Proc. Nat. Acad. Sci. USA Vol. 72, No. 9, pp. 3448-3452, September 1975 Biochemistry
Substrate specificity of the cyclic AMP-dependent protein kinase (caseins/protein sequence)
BRUCE E. KEMP, DAVID B. BYLUND, TUNG-SHIUH HUANG, AND EDWIN G. KREBS Department of Biological Chemistry, School of Medicine, University of California, Davis, Calif. 95616
Contributed by Edwin G. Krebs, July 7,1975
ABSTRACT The protein substrate specificity of the catalytic subunit of rabbit skeletal muscle cyclic AMP-dependent protein kinase (EC 2.7.1.37; ATP:protein phosphotransferase) has been studied using genetic variants of (3 casein. It was found that (3 casein-B was phosphorylated at a much greater rate than (3 caseins Al, A2, A3, or C. The enhanced phosphorylation of (3 casein-B, as compared with the most common variant A2, was attributed to an arginine substitution for a serine at position 122, which caused a nearby residue, serine 124, to become a phosphorylation site for the protein kinase. These results further support the concept that the local primary structure is important in specificity and that arginine may be a specific determinant common to all the local phosphorylation site sequences recognized by the cyclic AMPdependent protein kinase. The apparent lack of protein substrate specificity exhibited by the cyclic AMP-dependent protein kinase (EC 2.7.1.37; ATP:protein phosphotransferase) has been an enigma in the current concept of cyclic AMP-mediated hormonal regulation. Intuitively the physiological role of this enzyme would seem to demand considerable substrate specificity, and yet it has been shown to phosphorylate a wide variety of proteins in vitro (1). Several authors have considered the possibility that the primary sequence of amino acids around the phosphorylation site might be an important specificity determinant for the protein kinase (2, 3, 9). Precedent for this idea can be found in other protein modification reactions (4, 5). However, the protein kinase phosphorylation site sequences appeared to lack any obvious similarities (see Table 1). Accordingly, it was suggested that some aspect of the tertiary structure of the substrates might be involved (2, 3) which would not be apparent in the local primary sequence around the phosphorylation site. There are, however, several observations that are difficult to reconcile with the concept that some unique configuration of peptide chains is required for specificity. For instance, recent studies in this laboratory have shown that although native chicken lysosome is not a substrate for the protein kinase, it is phosphorylated by this enzyme at two specific sites, serine 24 to serine 50, after chemical denaturation. Daile and Carnegie (10) have reported that the cyclic AMP-dependent protein kinase will phosphorylate a 17-residue peptide isolated from human myelin basic protein which presumably no longer retains the tertiary structure of the native protein. The cyclic AMPdependent and cyclic AMP-independent protein kinases phosphorylate different sites in two substrates that have a comparatively uncomplicated tertiary structure, namely, histone F1 (2) and myelin basic protein (8). Even though all sites are presumably available for phosphorylation in these two substrates, each kinase is specific. In this communication we report studies on the specificity of the cyclic AMP-dependent protein kinase towards genetic
variants of $ casein. Since the amino-acid sequences of these variants are known, it has been possible to relate differences in substrate properties to specific substitutions in the primary sequence. The results support the concept that local primary protein structure plays a major role in determining the substrate specificity of the cyclic AMP-dependent protein kinase. METHODS AND MATERIALS Cyclic AMP-Dependent Protein Kinase. Homogeneous catalytic subunit of rabbit skeletal muscle cyclic AMP-dependent protein kinase (Peak I) prepared by the method of Beavo et al. (11) was used in this study. Phosphorylation of Protein Substrates by the Protein Kinase. The reaction mixture, with a final volume of 0.08 ml, contained 1.0 mM ['y-32P]ATP (10-200 cpm/pmol), 62.5 mM 2(N-morpholino)ethanesulfonic acid (pH 6.8), 12.5 mM magnesium acetate, 0.25 mM ethylene glycol bis-(f3-aminoethyl ether)-N-N'-tetraacetic acid, protein kinase, and substrate at concentrations as indicated in the appropriate figure legends. Incubations were carried out for 5 min at 30°. Reactions were terminated with glacial acetic acid to give a final concentration of 30%. Anion exchange columns (2 ml AG 1 X 8 resin, Bio-Rad) equilibrated with 30% acetic acid were used to separate the phosphoprotein from the [-y32P]ATP. Under these conditions proteins and peptides are fully protonated and elute from the column, whereas ATP and Pi bind tightly. The columns were eluted directly into liquid scintillation vials, and the excess acetic acid was removed by evaporation. Quantitative recoveries of phosphorylated f casein, histone, and lysozyme were obtained. Cyanogen Bromide (BrCN) and Enzymic Digestions. Phosphorylated (3 casein-B (48 mg) was lyophilized in the presence of 86 Atmol of 2-mercaptoethanol prior to digestion with cyanogen bromide (200-fold excess of BrCN over total thiol groups) in 70% formic acid (12). Enzymatic digestions with alpha-chymotrypsin (Worthington) and aminopeptidase M (Henley and Co., New York) were performed according to published procedures (13). The resultant peptideso were purified by gel filtration using Sephadex G-25 and G-50 (Pharmacia) columns (1.9 X 160 cm) in the presence of 30% acetic acid. Primary amine-containing peptides in the column effluent were detected using fluorescamine (Fluram, Roche Diagnostics Corp.) (14). High voltage electrophoresis and amino acid analysis were performed as described (9). Casein Variants. The purified casein fractions used in this study were very kindly provided by Mrs. E. W. Bingham and Dr. M. L. Groves of the USDA Eastern Regional Research Center. These fractions were isolated from the milk of individual cows homozygous for the respective casein variants (16, 23). 3448
Proc. Nat. Acad. Sci. USA 72 (1975)
Biochemistry: Kemp et al.
