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[18] Reversible autophosphorylation of type II cAMP-dependent protein kinase: Distinction between intramolecular and intermolecular reactions
176
SPECIFIC PROTEIN KINASES
[18]
[18] R e v e r s i b l e A u t o p h o s p h o r y l a t i o n o f T y p e II cAMP-Dependent Protein Kinase: Distinction between Intramolecular and Intermolecular Reactions By JACK ERLICHMAN, RAPHAEL RANGEL-ALDAO, and ORA M. ROSEN
Many proteins are subject to reversible covalent modification by phosphorylation and it is widely recognized that this modification is a critical component of the physiological response of mammalian cells to specific stimuli. The cAMP-dependent protein kinases (PK) catalyze the initial phosphorylation reaction in cAMP-mediated pathways. In most tissues and cells there are two classes of cAMP-dependent protein kinases, designated types I and II by their order of elution from DEAE-cellulose. Both classes are composed of four polypeptide chains, consisting of a regulatory (R) dimer and two catalytic (C) subunits. The R components of the two types of kinase are different from each other whereas the C components appear to be both identical or very similar. 1 Both types I and II PKs are themselves subject to covalent modification by phosphorylation. 2,3 Each R monomer may contain up to 2 mol of phosphate; however, the mechanism by which R subunits are phosphorylated is different for each class of kinase 4'5 and may also be different for each site present in a R monomer. 6 Phosphorylation of the single, readily exchangeable site on type II R enhances binding of cAMP to R 7 and slows the rate of reassociation of R and C subunits when cAMP levels are lowered. 8 In this chapter we describe techniques to measure the reversible phosphorylation of the exchangeable site on type II R and distinguish between intramolecular and intermolecular phosphorylation of this site. Preparation of Protein Kinase Type II cAMP-dependent protein kinase, the predominant form of cAMP-dependent PK in bovine cardiac muscle, was purified from this tissue by the method of Rubin et al. and had a specific activity of 800 nmol i D. B. Glass and E. G. K r e b s , Annu. Rev. Pharmacol. Toxicol. 20, 363 (1980). 2 j. Erlichman, R. Rosenfeld, and O. M. R o s e n , J. Biol. Chem. 249, 5000 (1974). 3 R. L. G e a h l e n and E. G. K r e b s , J. Biol. Chem. 255, 1164 (1980). 4 R. Rangel-Aldao and O. M. R o s e n , J. Biol. Chem. 251, 7526 (1976). 5 R. L. G e a h l e n and E. G. K r e b s , J. Biol. Chem. 255, 9375 (1980). 6 D. A. Flockhart, D. M. Watterson, and J. D. Corbin, J. Biol. Chem. 255, 4435 (1980). 7 F. H o f m a n n , J. A. Beavo, P. J. Bechtel, a n d E. G. K r e b s , J. Biol. Chem. 250, 7795 (1975). 8 R. Rangel-Aldao and O. M. Rosen, J. Biol. Chem. 251, 3375 (1976).
METHODS IN ENZYMOLOGY, VOL. 99
Copyright © 1983by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181999-X
[18]
AUTOPHOSPHORYLATION OF TYPE II PROTEIN KINASE
177
32p transferred to protamine /min/mg protein. 9 R and C subunits were prepared by dissociation of the holoenzyme in the presence of cGMP followed by chromatography on DEAE-cellulose 9 or to-aminohexylagarose. 8 Purified R and C subunits from heart can also be prepared by a combination of chromatography on DEAE-cellulose and affinity chromatography on 8-(6-aminohexyl)amino-cAMP-Sepharose 4B. 1° C subunits are then isolated from the material that does not adsorb to the affinity resin by chromatography on CM-Sephadex. 11The kinase holoenzyme and its subunits prepared by these methods are greater than 90% pure when assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Preparation of Protein Kinase Inhibitor Protein kinase inhibitor was purified according to the method described by Walsh et al.12 Its specific activity was 650 units/mg of protein. One unit is defined as the amount needed to inhibit the incorporation of 1 pmol of 3~p into histone when 0.08 /zg of purified protein kinase was incubated for 15 rain at 37° in 200/zl containing 50 mM potassium phosphate buffer pH 7.1, l0 mM MgSO,, 50 /zg of histone IIA, 0.5 mg of bovine serum albumin, and 2/zM cAMP. Phosphorylation of Protein Kinase Holoenzyme R2 • C2 + 2 A T P ~ [R-P]2 • C2 + 2ADP
Purified protein kinase (10-100/xg) is incubated for 15 min at 23 ° in 100 /zl of 50 mM potassium phosphate buffer, pH 7.0, containing 50/.~M [y32p]ATP (>1000 epm/pmol), 10 mM MgSO4, and 1 mM dithiothreitol. Upon completion of the reaction the amount of 32p incorporated into the enzyme is determined by one of two methods: (1) An aliquot of the enzyme is precipitated with cold 10% trichloroacetic acid, the precipitate is collected on glass fiber filters, and assayed for radioactivity by liquid scintillation spectrometry. (2) An aliquot is transferred to another tube containing 200/zl of 50 mM potassium phosphate buffer (pH 7.1), containing 0.1 M EDTA and 2 /xM [3H]cAMP. The contents are poured onto Millipore filters, washed with 10 ml of the same buffer, dried, and assayed f o r 32p and 3H. Phosphorylation is expressed as moles 32p incorporated per mole [3H]cAMP bound at saturation. Unincorporated 32p is removed 9 C. S. Rubin, J. Erlichman, and O. M. Rosen, J. Biol. Chem. 247, 36 (1972). 10 W. Weber and H. Hilz, Eur. J. Biochem. 83, 215 (1978). ll p. j. Bechtel, J. A. Beavo, and E. G. Krebs, J. Biol. Chem. 252, 2691 (1977). 12 D. A. Walsh, C. D. Ashby, C. Gonzalez, D. Calkins, E. H. Fischer, and E. G. Krebs, J. Biol. Chem. 246, 1977 (1971).
