk

sites

Identified

In a few

because

as an example, the

,n viva

been

Only

also

phosphorylation

ephinephrine, first

the has

(PI-YSSSSGSLKPSLENLDVQV

to

be the

identified actions for

then

peptide

is rarely

signal

phosphorylation.

protein

site

lead

are that

well species

phosphorylation

to a single

to a non-CAMP

in vifro,

CAMP-dependent correlated or a highly

For the

to be

of in viva

restricted

can same

shown

there

still

remains

sites

are

with

CAMP related

second-

dependent

not

those action.

species

difficulties with

the

Several became

available.

states is not in fact ideal for the cell. One possibilit), wh!, proteins may have e\~lved to ditfering Ie~+i of substrate efficacy is to allow those proteins to be $Iosphorylated in a specifc order following a stimulator). signal. Thus, the variety. of phosphotylation sequences, and in consequence the broad range of substrate etficacies, may occur in order to bring about the correct order of substrate l~hos~~ho~lation, thus optimizing the coordinated physiological response to that which is needed. A second possibilinV is that some substrates interact with the active site of the protein kinase at sites other than those with which PKI interacts. It would seem most improbable that a substrate woulcl interact with 3 totall)

difkrent set of docking sites than PKI, but not at all unreasonable that there may be some sites on the protein kinase with which substrates interact but with which PKI does not. Further, even when interacting at the same site, the nature (or extent) of the interaction might be different because of surrounding residues. Insufftcient peptide substrate studies have been carried out with broadly diverse sequences to judge whether the binding affinities of some protein substrates are higher than could be accounted for simply by interaction with the sites in the enzyme that have been so far identified. This, therefore, remains an open question but if proteins have evolved to be better or worse substrates, depending upon the evolutionaq~ pressures upon the cell, there would appear

249

250

Cell regulation

to be no reason why binding would have been restricted only to those sites-so far ibentified. On the surface of the protein kinase and in the vicinity of the catalytic site there are several possibilities for alternate docking sites. The regulatory subunit of the tip-dependent protein kinase, in fact, interacts with residues on the catalytic subunit other than those used by PKI. The regulatory subunit binds with a higher affinity to the catalytic subunit than does PKI but apparently lacks any residue equivalent to PKI PhelO which makes such an important contribution to the binding of PKI. Data from mutation studies of the yeast enzyme [23*,2-+] would also lend support to this conclusion. Lysl89, Glyl93. Argl9-t. TJr1.196.Lys213, Lys217 and Thr241 of the C, yeast cAh4P-dependent pro tein kinase are important recognition determinants for the regulatory subunit, whereas they are not interaction sites for PKI. If the regulatory subunit binds to other sites at the catalytic site of the protein kinase then substrates may do so likewise. We have attempted to paint the picture of the catalytic site of the protein kinase that must be viewed quite differentl) from the bulk of all enzymes (i.e., those in the ‘unique substrate enzyme’ and ‘substrate-independent multi-substrate enzyme’ classes). What is suggested is that the catalytic site of the cAMP-dependent kinase, and other similar enzymes, will recognize substrates by ;I mosaic of different interactions. some albeit onl), subtle distinctions from those of PI(I and some probably involving diRerem/additional catalytic subunit residues. This mosaic of binding patterns and aRinities may have evolved to regulate the order of substmte phosphor)llatiori.

References

and recommended

Papers of particular interest. published view, have been highlighted as: . of special interest .. of outstanding interest 1.

within

MCKNK;HI- GS: Cyclic AMP Second Opitl Cell Hid 1‘99 1 3:2 13-2 17.

for the Catalytic nase. .1/o/ hdocriml

peritd

-I

1IANU SK. QI’INN Ahl. Ilr~rerr 7‘. The Protein Conserved Features and Deduced Phylogeny alytic Domain. .Sirc~i~~ 1988, 24 1 :-t-52.

x.

~NIGIITOS DR. ~IONG NH. T.4~1.0~ 55. SoW;WW JM: Crystallization Studies of CAMP-dependent Protein Kinase. ./ ,tlol Hiol 1991. 220:217-220

9.

\VIW~~IIO~‘~I’

tion of the tein Kinase 2SS.36X2-W2.

of

Kinase Family: of the Cat-

5. W’AI.~I~ 1% Mg x ATP2-dependent InterdcInhibitor Protein of the CAMP-dependent Prowith the Catalytic Subunit. ,/ Hid G!xv~r 19x3,

10.