3449
Table 1. Sites of phosphorylation by mammalian cyclic AMP-dependent protein kinase
/3 Casein-B Phosphorylase kinase
((3subunit) Phosphorylase kinase (az subunit) Pyruvate kinase Glycogen synthetase
Histone F,
This paper
Phe-Thr-Glu-Arg-Gln-Ser(P)-Leu-Thr-Leu-Thr-Asp 124 Val Arg* -Gln-Ser-Gly-Ser(P)- -Tyr-Pro-Leu-Lys Ile Lys Arg* -Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser Lys Leu-Arg-Arg-Ala-Ser(P)-Leu Arg*
Lys RCMM lysozymet
Ref.
Sequence
Substrate
(3) (3) (18) (19)
-Glx-Ile-Ser(P)-Val-Arg
(9)
Asn-Tyr-Arg-Gly-Tyr-Ser(P)-Leu-Gly-Asn-Trp-Val 24 Arg-Asn-Thr-Asp-Gly-Ser(P)-Thr-Asp-Tyr-Gly-Ile 50
(9)
(2, 6, 21)
Ala-Arg-Arg-Lys-Ala-Ser(P)-Gly-Pro-Pro-Val-Ser 38
Troponin Histone F2bt
(13, 20) (13, 20)
Arg-Gln-His-Leu-Lys-Ser(P)-Val-Met-Gln-Leu Val-Arg-Met-Ser(P)-Ala-Asp-Ala-Met-Leu Arg-Lys-Glu-Ser-Tyr-Ser-Val-Tyr-Val-Tyr-Lys
(22)
38 Myelin basic protein
(7, 8)
Lys-Gly-Arg-Gly-Leu-Ser(P)-Leu-Ser-Arg-Phe-Ser § 110
(8)
Pro-Arg-His-Arg-Asp-Thr(P)-Gly-Ile-Leu-Asp-Ser 34
(7)
Ser-Gln-Arg-His-Gly-Ser(P)-Lys-Tyr-Leu-Ala-Thrl 12
* Arginine or lysine expected since it is a tryptic phosphopeptide. t Reduced, carboxymethylated, and maleylated (RCMM-) chicken lysozyme. t It is not known which of the two serines in this peptide is phosphorylated. § Phosphorylation site identified in human, rat, and bovine myelin basic protein. Phosphorylation site identified in bovine myelin basic protein. Phosphorylation site identified in rat and human myelin basic protein.
RESULTS Comparison of casein variants as substrates for the protein kinase Several casein variants were tested as substrates for protein kinase. Since these variants differ by known amino-acid substitutions (Table 2), it was possible to correlate rates of phosphorylation with their primary structure differences. One variant, casein-B, was phosphorylated approximately 70fold more rapidly than the most common variant, casein A2 (Table 2). Compared with this variant, casein-B differs by two amino acids, having proline 67 replaced by a histidine and serine 122 replaced by an arginine. Even at substrate concentrations up to 8 mg/ml, the rates of phosphorylation of all the casein variants except casein-B were negligible compared with the rate obtained with whole calf thymus histone. Characteristics of the reaction product using ,B caseinB A time course of phosphorylation of casein-B showed that it was phosphorylated by the protein kinase to the extent of 0.8 mol of phosphate per mol of substrate in 60 min (Fig. 1A). Phosphate incorporation beyond this point proceeded very slowly, i.e., after 6 hr of incubation only 1.2 mol of phosphate was incorporated per mol. These observations suggest that the rapid phosphorylation of casein-B occurs at a single site.
The phosphorylated ,B casein-B migrated as a single band on sodium dodecyl sulfate/acrylamide gels, and 84% of the radioactivity applied to the gel was recovered from this band (see Fig. 1B). The radioactivity associated with the / casein-B was alkali-labile (96% release in 15 min in 0.1 M NaOH at 1000) and relatively stable in acid (92% remaining after 15 min in 0.1 M HCI at 100°). These properties are characteristic of either serine or threonine phosphoester linkages. After partial acid hydrolysis (6 M HCI, 1000, 2 hr Table 2. Phosphorylation of ( casein genetic variants Protein kinase activity
Substrate (3 casein (nmol/min per mg) variant Al A2
A3 B C
17.1 21.5 14.6 1425.6 37.5
Amino-acid residues in the variable positions* 37
67
106
122
Glu Glu Glu Glu Lys
His Pro Pro His His
His His Gln His His
Ser Ser Ser Arg Ser
Protein kinase assays were done as described in Methods and Materials with an incubation time of 5 min, a [y-32P]ATP concentration of 0.95 mM, and protein substrate and enzyme concentrations of 2.5 mg/ml and 16 Mg/ml, respectively. * Assignments according to Grosclaude et al. (15).
3450
Biochemistry: Kemp et al. Table 3. Phosphorylation of casein temperature-sensitive fragments
Proc. Nat. Acad. Sci. USA 72 (1975)
m 1.0 ._
(n
0
Substrate* TS.A2
Protein kinase activity (nmol/ min per mg) 0.0
c- 0.8 0
Corresponding amino-acid sequences in (3 casein
His-Lys-Glu. . Ser.. Val-OH 108 122.. .209 Gln-Lys-Glu. . .Ser. . .Val-OH 106 108 122 209 Glu. . .Arg... .Val-OH 108 122 209
106
TS.A3
0.2
TS.B
61.4
f3Casein-B Calf thymus histone
51.5 188.7
a, 0 m 1%
0.6
U.
00.4
ca. 0~ m
0.2
0
(n 0
0
10
Protein kinase assays were done as described in Methods and Materials and Table 1, except that the incubation temperature was 0.5°, the enzyme concentration 10 Ag/ml, and the final concentration of histone was 1 mg/ml. The casein fractions were all tested at a final concentration of 2.5 mg/ml. * Nomenclature for the temperature-sensitive fragments according to Groves et al. (16).
in an evacuated tube) and high voltage electrophoresis at pH 1.9, 34.3% and 36.6% of the radioactivity migrated with the phosphoserine and inorganic phosphate markers, respectively. No more than 0.2% of the radioactivity could be detected in the phosphothreonine position. The remaining 29% of the phosphorylated product was incompletely hydrolyzed and migrated towards the cathode. The total recovery of radioactivity applied to the electrophoretogram was 81%.