178
SPECIFIC
1.0
PROTEIN
KINASES
[18]
B
0.8 n"
-I
0.6
o.,
--
O
--
i"
I
I
2
--I 3
I
I
I
I
4
5
6
7
I
ATP (FM) FIG. 1. Autophosphorylation at different concentrations of ATP. The concentrations of protein kinase were 71.2 pmol/ml ( O - - O ) and 213.7 pmol/ml ( 0 - - 0 ) . The specific activity of the ATP was 2200 cpm/pmol. Reactions were carried out at 4° for 2 min in the absence of cAMP. From Rosen and Erlichman. ~4
from samples by dialysis or filtration on Sephadex G-25. Since only one of the two cAMP binding sites present on the R monomer is measured using this assay 13 the ratio of 32p/3H approaches one when unphosphorylated enzyme is fully labeled. 8 With most preparations of enzyme, phosphorylation is 80% complete in less than 1 min and 100% complete after 10 min of incubation in the presence or absence of cAMP. The enzyme incorporates 1.0-1.9 mol of phosphate per mol of holoenzyme. It is likely that this variation in s2p incorporation reflects endogenous phosphate present in the purified enzyme. 14The rate of 32p incorporation can be slowed by either lowering the temperature to 4 ° or decreasing the concentration of [y-32p]ATp.14 The apparent Km for ATP for autophosphorylation is 0.4/zM (Fig. 1) compared to 12/zM ATP when exogenous proteins are used as substrates. 9 Phosphorylation of Isolated R Subunits Rz + 2ATP
c ~ [R-P]~ + 2ADP
cAMP
The procedure for phosphorylating isolated regulatory subunits is identical to autophosphorylation of holoenzyme except that catalytic 13 j. D. Corbin, P. H. Sugden, L. West, D. A. Flockhart, T. M. Lincoln, and D. McCarthy, J. Biol. Chem. 253, 3997 (1978). 14 O. M. Rosen and J. Erlichman, J. Biol. Chem. 250, 7788 (1975).
[18]
AUTOPHOSPHORYLATION OF TYPE II PROTEIN KINASE
179
amounts of C can be used to phosphorylate excess R subunits. The reaction requires cAMP to prevent reassociation of R and C. Heat-inactivated holoenzyme may be used in place of purified R subunits as the substrate.~4 Since heat-inactivated R is no longer capable of interacting with C, 6 cAMP is not needed if purified C is used instead of holoenzyme. Distinction between Intramolecular and Intermolecular Autophosphorylation There are a number of ways by which the phosphorylated form of the protein kinase holoenzyme could be generated: (1) intermolecular phosphorylation of R by the catalytic subunit in the presence of cAMP followed by reassociation of the subunits; and (2) intermolecular or intramolecular phosphorylation catalyzed by the intact holoenzyme. If phosphorylation occurs by an intermolecular reaction involving free C then conditions that inhibit catalytic subunit activity (e.g., addition of protein kinase inhibitor protein) should prevent phosphorylation of R. The initial velocity of the phosphorylation reaction should be proportional to the enzyme concentration if an intermolecular mechanism is involved and independent of enzyme concentration if the reaction is intramolecular. Finally, addition of catalytic subunit to the holoenzyme should affect the rate of phosphorylation of R if intermolecular phosphorylation is occurring.
Effect of Protein Kinase Inhibitors on Protein Kinase Activity Holoenzyme is incubated in 50 /zl of 50 mM potassium phosphate buffer, pH 7.1 at 0° with various concentrations of either protein kinase inhibitor protein, R subunits, or nonsubstrate peptides. The mixtures are then assayed at 37° for 15 min in the presence or absence of cAMP for (1) protein kinase activity using histone as substrate or (2) autophosphorylation. Typical results are shown in Figs. 2 and 3 and Table I. Addition of protein kinase inhibitor blocks histone phosphorylation in the presence or absence of cAMP but has no effect on autophosphorylation in the absence of cAMP (Fig. 2). Similarly, the addition of an inhibitor peptide such as Lys-Tyr-Thr has no effect on autophosphorylation in the absence of cAMP but blocks phosphorylation of R in the presence of cAMP (Fig. 3). R subunits in excess of C are phosphorylated only when cAMP is added to dissociate the holoenzyme (Table I).
Effect of Enzyme Concentration on the Rate of Autophosphorylation Initial velocity of autophosphorylation is measured at several enzyme dilutions. Incubations are carried out in 50/xl of 50 mM potassium phos-
180
SPECIFIC PROTEIN KINASES
[18]
o 0
O
E
A
--
0.5 ~
o
(.3
0.4t~
4 0
~ 3
-\
O
0 . 2 ~O
Q.
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" Q. N
2
4
6
Protein I
I 0.2
I
8
I0 12 14 16 18 20
Kinase I
I
I
Inhibitor I
I
I
m
[/~g) I
I 2
R2 (/~g)
FIG. 2. Effect of cAMP-binding protein and protein kinase inhibitor on protein kinase activity. Holoenzyme (0.08 /xg) was incubated in 20 /zl of 50 mM potassium phosphate buffer, pH 7.1, for 5 min at 0° with either protein kinase inhibitor or cAMP-bindingprotein at the concentrations depicted on the abscissa. The mixtures were then assayed at 37° for 15 min for either (a) protein kinase activity using histone as substrate or (b) self-phosphorylation in the presence of 5 /zM [y-32P]ATP(9000 cpm/pmol). For assays using histone as substrate [see (a), above] the symbols are ( 0 - - 0 ) R2C2plus protein kinase inhibitor plus 2 /~M cAMP; (O--O) R2C2 plus protein kinase inhibitor; (0---0) R2C: plus R: plus 2/zM cAMP; (©---O) R2C2plus R2. For self-phosphorylationassays [see (b), above] the symbols used are (A--A) R2Cz plus protein kinase inhibitor, (A--&) R:C2 plus R2. From RangelAldao and Rosen.4
phate buffer p H 7.1 at 4 °. Lowering the temperature to 4 ° slows the reaction rate sufficiently to measure initial velocities when short incubation times are used (see Fig. 4). The velocity of autophosphorylation in the absence of c A M P is not affected by a 100-fold dilution of the e n z y m e (Fig. 4). Furthermore, when C subunits are added to excess R in the absence of c A M P , phosphorylation of R is stoichiometric with the a m o u n t of h o l o e n z y m e formed. In the presence of c A M P , maximal p h o s p h o r y lation of R is independent of the concentration of C and all o f the R subunits can be phosphorylated at each concentration of C (Fig. 5). When h o l o e n z y m e is used as a substrate for C the rate of autophosphorylation is unaffected by the addition of C subunits. 4 These studies indicate that the C subunit catalyzes the intramolecular autophosphorylation of its own R
[18]
AUTOPHOSPHORYLATION OF TYPE II PROTEIN KINASE
>-
181
c 50 E o
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.
.
.
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.
.