Hl:l;l) J. KIWIi \‘. ld3111 MC. <~III;NG II-C. W’~lsli DA: Circular Dichroic Evidence for an Ordered Sequence of Ligand/Binding Site interactions in the Catalytic Reaction of the CAMP-dependent Protein Kinase. Hir~-k~>r&q* 19X5. 24:296--29’.

11

V’IIITEIIO~‘S~~

II.

\WI>I I DA. Arxx~.<,> Kl.. \‘AN I’,\‘I-nis SM. GI>~s> Dl3, GAKI~I-W LP: The Inhibitor Protein of the CAMP-dependent Protein Kinase. In f’+tidcs oml I’rorc4tt I%w~~~~MII~CII:~I~. Edittd h> Kemp HF. Ilv~c~kl. Boc:c liut~>n: CRC Prc.ss: IWO: -13-x-1

13.

GLW DB. CHI!N(; 11-C. Mt~:l~li~ l&l. Kliel) J, WAI.WI DA: Pirnq Structural Determinants Essential for Potent inhibition of CAMP-dependent Protein Kinase by Inhibitory Peptides Corresponding to the Active Portion of the Heat Stable Inhibitor Protein. .I Bid Qwn 19X9. 264:XXO2-XX10

1-I.

(;I..<% DB. I.t’l’~x,?‘~sr 1,l. KATZ 13kl. WAI.\II DA: Protein Kinase Inhibitor-(6.22).amide Peptide Analogs with Standard and Nonstandard Amino Acid Substitutions for Phenylalanine 10. ./ Bid Wwtn 19X9. 2&:l-li79-1-r5X+

ii.

\‘.49 I’.4Trli~ SM. NC; DC. Tl I‘% JPI 1. AS<;I-1.0s W’,\l.\ll DA: Molecular Cloning of a Rat Testis Inhibitor Protein of CAMP-dependent Protein iWl/ :lirrr/ Scr I 5 A I99 I, 88:i.W3-55X’

16.

RolJIJ JS. TKew,ltlill.4 J. GI..\%s DI3. LttitxJi \VK. EM. tilN%lil. \‘. Wr.41u.\li I)A. Conformational Analysis of PKI(S-22).amide. the Active Inhibitory Fragment of the Inhibitor Protein of the CAMP-dependent Protein Kinase. Niochw ,/ 19x9. 2cA.37 1-3X0.

S. ITli~\~~x:~~ JR, C;~%l;~.t~li Jli. KHI:I~~ EG. W~I.SII I)A. Studies on the Kinetic Mechanism of the Catalytic Subunit of the CAMP-dependent Protein Kinase. .I Nid Cbrnr 19X.3, 258.3691-3’0 1.

Rtiet)

Kl.. SWTtI AI. Form of the Kinase. /+w

J. Ix

hAI~I1I’KI’

Messanger

Systems.

GUI-I

KNIGHTON DR. %Hl:h’c; J. Tli~: Ej’cK If. Xl’oNC N. T.4~l.o~ SS. SOWAIX~C~ JM: Structure of a Peptide Inhibitor Bound to the Catalytic Subunit of Cyclic Adenosine Monophosphatedependent Protein Kinase. Scic~cc~ 1‘991. 253:-1 I-1--120. Coupled with [.W], this paper presents the alignment of PKI c,n the catalytic subunit of the protein kinase. Poses the question of just to what extent this represents the site(s) of intemction of suhstmtes with the protein kinase. Krwtn’o;u DR. ZHKNC J. TtiN Exx If. ASIIFOW VA, XIONC; N. TAYLOR SS. SOWAIMI JM: Crystal Structure of the Catalytic Subunit of Cyclic Adenosine Monophosphate-dependent Protein Kinase. Ssicwcr 1991, 253:+07-11-1. For the first time the structure of a protein kinasr cdn he visualized From the structure of the cAMi’-dependent protein kina\e. others can t-x modeled.

I’. .

Gl..k\s DB. Flil.t.titi blJ. LI~VIS LR. W’;\t.~II DA. Structural Basis for the Low mnities of Yeast CAMP-dependent and Mammalian cCMP-dependent Protein Kineses for Protein Inhibitor Peptides. H~&~ri~/t~~ 1992. 31:-r?‘++? The ditfercnce in docking sites of PKI 10 c3ch of these kinases corrcspends well \\ith their vxiaticms in inhibitor) potc’m~~. Thi,\ illustrates ho\v \:uiatic)ns in substrate atrinities can lx determined IX.