20
30 40 Time (min)
60
50
5 4 CO
0 x
3
E 0. 0
0-
cmm
2
Phosphorylation of temperature-sensitive fragments Acid precipitates of whole bovine casein are known to contain temperature-sensitive protein fragments that precipitate at room temperature but are quite soluble at lower temperatures. The amino-acid sequences of these fragments are homologous with the carboxyl-terminal halves of native , caseins (16). Thus, the temperature-sensitive fragment related to # casein-B, TS.B, has an amino-acid sequence corresponding to the segment containing glutamate 108 through to the carboxyl-terminal residue valine 209 of this variant. Several of these temperature-sensitive fragments corresponding to different variants were tested as substrates for the protein kinase. Phosphorylation of both fragments TS.A2 and TS.A3 was negligible compared with either TS.B or native flcasein-B (Table 3). Insolubility of the /3 casein temperature-sensitive fragments at normal temperatures necessitated that the assay temperature be reduced to 0.50. As expected, the absolute rates of phosphorylation of other substrates were also depressed under these conditions (Table 3). Since fragment TS.B contains only the arginine substitution, of the two amino-acid substitutions that occur in native /3casein-B, it appeared probable that this substitution alone was responsible for the enhanced phosphorylation of both TS.B and (3 casein-B compared with the other variants.
Identification of the site(s) in 3 casein-B phosphorylated by protein kinase 3 Casein-B (48 mg), phosphorylated to the extent of 0.8 mole of phosphate per mol, was digested with BrCN. One major radioactive peak (representing 81% of the total 32p incorporated) was detected after gel chromatography on Se-
1
0
2
4 cm
'6
8
FIG. 1. f8 Casein-B phosphorylation. (A) Time course of ,B casein-B phosphorylation. f3 Casein-B (4 mg/ml) was incubated with 10 ,gg/ml of protein kinase in a large reaction mixture (12 ml) having the composition described in Methods and Materials. Aliquots (15 ul) were removed at intervals, and the incorporation of 32p was measured as described. (B) Sodium dodecyl sulfate/polyacrylamide (10%) gel electrophoresis of phosphorylated f, casein-B. Gels were stained with Coomassie blue, photographed, and sliced into 2-mm fractions. Radioactivity associated with the gel slices was determined by liquid scintillation counting after digestion with 30% H202 at 950.
phadex G-50. The amino-acid composition of the purified phosphopeptide (rechromatography on Sephadex G-50 and electrophoresis at pH 1.9) was found to correspond with one of the expected seven cyanogen bromide peptides reported by Ribadeau-Dumas et al. (17). The amino-acid composition of the phosphopeptide corresponded with the peptide BrCN-2 described by these authors and is derived from the amino-acid sequence between Pro-110 and Met-144. This region is located in the COOH-terminal half of f casein-B and contains a high proportion of hydrophobic residues (see Fig. 3).
Proc. Nat. Acad. Sci. USA 72 (1975)
Biochemistry: Kemp et al.
120
I
-6
.5
O,
0.5
4
C2
°> c 4
;r
-i4 "45
=
1
130 Arg-Gln-Ser(P)-Leu-Thr-Leu-Thr-Asp-Val-GluC-1
Co
co
0.3 @
C._
0.2 o
'C 4
w
Pro-Phe-Pro-Lys-Tyr-Pro-Val-Gln-Pro-Phe-Thr-GluS ~~~C-3-A C-3-A _~~~~~~~~~~~~_______
0.4 c
*4.C
o
ci
3451
~~~~~C-2
-
---C-4 -
_
0.1
Cl-
contains only two serine residues, namely, Ser-124 and Ser142. In order to determine which of these two sites was
140 Asn-Leu-His-Leu-Pro-Pro-Leu-Leu-Leu-Gln-Ser-Trp-Met * ** -C-5b e ** C~C-3-B FIG. 3. Phosphorylation site of fl casein-B incubated with protein kinase. The amino-acid sequence of peptide BrCN-2 is shown. This sequence corresponds with the region between Pro-110 and Met-144 in fl casein-B as designated by Ribadeau-Dumas et al. (17). The chymotryptic peptides derived from BrCN-2 were identified on the basis of their amino acid composition (see Table 4).
phosphorylated, peptide BrCN-2 was further hydrolyzed with chymotrypsin. The chymotryptic peptides were subsequently fractionated on Sephadex G-25 (Fig. 2) and purified by high voltage electrophoresis at pH 1.9. The amino-acid compositions of the purified chymotryptic peptides are shown in Table 4. Two chymotryptic peptides, BrCN-2-C-5 and BrCN-2-C-6, gave a positive Ehrlich reaction and had amino-acid compositions corresponding to the sequence containing residues 140 to 144 and 140 to 143, respectively. These peptides are derived from the COOH-terminal region of BrCN-2 and include Ser-142. The presence of tryptophan and serine in both these chymotryptic peptides was also demonstrated by amino-acid analysis after aminopeptidase M digestion. Since no 32P radioactivity was associated with
either of the chymotryptic peptides containing Ser-142, it follows that this residue was not phosphorylated. The radioactive peptides BrCN-2-C-1, BrCH-2-C-2, and BrCH-2C-4 had amino-acid compositions corresponding to the sequences containing residues 120 to 139, 120 to 133, and 120 to 125, respectively. All three of these phosphopeptides contained Ser-124. Since only phosphoserine and not phosphothreonine was detected in partial acid hydrolysates of either the intact phosphorylated casein-B or the phosphopeptide BrCN-2, it follows that serine 124 is the phosphorylation site. Near stoichiometric recovery of the radioactive 32P and the chymotryptic peptides was obtained (see Table 4). In Fig. 3 the primary structure of the phosphopeptide
l0
0
20
30
40
50
60
70
80
90
FRACTION NUMBER
FIG. 2. Sephadex G-25 column profile of chymotryptic peptides derived from phosphorylated peptide BrCN-2. Fractions (5 ml) were assayed for absorbance at 280 nm, 32P radioactivity, and primary amino containing peptides with fluorescamine.