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I
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I
I
I
50
75
150
CONCENTRATION OF LYS - T Y R - THR (mM_)
FIG. 3. Effect of Lys-Tyr-Thr on self-phosphorylation of protein kinase. Holoenzyme (2.85 pmol) was incubated at 4° for 1 min in 50/zl of 50 mM potassium phosphate buffer, pH 7.1, containing 10 mM MgSO4, 0.5 /xM [y-32P]ATP (13,180 cpm/pmol), 50 /xg of bovine serum albumin, with ( 0 - - 0 ) or without (O---Q) 1 /zM cAMP. The reaction was stopped by the addition of 200 txl of 0.1 M EDTA, pH 4.0 and the mixture filtered on Millipore filters. The filters were washed twice with 10 ml of the EDTA solution, dried, and assayed for 32p in a solution of Omnifluor/toluene, 4 g/liter. The total amount of 32p incorporated into protein kinase was 3.7 pmol. From Rosen e t a l ) 6
TABLE I PHOSPHORYLATION OF c A M P - B I N D I N G PROTEIN BY PROTEIN KINASE IN THE PRESENCE AND ABSENCE OF cAMP~,b
R2C2 (pmol)
R2 . (pmol)
cAMP
[32p]Phosphate incorporated (pmol)
5.70 5.70 5.70 5.70
--15 15
+ +
6.40 6.40 7.03 20.17
a Holoenzyme was incubated in 25/zl of 50 mM potassium phosphate buffer, pH 7.1, with or without cAMP-binding protein. The mixtures were then assayed for self-phosphorylation in the presence of 0.2 mM [3,-32p]ATP (1000 cpm/ pmol) and, where indicated, 8 ttM cAMP. Incubations were for 10 min at 37° in a final volume of 50 /xl. RzC2, holoenzyme; R2, cAMP-binding protein. b From Rangel-Aldao and Rosen. 4
182
SPECIFIC PROTEIN KINASES
[18]
)A
v--_ .D >
8-
•
_
< z
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4
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.~..~
z
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rr ~
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0
a.
I
I
50 45 TIME (see)
I
I
60
75
FIG. 4. Effect of protein kinase concentration on self-phosphorylation. Holoenzyme was phosphorylated at three different concentrations at 4 ° in 50/xl of 50 mM potassium phosphate, pH 7.1, containing 10 mM MgSO4, 50/~g of bovine serum albumin, 5/zM [7-32P]ATP (30,000 cpm/pmol), and, where indicated, 1.0/zM cAMP. The 32p incorporated into the holoenzyme at each concentration was multiplied by the dilution factor in order to obtain the values depicted on the ordinate. The symbols used are ( 0 - . - © ) , 22.8 pmol of R2C:; ( A - . - A ) , 1,14 pmol of R2C2, (?q-.-F3), 0.228 pmol of R2C2; ( e - - O ) , 22.8 pmol of R2C2 + cAMP; (O-nO), 0.228 pmol of R2C2 + cAMP. From Rangel-Aldao and Rosen. 4
5 -
~
4
o.
•o
iii o
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l
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/ O
/
2
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2 3 4 5 6 7 8
C (pmole) FIG. 5. Phosphorylation of cAMP-binding protein by catalytic subunit in the presence or absence of cAMP. cAMP-binding protein (4 pmol) was incubated at 4 ° for 1 min with the indicated amounts of catalytic subunit in 40/~1 of 50 mM potassium phosphate buffer, pH 7.1, containing 10 mM MgSO4, with ( O - - O ) or without (©---O) 1.0/~M cAMP. The reaction was initiated by the addition of 100 pmol of [y-32p]ATP (21,000 cpm/pmol) and incubations were carded out at 4 ° for 20 min. From Rangel and Rosen. 4
[18]
A U T O P H O S P H O R Y L A T I O N OF TYPE I I PROTEIN KINASE
183
subunit when present in the intact holoenzyme but can also catalyze the intermolecular phosphorylation of R when the holoenzyme is dissociated by cAMP. The kinetic parameters of the intramolecular autophosphorylation are distinguishable from those characteristic of the intermolecular phosphorylation. The Km for ATP is 30-fold lower. The velocity is significantly greater for the intramolecular phosphorylation. In the absence of cAMP, only those R subunits associated with C can serve as a substrate. In contrast, the intermolecular phosphorylation of R is analogous to phosphorylation of exogenous protein substrates such as histones or protamine. Phosphorylation is blocked by inhibitors of catalytic activity such as the protein kinase inhibitor. The rate of phosphorylation of excess R is stimulated by cAMP and proportional to the concentration of C. Since the total concentration of ATP in mammalian cells is greater than 1 mM and the kinase exists principally as a holoenzyme in the unstimulated cell, it is likely that the intramolecular reaction is responsible for maintaining the holoenzyme in the phosphoform. Reversal of Autophosphorylation and Dephosphorylation [R-P]2 " C2 + 2 A D P - - > R2C2 + 2 A T P JR-P]2 " C2
phosphoprotein phosphatase ~ R2 • C2 + 2Pi
There are two mechanisms for dephosphorylating phospho-R. The first is reversal of the autophosphorylation reaction. This reaction is monitored by measuring the disappearance of radioactivity from 32p-labeled protein kinase concomitant with the formation of charcoal-adsorbable radioactivity which is identified chromatographically as [32p]ATP.14 The standard reaction mixture (0.1 ml) contains 50 mM Tris-maleate buffer, pH 5.5, 5 mM MgSO4, 20 p~M cAMP (where indicated), and 32p_ labeled protein kinase containing approximately 1 pmol of 32p/pmol of enzyme. Reactions are generally incubated for 1 min at 23 ° or for 30 min in ice and are terminated by the rapid addition of 2.0 ml of cold, 0.2 mM Tris-HCl buffer, pH 7.5, followed by filtration on Millipore filters. The filters quantitatively retain the protein kinase. The filtrates (including a 3ml wash with the Tris-HCl buffer) are collected into tubes containing 10 ~1 of 1.0 N HC1. Norit (0.3 ml of a 1/10 suspension in water) is added with shaking and then collected on Whatman glass fiber (GF/C) filters. Controls in which either MgSO4 or ADP are omitted from the reaction mixture are always less than 10% of the experimentally determined values. Millipore and glass fiber filters are counted by liquid scintillation spectrometry. The reaction is dependent upon the presence of active protein
184
SPECIFIC PROTEIN KINASES
[18] la.i t.o
A X
I 15
x
0:: ~
9
Zz~
6
0_ I¢)
v
o Iz
f \x X--
o Q.. o.