3. ..

5.

Ki-

Kwlitcs EG. BI:AVO JA: Phosphorylation-dephosphorylation Enzymes. .4irf/ri h’~r, Hidwm 1979. #:923-959.

of re

2. ..

4.

Protein

6.

reading the annual

Subunit of CAMP-dependent 1990. 4~65-175.

LIHIEH MD, MCKNIWT GS: Expression of cDNAs for Two Isoforms of the Catalytic Subunit of CAMP-dependent Protein Kinase. J Hid C%VII 19X7, 262:15202-15207. BEEBE SJ, OWN 0. S,\~rmeKc; M. FROYAS A. N,WS~ON V. J.4tr~stis T: Molecular Cloning of a Tissue-specific Protein Kinase (C Gamma) f?om Human Testis Representing a Third lsofrom

19. L

so.

Gl,t\a DB. Clllixc; I I-C. l&w BE. W’,\t.srl DA. Differential and Common Recognition of the Catalytic Sites of the cGMPdependent and CAMP-dependent Protein Kinases by Inhibitory Peptides Derived from the Heat-stable Inhibitor Protein. .I Nid C&W 19x6. 261: 1216Gl2171. KerW BE. I’EAKWN RH, Protein Kinase Recognition Motifs. 7i-catrrIs Moclwtr Sci 1990. 15:3+2-346. I20*I.

Sequence

Klih’Nlil.l.\’ I’J, tiKI:W EG: Consensus Sequences as Substrate Specilicity Determinants for Protein Kinases and Phosphatases. .I Hid G%wm 1991, 266: 1iiiiI iiiX. This paper and [ 19.1 summarize our current unclcrstandin~ of the motifs that app~dr to dictate sutxtctte apeciliciy for protein kim1sc.s and pht)sphatases. Two major points arc highlighted hy this work: first. differences exist hemeen the en?mes. although for some the? arc re.

Substrate

diver&v

of the

Li.

71. .

see 22. .

GI~W (15. a Protcin in Catalysis 266:x,:x0,-x05 [2.3*1

%OI.I.I:R XIJ: Rational Scanning Mutagenesis of Kinasc Identilics Functional Regions Involved and Suhstratc Interactions. ,I /Qo/ OW~I 1991, I

Gl111:4 (3. Zol.rl;i~ Ml. tions that Dctcrmine of the cAMP-dcpcndcnt 30-i.329 ~i3.Gi SLY 17fPl

Idcntitication of Electrostatic Intcracthe Phosphoq4ation Site Spcciticiq Protein Kinasc. /~ir~/~wris/~~~ I99 I,

CAMP-deDendent

orotein

kinase

Walsh,

MCCI ILI.OM;I~ TE. W~I.~II DA: Phosphotylation phorylation of Phosphotylase Kinase in Heart. .I Hid Chw: 19-‘9. 254:‘3ii-32.

Glass,

the

tillll~\ (3. KSlC~llTC~~ I)R. ho\\hlhtil J\l. I‘.\\‘IOI< 55. %Olil.l.liH NJ. Systematic Mutational Analysis of cAMP-dependent Protein Kinax Idcntitics llnrcgulatcd (:atalytic Subunit and Dcfincs Regions for the Recognition of the Regulatory Subunit. ./ H/o/ Chwr IW2, 267:-1SOh -IX I-,

and DephosPerfused Rat

Ascx~o~ Kl.. R\MN:I IANI)KW C. WALVI DA: Subunit Phosphorylation and Activation of Phosphorylase Kinase in Perfused Rat Hearts. ,I /Co/ C!wm 19X7. 26213219~3226. U;‘hlhl I I)A.

CUI’I’IN~;i3i

MS. Sh’,\K;L\l,\KRl~IlNAN

S. M(:CI:lLOtGH

Tli: Cyclic Adenosine Monophosphate Dependent and Independent Phosphorylation of Sarcolemma Membrane Proteins in Perfused Rat Heart. /ficnkf~r~~-)~ 1‘GE). 18:X7l-t(77 EN~;IAW I’J: Correlation Between Contraction rylation of the Inhibitory Subunit of Troponin Rat Heart. /;/%S’ /.rl/ IV?i. %ki’-(,O.

24 ..

Mitchell

and Phosphoin Perfussed

251