The amino-acid
sequence
between Pro-110 and Met-144
Table 4. Amino-acid composition of chymotryptic peptides derived from peptide BrCN-2 C-1 0
Lys His Arg Asp Thr* Sert Glu Pro Gly Ala Val Ile Leu Tyr* Phe HL Trp §
32P
(0.06) 0.78 0.96 2.09 2.38 0.90 3.25 2.46 (0.2) -
1.24 -
E
0
-
(0.05) (0.27) 0.81 2.05 2.79 0.98 2.87 (0.62) (0.27) (0.08)
1
1.12
-
1 1 2 3 1 3 2
-
-
E
0
-
0.91
-
-
1 2 3 1 3
-
-
1
(0.02) 0.76 (0.48) 1.09 0.83
-
-
-
-
-
-
-
(0.02) 1.01
-
1 4 -
-
1.01 -
6
-
-
-
(0.18)
-
0.08
-
(0.05) 0.97 1.97
-
-
-
-
-
-
-
1.02
-
-
Total residues 20 Peptide (nmol) 230.2
-
0.8 (0.11)
-
3
1 -
-
-
-
E
-
4.12 (0.03)
1
0
-
-
-
E
C-4
(0.02)
5.93
3.39
C-3-B
C-3-A
C-2
-
-
-
-
2.12 (0.11) -
1
-
-
-
-
3.08
2 -
-
-
3
E
0
1.94 (0.27) (0.09) (0.05)
E
0
-
-
-
-
(0.01)
-
-
-
-
-
-
-
-
(0.03)
-
-
_
1 1
1.03 1.05
1 1
1
1 1 2
1.03
1.06 -
-
-
-
-
-
(0.07)
-
(0.06)
-
(0.31)
-
-
-
-
-
-
-
-
-
-
-
0.91
1
1.4
1
0.91
-
-
-
-
-
-
-
-
-
-
-
-
-
0.82
-
-
-
-
0.77
6 145
6 83.97
10 345.6
E
-
-
14 83.2
0
-
1 2
-
C-6
C-5
(0.13)
-
1
(0.06)
D 0.67
1
-1
-
0.87
-
1 -
5 151.4
4 274
Values given are moles of amino-acid residue per mole of peptide. Those in parentheses are presumed to be impurities. The quantities of amino acids expected from the primary sequence of BrCN-2 reported by Grosclaude et al. (18) are given as integral values; 0 and E refer to observed and expected values, respectively. * Corrected by 5% for destruction during hydrolysis. t Corrected by 10% for destruction during hydrolysis. Homoserine lactone, detected but not quantitated. § Tryptophan determination after aminopeptidase M digestion. t
3452
Biochemistry: Kemp et al.
BrCN-2 is shown with the segments corresponding. to the various chymotryptic peptides derived from it.
DISCUSSION It is clear from the comparison of the j3 casein genetic variants reported here that amino-acid substitutions may have profound effects on the phosphorylation of these variants. (3 Casein-B is the most effective substrate, differing from the common variant A2 by two amino-acid substitutions, namely, histidine instead of proline at position 67 and arginine instead of serine at position 122. The latter substitution accounts for the ability of variant B to serve as a substrate. This follows since variant A', which contains only the histidine substitution, is a poor substrate (see Table 2). Moreover, the results of studies with temperature-sensitive fragments of the casein variants indicated that the arginine substitution alone is responsible for the enhanced phosphorylation. Apparently, the presence of this particular amino acid residue causes a nearby serine, i.e., serine 124, to become a phosphorylation site for the protein kinase. Re-examination of the reported partial sequences around the phosphorylation sites of a number of the protein kinase substrates (see Table 1) indicates that an arginine residue occurs on the NH2-terminal side of most phosphorylation sites. In the two cases where an arginine residue has not been observed the sequences are incomplete, and since they are derived from tryptic phosphopeptides, it can be expected that either arginine or lysine will occupy the next position in the sequence towards the NH2-terminus. Initially, the apparent variability in the number of residues between the phosphorylated serine and the arginine was puzzling. However, preliminary consideration of the size of the guanidine side chain compared with the length of a peptide residue suggested that arginine could occupy a number of positions, perhaps between two and five residues from the serine. Although this discussion has emphasized the importance of the local primary sequence in determining substrate specificity, it is evident from studies done in this laboratory on chicken lysozyme that the tertiary structure of a protein may be important, at least in a negative sense, in that it can mask the specificity determinants present in the primary structure (9). A similar effect appears to be responsible for preventing glycogen phosphorylase from being phosphorylated by the cyclic AMP-dependent protein kinase even though the phosphorylation site sequence in phosphorylase contains an arginine in the appropriate position (24). However, D. J. Graves of the 'owa State University (personal communication) has found that a synthetic peptide [Ser-
Asp-Gln-Glu-Lys-Arg-Lys-Gln-Ile-Ser(P)-Val-Arg-Gly-Leul
corresponding to the phosphorylation site from glycogen phosphorylase can be phosphorylated by the cyclic AMPdependent protein kinase. Thus, even though the phosphorylase sequence contains the appropriate specificity determinants, these are not accessible in native phosphorylase. Not unexpectedly, the specificity determinants of the cyclic AMP-dependent protein kinase appear to be quite distinct from those of other protein kinases. Ribadeau-Dumas et al. (17) have proposed that the casein kinase thought to be responsible for the five naturally occurring phosphoserine residues in ,B casein-B (Ser-15, Ser-17, Ser-18, Ser-19, and Ser-35) recognizes either glutamate or phosphoserine residues as specificity determinants. On the other hand, phosphorylase kinase appears to have a requirement for basic residues on both sides of the phosphorylation site (24). This
Proc. Nat. Acad. Sci. USA 72 (1975)
observation is in accord with the greater substrate specificity of phosphorylase kinase as compared with that displayed by protein kinase in vitro. Further studies are in progress using synthetic peptides and chemical modification of protein substrates to examine the role of arginine and other residues in substrate specificity. A synthetic peptide with the sequence Arg-Gly-Tyr-Ser-Leu-Gly corresponding to the region around Ser-24 in chicken lysozyme is a substrate, whereas the nitroarginine derivative of this peptide is poorly phosphorylated, if.at all (Kemp et al., unpublished observations) by the protein kinase. This study could not have been undertaken without the generous gift of ( casein genetic variants from Mrs. E. W. Bingham and Dr. M. L. Groves of the USDA Eastern Regional Research Center. B.E.K. was a recipient of an Australian CSIRO Postdoctoral Studentship. This work was supported by grants from the National Institutes of Health (AM 16716, HL 14780, and AM 12842). 1.