5
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X
J 5
I 6
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~o
5
z
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I 8
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tr Q. I¢)
pH FIG. 6. Activity of the forward and reverse reactions at different pH values. The forward reaction (X--X) was carried out using 28 pmol of protein kinase and 4/xM ATP (1300 cpm/ pmol). Incubations were for 1 min at 0°. The reverse reaction ( O - - O ) was performed with 0.02 mM ADP, 5 mM Mg2÷, and 14 pmol of enzyme containing 0.74 pmol of 32p/pmol of enzyme, 341 cpm/pmol of 32p. Incubations were for 30 min at 0°. Both reactions were carried out in the absence of cAMP. Buffers were 0.05 M Tris-maleate, adjusted to give the indicated pH at 0°. From Rosen and Edichman) 4
kinase, Mg2+, and ADP. Other nucleotides including GDP cannot substitute for ADP. Although the forward autophosphorylation reaction is optimal between pH 6 and 8.5, the reverse reaction has a sharp pH optimum at pH 5.5 (Fig. 6). The rate of the reverse reaction, like the forward reaction, is extremely rapid and only linear (in the presence of cAMP) for 2 to 3 min at 23 °. The rate is dramatically enhanced over a wide range of ADP concentrations by the addition of cAMP (Fig. 7). The concentration of ADP required for half-maximal activation of the reverse reaction (in the presence of cAMP) is about 15/xM, only 35- to 40fold greater than the Km for ATP in the forward reaction. The concentration of ADP required to reverse the autophosphorylation reaction at more physiological pH values may, however, be substantially greater than that needed at pH 5.5. The second mechanism, a phosphoprotein phosphatase reaction, is in keeping with the precedent established for other proteins whose biological activity is regulated by reversible phosphorylation. A phosphoprotein phosphatase was purified from bovine cardiac muscle by the procedure of Chou et al.15 and used to study dephosphorylation of phospho-PK. Reac15 C. K. Chou, J. Alfano, and O. M. Rosen, J. Biol. Chem. 252, 2855 (1977).
[18]
AUTOPHOSPHORYLATION
OF
TYPE
II
PROTEIN
185
KINASE
,o A
03
w .J
24
- /,,of -/ 12 o
la.I tY
a.
°"
8
I
tM I¢)
J
~b,""'~ 40
I
I 80
I
I I I I I 120 160 200
I
I 240
I
I 280
I
I //I 320 640
ADP ( M.M )
FIG. 7. Effect of ADP on the reverse reaction. Phosphorylated protein kinase (54 pmol containing 0.74 pmol of 32P/pmol of enzyme, 85.2 cpm/pmol of 32p) was incubated with 5 mM Mg2÷and the indicated concentration of ADP for 1 min at 23° in the presence ((3--0) or absence ( 0 - - 0 ) of 20/xM cAMP. From Rosen and Edichman) 4 tions are carried out in 50 ~1 o f 50 m M T r i s - H C l buffer p H 7.4 containing 5 m M dithiothreitol, 90 ng purified p h o s p h o p r o t e i n p h o s p h a t a s e , and either 32p-R2 or 32p-RECz. A s shown in Table II, the p h o s p h a t a s e is far m o r e effective in r e m o v i n g 32p f r o m R2 than f r o m h o l o e n z y m e . Reassociation of P K with C a p p e a r s to block accessibility of the p h o s p h a t e group to the phosphatase. Other p h o s p h o p r o t e i n p h o s p h a t a s e s such as potato acid p h o s p h a t a s e ]6 also prefer p h o s p h o - R to p h o s p h o R2C2 as a substrate for dephosphorylation. Application of Autophosphorylation to the Study of Protein Kinase Structure and Regulation Studies f r o m several laboratories h a v e used a u t o p h o s p h o r y l a t i o n as a p r o b e to elucidate the structure and regulation of type II c A M P - d e p e n dent PKs. Early studies f r o m this laboratory showed that the phosphoform of P K was m o r e susceptible to net dissociation by c A M P than dep h o s p h o - P K . 2 Rangel and R o s e n 8,17 extended those studies to show that the degree o f p h o s p h o r y l a t i o n of R m a r k e d l y affects its ability to r e c o m bine with C to regenerate h o l o e n z y m e . The sequence of the autophos16o. M. Rosen, R. Rangel-Aldao, and J. Erlichman, Curr. Top. Cell. Regul. 12, 39 (1977). 17R. Rangel-Aldao and O. M. Rosen, J. Biol. Chem. 252, 7140 (1977).
186
[18]
SPECIFIC PROTEIN KINASES TABLE II EFFECT OF cAMP ON PHOSPHOPROTEIN PHOSPHATASE ACTIVITY ON THE PHOSPHORYLATED FORMS OF PROTEIN KINASE AND cAMP-BINDING PROTEINa'b
Substrate 1. Phosphoprotein kinase (3/~M)
cAMP (0.1 mM) -
+ 2. Phosphoprotein kinase (3/~M) + catalytic subunit (6.6 p.M) 3. Phospho-cAMP-binding protein (5.7/~M)
-
+ -
+ 4. Phospho-cAMP-binding protein (5.7/xM) + catalytic subunit (0.67/xM) 5. Phospho-cAMP-binding protein (5.7/zM) + catalytic subunit (3.3 p.M) 6. Phospho-cAMP-binding protein (5.7 p.M) + catalytic subunit (6.6/zM)
-
+ -
+ -
+
Phosphatase activity (pmol 32p released/5 min) 2.7 13.7 2.2 12.7 13.5 14.0 12.9 16.6 3.2 15.5 1.8 13.8
The reactions were performed with 90 ng of purified ethanol-treated phosphatase in a final volume of 25 /zl. Incubations were for 5 min at 37°. Protein kinase and cAMPbinding protein contained 7.8 nmol 32P/mg protein and 12.1 nmol 32P/mg protein, respectively. The specific activity of the 32p-labeled substrates was 965 cpm/pmol 32p. b From Chou et al. 15
phorylation site in bovine heart PK has been determined by Krebs and Beavo 18 and is homologous to the sequence present in the cAMP binding fragment in porcine skeletal muscle. 19 Studies by Flockhart e t al. 6 have shown that this autophosphorylation site is located within the catalytic subunit binding domain. Many investigators have used autophosphorylation of type II PKs to covalently radioactively label the enzyme so that the subunits can be easily identified during various procedures such as immunoprecipitation and polyacrylamide gel electrophoresis. 2°
18 E. G. Krebs and J. A. Beavo, Annu. Rev. Biochem. 48, 923 (1979). 19 R. L. Potter and S. S. Taylor, J. Biol. Chem. 254, 9000 (1979). 20 C. S. Rubin, R. Rangel-Aldao, D. Sarkar, J. Erlichman, and N. Fleischer, J. Biol. Chem. 254, 3797 (1979).