2. 3.
4. 5. 6. 7. 8. 9.
10. 11. 12. 13.
Soderling, T. R. & Park, C. R. (1974) in Advances in Cyplic
Nucleotide Research, eds. Greengard, P. & Robison, G. A. (Raven Press, New York), Vol. 4, pp. 283-333. Langan, T. A. (1973) in Advances in Cyclic Nucleotide Research, eds. Greengard, P. & Robison, G. A. (Raven Press, New York), Vol. 3, pp. 99-153. Cohen, P., Watson, D. C. & Dixon, G. H. (1975) Eur. J. Biochem. 51,79-92. Cardinale, C. J. & Udenfriend, S. (1974) Adv. Enzymol. 41, 245-300. Heide, K. & Schwick, H. G. (1973) Angew. Chem. Int. Ed. Engl. 12, 721-733. Langan, T. A. (1971) Ann. N.Y. Acad. Sci. 185, 166-180. Carnegie, P. R., Kemp, B. E., Dunkley, P. R. & Murray, A. W. (1973) Biochem. J. 135,569-572. Carnegie, P. R., Dunkley, P. R., Kemp, B. E. & Murray, A. W. (1974) Nature 249, 147-150. Bylund, D. B. & Krebs, E. G. (1975) J. Biol. Chem., in press. Daile, P. & Carnegie, P. R. (1974) Biochem. Bwphys. Res. Commun. 61, 852-858. Beavo, J. A., Bechtel, P. J. & Krebs, E. G. (1974) in Methods in Enzymology, eds. O'Malley, B. W. & Hardman, J. G. (Academic Press, New York), Vol. 38, Part C, pp. 299-308. Steers, E., Jr., Graven, G. R., Anfinsen, C. B. & Bethune, J. L. (1965) J. Biol. Chem. 240,2478-2484. Huang, T. S., Bylund, D. B., Stull, J. T. & Krebs, E. G. (1974)
FEBS Lett. 42,249-252. 14. Weigele, M., De Bernardo, S. L., Tengi, J. A. & Leimgruber, W. (1972) J. Am. Chem. Soc. 94,5927-5928. 15. Grosclaude, F., Mahe, M. F., Mercier, J. C. & RibadeauDumas, B. (1972) Eur. J. Biochem. 26,328-337. 16. Groves, M. L., Gordon, W. G., Kalan, E. B. & Jones, S. B. (1973) J. Dairy Sci. 56,558-568. 17. Ribadeau-Dumas, B., Grosclaude, F. & Mercier, J. C. (1970) Eur. J. Biochem. 14,451-459. 18. Hjelmquist, G., Andersson, J., Edlund, B. & Engstrom, L. (1974) Biochem. Biophys. Res. Commun. 61,559-563. 19. Larner, J. & Sanger, F. (1965) J. Mol. Biol. 11,491-500. 20. Moir, A. J. G., Wilkinson, J. M. & Perry, S. V. (1974) FEBS Lett. 42, 253-256. 21. DeLange, R. J. & Smith, E. L. (1971) Annu. Rev. Biochem. 40,279-314. 22. Farago, A., Romhanyi, T., Antoni, F., Takats, A. & Fabian, F. (1975) Nature 254,88. 23. Groves, M. L. & Gordon, W. G. (1969) Biochim. Biophys. Acta 194, 421-432. 24. Graves, D. J., Martensen, T. M., Tu, J. I. & Tessmer, G. M. (i974) in Metabolic Interconversion of Enzymes, eds. Fischer, E. H., Krebs, E. G., Neurath, H. & Stadtman, E. R. (Springer-Verlag, New York), pp. 53-61.
Substrate specificity of the cyclic AMP-dependent protein kinase (caseins/protein sequence)
BRUCE E. KEMP, DAVID B. BYLUND, TUNG-SHIUH HUANG, AND EDWIN G. KREBS Department of Biological Chemistry, School of Medicine, University of California, Davis, Calif. 95616
Contributed by Edwin G. Krebs, July 7,1975
ABSTRACT The protein substrate specificity of the catalytic subunit of rabbit skeletal muscle cyclic AMP-dependent protein kinase (EC 2.7.1.37; ATP:protein phosphotransferase) has been studied using genetic variants of (3 casein. It was found that (3 casein-B was phosphorylated at a much greater rate than (3 caseins Al, A2, A3, or C. The enhanced phosphorylation of (3 casein-B, as compared with the most common variant A2, was attributed to an arginine substitution for a serine at position 122, which caused a nearby residue, serine 124, to become a phosphorylation site for the protein kinase. These results further support the concept that the local primary structure is important in specificity and that arginine may be a specific determinant common to all the local phosphorylation site sequences recognized by the cyclic AMPdependent protein kinase. The apparent lack of protein substrate specificity exhibited by the cyclic AMP-dependent protein kinase (EC 2.7.1.37; ATP:protein phosphotransferase) has been an enigma in the current concept of cyclic AMP-mediated hormonal regulation. Intuitively the physiological role of this enzyme would seem to demand considerable substrate specificity, and yet it has been shown to phosphorylate a wide variety of proteins in vitro (1). Several authors have considered the possibility that the primary sequence of amino acids around the phosphorylation site might be an important specificity determinant for the protein kinase (2, 3, 9). Precedent for this idea can be found in other protein modification reactions (4, 5). However, the protein kinase phosphorylation site sequences appeared to lack any obvious similarities (see Table 1). Accordingly, it was suggested that some aspect of the tertiary structure of the substrates might be involved (2, 3) which would not be apparent in the local primary sequence around the phosphorylation site. There are, however, several observations that are difficult to reconcile with the concept that some unique configuration of peptide chains is required for specificity. For instance, recent studies in this laboratory have shown that although native chicken lysosome is not a substrate for the protein kinase, it is phosphorylated by this enzyme at two specific sites, serine 24 to serine 50, after chemical denaturation. Daile and Carnegie (10) have reported that the cyclic AMP-dependent protein kinase will phosphorylate a 17-residue peptide isolated from human myelin basic protein which presumably no longer retains the tertiary structure of the native protein. The cyclic AMPdependent and cyclic AMP-independent protein kinases phosphorylate different sites in two substrates that have a comparatively uncomplicated tertiary structure, namely, histone F1 (2) and myelin basic protein (8). Even though all sites are presumably available for phosphorylation in these two substrates, each kinase is specific. In this communication we report studies on the specificity of the cyclic AMP-dependent protein kinase towards genetic
variants of $ casein. Since the amino-acid sequences of these variants are known, it has been possible to relate differences in substrate properties to specific substitutions in the primary sequence. The results support the concept that local primary protein structure plays a major role in determining the substrate specificity of the cyclic AMP-dependent protein kinase. METHODS AND MATERIALS Cyclic AMP-Dependent Protein Kinase. Homogeneous catalytic subunit of rabbit skeletal muscle cyclic AMP-dependent protein kinase (Peak I) prepared by the method of Beavo et al. (11) was used in this study. Phosphorylation of Protein Substrates by the Protein Kinase. The reaction mixture, with a final volume of 0.08 ml, contained 1.0 mM ['y-32P]ATP (10-200 cpm/pmol), 62.5 mM 2(N-morpholino)ethanesulfonic acid (pH 6.8), 12.5 mM magnesium acetate, 0.25 mM ethylene glycol bis-(f3-aminoethyl ether)-N-N'-tetraacetic acid, protein kinase, and substrate at concentrations as indicated in the appropriate figure legends. Incubations were carried out for 5 min at 30°. Reactions were terminated with glacial acetic acid to give a final concentration of 30%. Anion exchange columns (2 ml AG 1 X 8 resin, Bio-Rad) equilibrated with 30% acetic acid were used to separate the phosphoprotein from the [-y32P]ATP. Under these conditions proteins and peptides are fully protonated and elute from the column, whereas ATP and Pi bind tightly. The columns were eluted directly into liquid scintillation vials, and the excess acetic acid was removed by evaporation. Quantitative recoveries of phosphorylated f casein, histone, and lysozyme were obtained. Cyanogen Bromide (BrCN) and Enzymic Digestions. Phosphorylated (3 casein-B (48 mg) was lyophilized in the presence of 86 Atmol of 2-mercaptoethanol prior to digestion with cyanogen bromide (200-fold excess of BrCN over total thiol groups) in 70% formic acid (12). Enzymatic digestions with alpha-chymotrypsin (Worthington) and aminopeptidase M (Henley and Co., New York) were performed according to published procedures (13). The resultant peptideso were purified by gel filtration using Sephadex G-25 and G-50 (Pharmacia) columns (1.9 X 160 cm) in the presence of 30% acetic acid. Primary amine-containing peptides in the column effluent were detected using fluorescamine (Fluram, Roche Diagnostics Corp.) (14). High voltage electrophoresis and amino acid analysis were performed as described (9). Casein Variants. The purified casein fractions used in this study were very kindly provided by Mrs. E. W. Bingham and Dr. M. L. Groves of the USDA Eastern Regional Research Center. These fractions were isolated from the milk of individual cows homozygous for the respective casein variants (16, 23). 3448
Proc. Nat. Acad. Sci. USA 72 (1975)
Biochemistry: Kemp et al.
3449
Table 1. Sites of phosphorylation by mammalian cyclic AMP-dependent protein kinase
/3 Casein-B Phosphorylase kinase
((3subunit) Phosphorylase kinase (az subunit) Pyruvate kinase Glycogen synthetase
Histone F,
This paper
Phe-Thr-Glu-Arg-Gln-Ser(P)-Leu-Thr-Leu-Thr-Asp 124 Val Arg* -Gln-Ser-Gly-Ser(P)- -Tyr-Pro-Leu-Lys Ile Lys Arg* -Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser Lys Leu-Arg-Arg-Ala-Ser(P)-Leu Arg*
Lys RCMM lysozymet
Ref.
Sequence
Substrate
(3) (3) (18) (19)
-Glx-Ile-Ser(P)-Val-Arg
(9)
Asn-Tyr-Arg-Gly-Tyr-Ser(P)-Leu-Gly-Asn-Trp-Val 24 Arg-Asn-Thr-Asp-Gly-Ser(P)-Thr-Asp-Tyr-Gly-Ile 50
(9)
(2, 6, 21)
Ala-Arg-Arg-Lys-Ala-Ser(P)-Gly-Pro-Pro-Val-Ser 38
Troponin Histone F2bt
(13, 20) (13, 20)
Arg-Gln-His-Leu-Lys-Ser(P)-Val-Met-Gln-Leu Val-Arg-Met-Ser(P)-Ala-Asp-Ala-Met-Leu Arg-Lys-Glu-Ser-Tyr-Ser-Val-Tyr-Val-Tyr-Lys
(22)
38 Myelin basic protein
(7, 8)
Lys-Gly-Arg-Gly-Leu-Ser(P)-Leu-Ser-Arg-Phe-Ser § 110
(8)
Pro-Arg-His-Arg-Asp-Thr(P)-Gly-Ile-Leu-Asp-Ser 34
(7)
Ser-Gln-Arg-His-Gly-Ser(P)-Lys-Tyr-Leu-Ala-Thrl 12
* Arginine or lysine expected since it is a tryptic phosphopeptide. t Reduced, carboxymethylated, and maleylated (RCMM-) chicken lysozyme. t It is not known which of the two serines in this peptide is phosphorylated. § Phosphorylation site identified in human, rat, and bovine myelin basic protein. Phosphorylation site identified in bovine myelin basic protein. Phosphorylation site identified in rat and human myelin basic protein.