SPECIFIC PROTEIN KINASES
[18]
[18] R e v e r s i b l e A u t o p h o s p h o r y l a t i o n o f T y p e II cAMP-Dependent Protein Kinase: Distinction between Intramolecular and Intermolecular Reactions By JACK ERLICHMAN, RAPHAEL RANGEL-ALDAO, and ORA M. ROSEN
Many proteins are subject to reversible covalent modification by phosphorylation and it is widely recognized that this modification is a critical component of the physiological response of mammalian cells to specific stimuli. The cAMP-dependent protein kinases (PK) catalyze the initial phosphorylation reaction in cAMP-mediated pathways. In most tissues and cells there are two classes of cAMP-dependent protein kinases, designated types I and II by their order of elution from DEAE-cellulose. Both classes are composed of four polypeptide chains, consisting of a regulatory (R) dimer and two catalytic (C) subunits. The R components of the two types of kinase are different from each other whereas the C components appear to be both identical or very similar. 1 Both types I and II PKs are themselves subject to covalent modification by phosphorylation. 2,3 Each R monomer may contain up to 2 mol of phosphate; however, the mechanism by which R subunits are phosphorylated is different for each class of kinase 4'5 and may also be different for each site present in a R monomer. 6 Phosphorylation of the single, readily exchangeable site on type II R enhances binding of cAMP to R 7 and slows the rate of reassociation of R and C subunits when cAMP levels are lowered. 8 In this chapter we describe techniques to measure the reversible phosphorylation of the exchangeable site on type II R and distinguish between intramolecular and intermolecular phosphorylation of this site. Preparation of Protein Kinase Type II cAMP-dependent protein kinase, the predominant form of cAMP-dependent PK in bovine cardiac muscle, was purified from this tissue by the method of Rubin et al. and had a specific activity of 800 nmol i D. B. Glass and E. G. K r e b s , Annu. Rev. Pharmacol. Toxicol. 20, 363 (1980). 2 j. Erlichman, R. Rosenfeld, and O. M. R o s e n , J. Biol. Chem. 249, 5000 (1974). 3 R. L. G e a h l e n and E. G. K r e b s , J. Biol. Chem. 255, 1164 (1980). 4 R. Rangel-Aldao and O. M. R o s e n , J. Biol. Chem. 251, 7526 (1976). 5 R. L. G e a h l e n and E. G. K r e b s , J. Biol. Chem. 255, 9375 (1980). 6 D. A. Flockhart, D. M. Watterson, and J. D. Corbin, J. Biol. Chem. 255, 4435 (1980). 7 F. H o f m a n n , J. A. Beavo, P. J. Bechtel, a n d E. G. K r e b s , J. Biol. Chem. 250, 7795 (1975). 8 R. Rangel-Aldao and O. M. Rosen, J. Biol. Chem. 251, 3375 (1976).
METHODS IN ENZYMOLOGY, VOL. 99
Copyright © 1983by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181999-X
[18]
AUTOPHOSPHORYLATION OF TYPE II PROTEIN KINASE
177
32p transferred to protamine /min/mg protein. 9 R and C subunits were prepared by dissociation of the holoenzyme in the presence of cGMP followed by chromatography on DEAE-cellulose 9 or to-aminohexylagarose. 8 Purified R and C subunits from heart can also be prepared by a combination of chromatography on DEAE-cellulose and affinity chromatography on 8-(6-aminohexyl)amino-cAMP-Sepharose 4B. 1° C subunits are then isolated from the material that does not adsorb to the affinity resin by chromatography on CM-Sephadex. 11The kinase holoenzyme and its subunits prepared by these methods are greater than 90% pure when assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Preparation of Protein Kinase Inhibitor Protein kinase inhibitor was purified according to the method described by Walsh et al.12 Its specific activity was 650 units/mg of protein. One unit is defined as the amount needed to inhibit the incorporation of 1 pmol of 3~p into histone when 0.08 /zg of purified protein kinase was incubated for 15 rain at 37° in 200/zl containing 50 mM potassium phosphate buffer pH 7.1, l0 mM MgSO,, 50 /zg of histone IIA, 0.5 mg of bovine serum albumin, and 2/zM cAMP. Phosphorylation of Protein Kinase Holoenzyme R2 • C2 + 2 A T P ~ [R-P]2 • C2 + 2ADP
Purified protein kinase (10-100/xg) is incubated for 15 min at 23 ° in 100 /zl of 50 mM potassium phosphate buffer, pH 7.0, containing 50/.~M [y32p]ATP (>1000 epm/pmol), 10 mM MgSO4, and 1 mM dithiothreitol. Upon completion of the reaction the amount of 32p incorporated into the enzyme is determined by one of two methods: (1) An aliquot of the enzyme is precipitated with cold 10% trichloroacetic acid, the precipitate is collected on glass fiber filters, and assayed for radioactivity by liquid scintillation spectrometry. (2) An aliquot is transferred to another tube containing 200/zl of 50 mM potassium phosphate buffer (pH 7.1), containing 0.1 M EDTA and 2 /xM [3H]cAMP. The contents are poured onto Millipore filters, washed with 10 ml of the same buffer, dried, and assayed f o r 32p and 3H. Phosphorylation is expressed as moles 32p incorporated per mole [3H]cAMP bound at saturation. Unincorporated 32p is removed 9 C. S. Rubin, J. Erlichman, and O. M. Rosen, J. Biol. Chem. 247, 36 (1972). 10 W. Weber and H. Hilz, Eur. J. Biochem. 83, 215 (1978). ll p. j. Bechtel, J. A. Beavo, and E. G. Krebs, J. Biol. Chem. 252, 2691 (1977). 12 D. A. Walsh, C. D. Ashby, C. Gonzalez, D. Calkins, E. H. Fischer, and E. G. Krebs, J. Biol. Chem. 246, 1977 (1971).