RESULTS Comparison of casein variants as substrates for the protein kinase Several casein variants were tested as substrates for protein kinase. Since these variants differ by known amino-acid substitutions (Table 2), it was possible to correlate rates of phosphorylation with their primary structure differences. One variant, casein-B, was phosphorylated approximately 70fold more rapidly than the most common variant, casein A2 (Table 2). Compared with this variant, casein-B differs by two amino acids, having proline 67 replaced by a histidine and serine 122 replaced by an arginine. Even at substrate concentrations up to 8 mg/ml, the rates of phosphorylation of all the casein variants except casein-B were negligible compared with the rate obtained with whole calf thymus histone. Characteristics of the reaction product using ,B caseinB A time course of phosphorylation of casein-B showed that it was phosphorylated by the protein kinase to the extent of 0.8 mol of phosphate per mol of substrate in 60 min (Fig. 1A). Phosphate incorporation beyond this point proceeded very slowly, i.e., after 6 hr of incubation only 1.2 mol of phosphate was incorporated per mol. These observations suggest that the rapid phosphorylation of casein-B occurs at a single site.
The phosphorylated ,B casein-B migrated as a single band on sodium dodecyl sulfate/acrylamide gels, and 84% of the radioactivity applied to the gel was recovered from this band (see Fig. 1B). The radioactivity associated with the / casein-B was alkali-labile (96% release in 15 min in 0.1 M NaOH at 1000) and relatively stable in acid (92% remaining after 15 min in 0.1 M HCI at 100°). These properties are characteristic of either serine or threonine phosphoester linkages. After partial acid hydrolysis (6 M HCI, 1000, 2 hr Table 2. Phosphorylation of ( casein genetic variants Protein kinase activity
Substrate (3 casein (nmol/min per mg) variant Al A2
A3 B C
17.1 21.5 14.6 1425.6 37.5
Amino-acid residues in the variable positions* 37
67
106
122
Glu Glu Glu Glu Lys
His Pro Pro His His
His His Gln His His
Ser Ser Ser Arg Ser
Protein kinase assays were done as described in Methods and Materials with an incubation time of 5 min, a [y-32P]ATP concentration of 0.95 mM, and protein substrate and enzyme concentrations of 2.5 mg/ml and 16 Mg/ml, respectively. * Assignments according to Grosclaude et al. (15).
3450
Biochemistry: Kemp et al. Table 3. Phosphorylation of casein temperature-sensitive fragments
Proc. Nat. Acad. Sci. USA 72 (1975)
m 1.0 ._
(n
0
Substrate* TS.A2
Protein kinase activity (nmol/ min per mg) 0.0
c- 0.8 0
Corresponding amino-acid sequences in (3 casein
His-Lys-Glu. . Ser.. Val-OH 108 122.. .209 Gln-Lys-Glu. . .Ser. . .Val-OH 106 108 122 209 Glu. . .Arg... .Val-OH 108 122 209
106
TS.A3
0.2
TS.B
61.4
f3Casein-B Calf thymus histone
51.5 188.7
a, 0 m 1%
0.6
U.
00.4
ca. 0~ m
0.2
0
(n 0
0
10
Protein kinase assays were done as described in Methods and Materials and Table 1, except that the incubation temperature was 0.5°, the enzyme concentration 10 Ag/ml, and the final concentration of histone was 1 mg/ml. The casein fractions were all tested at a final concentration of 2.5 mg/ml. * Nomenclature for the temperature-sensitive fragments according to Groves et al. (16).
in an evacuated tube) and high voltage electrophoresis at pH 1.9, 34.3% and 36.6% of the radioactivity migrated with the phosphoserine and inorganic phosphate markers, respectively. No more than 0.2% of the radioactivity could be detected in the phosphothreonine position. The remaining 29% of the phosphorylated product was incompletely hydrolyzed and migrated towards the cathode. The total recovery of radioactivity applied to the electrophoretogram was 81%.
20
30 40 Time (min)
60
50
5 4 CO
0 x
3
E 0. 0
0-
cmm
2
Phosphorylation of temperature-sensitive fragments Acid precipitates of whole bovine casein are known to contain temperature-sensitive protein fragments that precipitate at room temperature but are quite soluble at lower temperatures. The amino-acid sequences of these fragments are homologous with the carboxyl-terminal halves of native , caseins (16). Thus, the temperature-sensitive fragment related to # casein-B, TS.B, has an amino-acid sequence corresponding to the segment containing glutamate 108 through to the carboxyl-terminal residue valine 209 of this variant. Several of these temperature-sensitive fragments corresponding to different variants were tested as substrates for the protein kinase. Phosphorylation of both fragments TS.A2 and TS.A3 was negligible compared with either TS.B or native flcasein-B (Table 3). Insolubility of the /3 casein temperature-sensitive fragments at normal temperatures necessitated that the assay temperature be reduced to 0.50. As expected, the absolute rates of phosphorylation of other substrates were also depressed under these conditions (Table 3). Since fragment TS.B contains only the arginine substitution, of the two amino-acid substitutions that occur in native /3casein-B, it appeared probable that this substitution alone was responsible for the enhanced phosphorylation of both TS.B and (3 casein-B compared with the other variants.