178
SPECIFIC
1.0
PROTEIN
KINASES
[18]
B
0.8 n"
-I
0.6
o.,
--
O
--
i"
I
I
2
--I 3
I
I
I
I
4
5
6
7
I
ATP (FM) FIG. 1. Autophosphorylation at different concentrations of ATP. The concentrations of protein kinase were 71.2 pmol/ml ( O - - O ) and 213.7 pmol/ml ( 0 - - 0 ) . The specific activity of the ATP was 2200 cpm/pmol. Reactions were carried out at 4° for 2 min in the absence of cAMP. From Rosen and Erlichman. ~4
from samples by dialysis or filtration on Sephadex G-25. Since only one of the two cAMP binding sites present on the R monomer is measured using this assay 13 the ratio of 32p/3H approaches one when unphosphorylated enzyme is fully labeled. 8 With most preparations of enzyme, phosphorylation is 80% complete in less than 1 min and 100% complete after 10 min of incubation in the presence or absence of cAMP. The enzyme incorporates 1.0-1.9 mol of phosphate per mol of holoenzyme. It is likely that this variation in s2p incorporation reflects endogenous phosphate present in the purified enzyme. 14The rate of 32p incorporation can be slowed by either lowering the temperature to 4 ° or decreasing the concentration of [y-32p]ATp.14 The apparent Km for ATP for autophosphorylation is 0.4/zM (Fig. 1) compared to 12/zM ATP when exogenous proteins are used as substrates. 9 Phosphorylation of Isolated R Subunits Rz + 2ATP
c ~ [R-P]~ + 2ADP
cAMP
The procedure for phosphorylating isolated regulatory subunits is identical to autophosphorylation of holoenzyme except that catalytic 13 j. D. Corbin, P. H. Sugden, L. West, D. A. Flockhart, T. M. Lincoln, and D. McCarthy, J. Biol. Chem. 253, 3997 (1978). 14 O. M. Rosen and J. Erlichman, J. Biol. Chem. 250, 7788 (1975).
[18]
AUTOPHOSPHORYLATION OF TYPE II PROTEIN KINASE
179
amounts of C can be used to phosphorylate excess R subunits. The reaction requires cAMP to prevent reassociation of R and C. Heat-inactivated holoenzyme may be used in place of purified R subunits as the substrate.~4 Since heat-inactivated R is no longer capable of interacting with C, 6 cAMP is not needed if purified C is used instead of holoenzyme. Distinction between Intramolecular and Intermolecular Autophosphorylation There are a number of ways by which the phosphorylated form of the protein kinase holoenzyme could be generated: (1) intermolecular phosphorylation of R by the catalytic subunit in the presence of cAMP followed by reassociation of the subunits; and (2) intermolecular or intramolecular phosphorylation catalyzed by the intact holoenzyme. If phosphorylation occurs by an intermolecular reaction involving free C then conditions that inhibit catalytic subunit activity (e.g., addition of protein kinase inhibitor protein) should prevent phosphorylation of R. The initial velocity of the phosphorylation reaction should be proportional to the enzyme concentration if an intermolecular mechanism is involved and independent of enzyme concentration if the reaction is intramolecular. Finally, addition of catalytic subunit to the holoenzyme should affect the rate of phosphorylation of R if intermolecular phosphorylation is occurring.
Effect of Protein Kinase Inhibitors on Protein Kinase Activity Holoenzyme is incubated in 50 /zl of 50 mM potassium phosphate buffer, pH 7.1 at 0° with various concentrations of either protein kinase inhibitor protein, R subunits, or nonsubstrate peptides. The mixtures are then assayed at 37° for 15 min in the presence or absence of cAMP for (1) protein kinase activity using histone as substrate or (2) autophosphorylation. Typical results are shown in Figs. 2 and 3 and Table I. Addition of protein kinase inhibitor blocks histone phosphorylation in the presence or absence of cAMP but has no effect on autophosphorylation in the absence of cAMP (Fig. 2). Similarly, the addition of an inhibitor peptide such as Lys-Tyr-Thr has no effect on autophosphorylation in the absence of cAMP but blocks phosphorylation of R in the presence of cAMP (Fig. 3). R subunits in excess of C are phosphorylated only when cAMP is added to dissociate the holoenzyme (Table I).
Effect of Enzyme Concentration on the Rate of Autophosphorylation Initial velocity of autophosphorylation is measured at several enzyme dilutions. Incubations are carried out in 50/xl of 50 mM potassium phos-
180
SPECIFIC PROTEIN KINASES
[18]
o 0
O
E
A
--
0.5 ~
o
(.3
0.4t~
4 0
~ 3
-\
O
0 . 2 ~O
Q.
0.1
" Q. N
2
4
6
Protein I
I 0.2
I
8
I0 12 14 16 18 20
Kinase I
I
I
Inhibitor I
I
I
m
[/~g) I
I 2
R2 (/~g)
FIG. 2. Effect of cAMP-binding protein and protein kinase inhibitor on protein kinase activity. Holoenzyme (0.08 /xg) was incubated in 20 /zl of 50 mM potassium phosphate buffer, pH 7.1, for 5 min at 0° with either protein kinase inhibitor or cAMP-bindingprotein at the concentrations depicted on the abscissa. The mixtures were then assayed at 37° for 15 min for either (a) protein kinase activity using histone as substrate or (b) self-phosphorylation in the presence of 5 /zM [y-32P]ATP(9000 cpm/pmol). For assays using histone as substrate [see (a), above] the symbols are ( 0 - - 0 ) R2C2plus protein kinase inhibitor plus 2 /~M cAMP; (O--O) R2C2 plus protein kinase inhibitor; (0---0) R2C: plus R: plus 2/zM cAMP; (©---O) R2C2plus R2. For self-phosphorylationassays [see (b), above] the symbols used are (A--A) R2Cz plus protein kinase inhibitor, (A--&) R:C2 plus R2. From RangelAldao and Rosen.4
phate buffer p H 7.1 at 4 °. Lowering the temperature to 4 ° slows the reaction rate sufficiently to measure initial velocities when short incubation times are used (see Fig. 4). The velocity of autophosphorylation in the absence of c A M P is not affected by a 100-fold dilution of the e n z y m e (Fig. 4). Furthermore, when C subunits are added to excess R in the absence of c A M P , phosphorylation of R is stoichiometric with the a m o u n t of h o l o e n z y m e formed. In the presence of c A M P , maximal p h o s p h o r y lation of R is independent of the concentration of C and all o f the R subunits can be phosphorylated at each concentration of C (Fig. 5). When h o l o e n z y m e is used as a substrate for C the rate of autophosphorylation is unaffected by the addition of C subunits. 4 These studies indicate that the C subunit catalyzes the intramolecular autophosphorylation of its own R
[18]
AUTOPHOSPHORYLATION OF TYPE II PROTEIN KINASE
>-
181
c 50 E o
-- ~ 4 0 N
I.u
30,
.
.
.
.
.
.
=
z $a - 2 0
5 1o_ I
I
10 20
I
I
I
50
75
150
CONCENTRATION OF LYS - T Y R - THR (mM_)
FIG. 3. Effect of Lys-Tyr-Thr on self-phosphorylation of protein kinase. Holoenzyme (2.85 pmol) was incubated at 4° for 1 min in 50/zl of 50 mM potassium phosphate buffer, pH 7.1, containing 10 mM MgSO4, 0.5 /xM [y-32P]ATP (13,180 cpm/pmol), 50 /xg of bovine serum albumin, with ( 0 - - 0 ) or without (O---Q) 1 /zM cAMP. The reaction was stopped by the addition of 200 txl of 0.1 M EDTA, pH 4.0 and the mixture filtered on Millipore filters. The filters were washed twice with 10 ml of the EDTA solution, dried, and assayed for 32p in a solution of Omnifluor/toluene, 4 g/liter. The total amount of 32p incorporated into protein kinase was 3.7 pmol. From Rosen e t a l ) 6
TABLE I PHOSPHORYLATION OF c A M P - B I N D I N G PROTEIN BY PROTEIN KINASE IN THE PRESENCE AND ABSENCE OF cAMP~,b
R2C2 (pmol)
R2 . (pmol)
cAMP
[32p]Phosphate incorporated (pmol)
5.70 5.70 5.70 5.70
--15 15
+ +
6.40 6.40 7.03 20.17
a Holoenzyme was incubated in 25/zl of 50 mM potassium phosphate buffer, pH 7.1, with or without cAMP-binding protein. The mixtures were then assayed for self-phosphorylation in the presence of 0.2 mM [3,-32p]ATP (1000 cpm/ pmol) and, where indicated, 8 ttM cAMP. Incubations were for 10 min at 37° in a final volume of 50 /xl. RzC2, holoenzyme; R2, cAMP-binding protein. b From Rangel-Aldao and Rosen. 4
182
SPECIFIC PROTEIN KINASES
[18]
)A
v--_ .D >
8-
•
_
< z
"o_
• / °
4
--~
.~..~
z
2
rr ~
0
" I " ~'~' '~I--9-15
0
a.
I
I
50 45 TIME (see)
I
I
60
75
FIG. 4. Effect of protein kinase concentration on self-phosphorylation. Holoenzyme was phosphorylated at three different concentrations at 4 ° in 50/xl of 50 mM potassium phosphate, pH 7.1, containing 10 mM MgSO4, 50/~g of bovine serum albumin, 5/zM [7-32P]ATP (30,000 cpm/pmol), and, where indicated, 1.0/zM cAMP. The 32p incorporated into the holoenzyme at each concentration was multiplied by the dilution factor in order to obtain the values depicted on the ordinate. The symbols used are ( 0 - . - © ) , 22.8 pmol of R2C:; ( A - . - A ) , 1,14 pmol of R2C2, (?q-.-F3), 0.228 pmol of R2C2; ( e - - O ) , 22.8 pmol of R2C2 + cAMP; (O-nO), 0.228 pmol of R2C2 + cAMP. From Rangel-Aldao and Rosen. 4
5 -
~
4
o.
•o
iii o
"
l
5 /
/ O
/
2
/ / / I
I I
P
0 s" I 0 I
I
I
I
I
I
I
I
2 3 4 5 6 7 8
C (pmole) FIG. 5. Phosphorylation of cAMP-binding protein by catalytic subunit in the presence or absence of cAMP. cAMP-binding protein (4 pmol) was incubated at 4 ° for 1 min with the indicated amounts of catalytic subunit in 40/~1 of 50 mM potassium phosphate buffer, pH 7.1, containing 10 mM MgSO4, with ( O - - O ) or without (©---O) 1.0/~M cAMP. The reaction was initiated by the addition of 100 pmol of [y-32p]ATP (21,000 cpm/pmol) and incubations were carded out at 4 ° for 20 min. From Rangel and Rosen. 4
[18]
A U T O P H O S P H O R Y L A T I O N OF TYPE I I PROTEIN KINASE
183
subunit when present in the intact holoenzyme but can also catalyze the intermolecular phosphorylation of R when the holoenzyme is dissociated by cAMP. The kinetic parameters of the intramolecular autophosphorylation are distinguishable from those characteristic of the intermolecular phosphorylation. The Km for ATP is 30-fold lower. The velocity is significantly greater for the intramolecular phosphorylation. In the absence of cAMP, only those R subunits associated with C can serve as a substrate. In contrast, the intermolecular phosphorylation of R is analogous to phosphorylation of exogenous protein substrates such as histones or protamine. Phosphorylation is blocked by inhibitors of catalytic activity such as the protein kinase inhibitor. The rate of phosphorylation of excess R is stimulated by cAMP and proportional to the concentration of C. Since the total concentration of ATP in mammalian cells is greater than 1 mM and the kinase exists principally as a holoenzyme in the unstimulated cell, it is likely that the intramolecular reaction is responsible for maintaining the holoenzyme in the phosphoform. Reversal of Autophosphorylation and Dephosphorylation [R-P]2 " C2 + 2 A D P - - > R2C2 + 2 A T P JR-P]2 " C2
phosphoprotein phosphatase ~ R2 • C2 + 2Pi
There are two mechanisms for dephosphorylating phospho-R. The first is reversal of the autophosphorylation reaction. This reaction is monitored by measuring the disappearance of radioactivity from 32p-labeled protein kinase concomitant with the formation of charcoal-adsorbable radioactivity which is identified chromatographically as [32p]ATP.14 The standard reaction mixture (0.1 ml) contains 50 mM Tris-maleate buffer, pH 5.5, 5 mM MgSO4, 20 p~M cAMP (where indicated), and 32p_ labeled protein kinase containing approximately 1 pmol of 32p/pmol of enzyme. Reactions are generally incubated for 1 min at 23 ° or for 30 min in ice and are terminated by the rapid addition of 2.0 ml of cold, 0.2 mM Tris-HCl buffer, pH 7.5, followed by filtration on Millipore filters. The filters quantitatively retain the protein kinase. The filtrates (including a 3ml wash with the Tris-HCl buffer) are collected into tubes containing 10 ~1 of 1.0 N HC1. Norit (0.3 ml of a 1/10 suspension in water) is added with shaking and then collected on Whatman glass fiber (GF/C) filters. Controls in which either MgSO4 or ADP are omitted from the reaction mixture are always less than 10% of the experimentally determined values. Millipore and glass fiber filters are counted by liquid scintillation spectrometry. The reaction is dependent upon the presence of active protein
184
SPECIFIC PROTEIN KINASES
[18] la.i t.o
A X
I 15
x
0:: ~
9
Zz~
6
0_ I¢)
v
o Iz
f \x X--
o Q.. o.
5
5E
X
X
J 5
I 6
I 7
"j~
2-_ IM _.j LtJ
~o
5
z
I
I 8
o-
tr Q. I¢)
pH FIG. 6. Activity of the forward and reverse reactions at different pH values. The forward reaction (X--X) was carried out using 28 pmol of protein kinase and 4/xM ATP (1300 cpm/ pmol). Incubations were for 1 min at 0°. The reverse reaction ( O - - O ) was performed with 0.02 mM ADP, 5 mM Mg2÷, and 14 pmol of enzyme containing 0.74 pmol of 32p/pmol of enzyme, 341 cpm/pmol of 32p. Incubations were for 30 min at 0°. Both reactions were carried out in the absence of cAMP. Buffers were 0.05 M Tris-maleate, adjusted to give the indicated pH at 0°. From Rosen and Edichman) 4
kinase, Mg2+, and ADP. Other nucleotides including GDP cannot substitute for ADP. Although the forward autophosphorylation reaction is optimal between pH 6 and 8.5, the reverse reaction has a sharp pH optimum at pH 5.5 (Fig. 6). The rate of the reverse reaction, like the forward reaction, is extremely rapid and only linear (in the presence of cAMP) for 2 to 3 min at 23 °. The rate is dramatically enhanced over a wide range of ADP concentrations by the addition of cAMP (Fig. 7). The concentration of ADP required for half-maximal activation of the reverse reaction (in the presence of cAMP) is about 15/xM, only 35- to 40fold greater than the Km for ATP in the forward reaction. The concentration of ADP required to reverse the autophosphorylation reaction at more physiological pH values may, however, be substantially greater than that needed at pH 5.5. The second mechanism, a phosphoprotein phosphatase reaction, is in keeping with the precedent established for other proteins whose biological activity is regulated by reversible phosphorylation. A phosphoprotein phosphatase was purified from bovine cardiac muscle by the procedure of Chou et al.15 and used to study dephosphorylation of phospho-PK. Reac15 C. K. Chou, J. Alfano, and O. M. Rosen, J. Biol. Chem. 252, 2855 (1977).
[18]
AUTOPHOSPHORYLATION
OF
TYPE
II
PROTEIN
185
KINASE
,o A
03
w .J
24
- /,,of -/ 12 o
la.I tY
a.
°"
8
I
tM I¢)
J
~b,""'~ 40
I
I 80
I
I I I I I 120 160 200
I
I 240
I
I 280
I
I //I 320 640
ADP ( M.M )
FIG. 7. Effect of ADP on the reverse reaction. Phosphorylated protein kinase (54 pmol containing 0.74 pmol of 32P/pmol of enzyme, 85.2 cpm/pmol of 32p) was incubated with 5 mM Mg2÷and the indicated concentration of ADP for 1 min at 23° in the presence ((3--0) or absence ( 0 - - 0 ) of 20/xM cAMP. From Rosen and Edichman) 4 tions are carried out in 50 ~1 o f 50 m M T r i s - H C l buffer p H 7.4 containing 5 m M dithiothreitol, 90 ng purified p h o s p h o p r o t e i n p h o s p h a t a s e , and either 32p-R2 or 32p-RECz. A s shown in Table II, the p h o s p h a t a s e is far m o r e effective in r e m o v i n g 32p f r o m R2 than f r o m h o l o e n z y m e . Reassociation of P K with C a p p e a r s to block accessibility of the p h o s p h a t e group to the phosphatase. Other p h o s p h o p r o t e i n p h o s p h a t a s e s such as potato acid p h o s p h a t a s e ]6 also prefer p h o s p h o - R to p h o s p h o R2C2 as a substrate for dephosphorylation. Application of Autophosphorylation to the Study of Protein Kinase Structure and Regulation Studies f r o m several laboratories h a v e used a u t o p h o s p h o r y l a t i o n as a p r o b e to elucidate the structure and regulation of type II c A M P - d e p e n dent PKs. Early studies f r o m this laboratory showed that the phosphoform of P K was m o r e susceptible to net dissociation by c A M P than dep h o s p h o - P K . 2 Rangel and R o s e n 8,17 extended those studies to show that the degree o f p h o s p h o r y l a t i o n of R m a r k e d l y affects its ability to r e c o m bine with C to regenerate h o l o e n z y m e . The sequence of the autophos16o. M. Rosen, R. Rangel-Aldao, and J. Erlichman, Curr. Top. Cell. Regul. 12, 39 (1977). 17R. Rangel-Aldao and O. M. Rosen, J. Biol. Chem. 252, 7140 (1977).
186
[18]
SPECIFIC PROTEIN KINASES TABLE II EFFECT OF cAMP ON PHOSPHOPROTEIN PHOSPHATASE ACTIVITY ON THE PHOSPHORYLATED FORMS OF PROTEIN KINASE AND cAMP-BINDING PROTEINa'b
Substrate 1. Phosphoprotein kinase (3/~M)
cAMP (0.1 mM) -
+ 2. Phosphoprotein kinase (3/~M) + catalytic subunit (6.6 p.M) 3. Phospho-cAMP-binding protein (5.7/~M)
-
+ -
+ 4. Phospho-cAMP-binding protein (5.7/xM) + catalytic subunit (0.67/xM) 5. Phospho-cAMP-binding protein (5.7/zM) + catalytic subunit (3.3 p.M) 6. Phospho-cAMP-binding protein (5.7 p.M) + catalytic subunit (6.6/zM)
-
+ -
+ -
+
Phosphatase activity (pmol 32p released/5 min) 2.7 13.7 2.2 12.7 13.5 14.0 12.9 16.6 3.2 15.5 1.8 13.8
The reactions were performed with 90 ng of purified ethanol-treated phosphatase in a final volume of 25 /zl. Incubations were for 5 min at 37°. Protein kinase and cAMPbinding protein contained 7.8 nmol 32P/mg protein and 12.1 nmol 32P/mg protein, respectively. The specific activity of the 32p-labeled substrates was 965 cpm/pmol 32p. b From Chou et al. 15
phorylation site in bovine heart PK has been determined by Krebs and Beavo 18 and is homologous to the sequence present in the cAMP binding fragment in porcine skeletal muscle. 19 Studies by Flockhart e t al. 6 have shown that this autophosphorylation site is located within the catalytic subunit binding domain. Many investigators have used autophosphorylation of type II PKs to covalently radioactively label the enzyme so that the subunits can be easily identified during various procedures such as immunoprecipitation and polyacrylamide gel electrophoresis. 2°
18 E. G. Krebs and J. A. Beavo, Annu. Rev. Biochem. 48, 923 (1979). 19 R. L. Potter and S. S. Taylor, J. Biol. Chem. 254, 9000 (1979). 20 C. S. Rubin, R. Rangel-Aldao, D. Sarkar, J. Erlichman, and N. Fleischer, J. Biol. Chem. 254, 3797 (1979).