Identification of the site(s) in 3 casein-B phosphorylated by protein kinase 3 Casein-B (48 mg), phosphorylated to the extent of 0.8 mole of phosphate per mol, was digested with BrCN. One major radioactive peak (representing 81% of the total 32p incorporated) was detected after gel chromatography on Se-
1
0
2
4 cm
'6
8
FIG. 1. f8 Casein-B phosphorylation. (A) Time course of ,B casein-B phosphorylation. f3 Casein-B (4 mg/ml) was incubated with 10 ,gg/ml of protein kinase in a large reaction mixture (12 ml) having the composition described in Methods and Materials. Aliquots (15 ul) were removed at intervals, and the incorporation of 32p was measured as described. (B) Sodium dodecyl sulfate/polyacrylamide (10%) gel electrophoresis of phosphorylated f, casein-B. Gels were stained with Coomassie blue, photographed, and sliced into 2-mm fractions. Radioactivity associated with the gel slices was determined by liquid scintillation counting after digestion with 30% H202 at 950.
phadex G-50. The amino-acid composition of the purified phosphopeptide (rechromatography on Sephadex G-50 and electrophoresis at pH 1.9) was found to correspond with one of the expected seven cyanogen bromide peptides reported by Ribadeau-Dumas et al. (17). The amino-acid composition of the phosphopeptide corresponded with the peptide BrCN-2 described by these authors and is derived from the amino-acid sequence between Pro-110 and Met-144. This region is located in the COOH-terminal half of f casein-B and contains a high proportion of hydrophobic residues (see Fig. 3).
Proc. Nat. Acad. Sci. USA 72 (1975)
Biochemistry: Kemp et al.
120
I
-6
.5
O,
0.5
4
C2
°> c 4
;r
-i4 "45
=
1
130 Arg-Gln-Ser(P)-Leu-Thr-Leu-Thr-Asp-Val-GluC-1
Co
co
0.3 @
C._
0.2 o
'C 4
w
Pro-Phe-Pro-Lys-Tyr-Pro-Val-Gln-Pro-Phe-Thr-GluS ~~~C-3-A C-3-A _~~~~~~~~~~~~_______
0.4 c
*4.C
o
ci
3451
~~~~~C-2
-
---C-4 -
_
0.1
Cl-
contains only two serine residues, namely, Ser-124 and Ser142. In order to determine which of these two sites was
140 Asn-Leu-His-Leu-Pro-Pro-Leu-Leu-Leu-Gln-Ser-Trp-Met * ** -C-5b e ** C~C-3-B FIG. 3. Phosphorylation site of fl casein-B incubated with protein kinase. The amino-acid sequence of peptide BrCN-2 is shown. This sequence corresponds with the region between Pro-110 and Met-144 in fl casein-B as designated by Ribadeau-Dumas et al. (17). The chymotryptic peptides derived from BrCN-2 were identified on the basis of their amino acid composition (see Table 4).
phosphorylated, peptide BrCN-2 was further hydrolyzed with chymotrypsin. The chymotryptic peptides were subsequently fractionated on Sephadex G-25 (Fig. 2) and purified by high voltage electrophoresis at pH 1.9. The amino-acid compositions of the purified chymotryptic peptides are shown in Table 4. Two chymotryptic peptides, BrCN-2-C-5 and BrCN-2-C-6, gave a positive Ehrlich reaction and had amino-acid compositions corresponding to the sequence containing residues 140 to 144 and 140 to 143, respectively. These peptides are derived from the COOH-terminal region of BrCN-2 and include Ser-142. The presence of tryptophan and serine in both these chymotryptic peptides was also demonstrated by amino-acid analysis after aminopeptidase M digestion. Since no 32P radioactivity was associated with
either of the chymotryptic peptides containing Ser-142, it follows that this residue was not phosphorylated. The radioactive peptides BrCN-2-C-1, BrCH-2-C-2, and BrCH-2C-4 had amino-acid compositions corresponding to the sequences containing residues 120 to 139, 120 to 133, and 120 to 125, respectively. All three of these phosphopeptides contained Ser-124. Since only phosphoserine and not phosphothreonine was detected in partial acid hydrolysates of either the intact phosphorylated casein-B or the phosphopeptide BrCN-2, it follows that serine 124 is the phosphorylation site. Near stoichiometric recovery of the radioactive 32P and the chymotryptic peptides was obtained (see Table 4). In Fig. 3 the primary structure of the phosphopeptide
l0
0
20
30
40
50
60
70
80
90
FRACTION NUMBER
FIG. 2. Sephadex G-25 column profile of chymotryptic peptides derived from phosphorylated peptide BrCN-2. Fractions (5 ml) were assayed for absorbance at 280 nm, 32P radioactivity, and primary amino containing peptides with fluorescamine.
The amino-acid
sequence
between Pro-110 and Met-144
Table 4. Amino-acid composition of chymotryptic peptides derived from peptide BrCN-2 C-1 0
Lys His Arg Asp Thr* Sert Glu Pro Gly Ala Val Ile Leu Tyr* Phe HL Trp §
32P
(0.06) 0.78 0.96 2.09 2.38 0.90 3.25 2.46 (0.2) -
1.24 -
E
0
-
(0.05) (0.27) 0.81 2.05 2.79 0.98 2.87 (0.62) (0.27) (0.08)
1
1.12
-
1 1 2 3 1 3 2
-
-
E
0
-
0.91
-
-
1 2 3 1 3
-
-
1
(0.02) 0.76 (0.48) 1.09 0.83
-
-
-
-
-
-
-
(0.02) 1.01
-
1 4 -
-
1.01 -
6
-
-
-
(0.18)
-
0.08
-
(0.05) 0.97 1.97
-
-
-
-
-
-
-
1.02
-
-
Total residues 20 Peptide (nmol) 230.2
-
0.8 (0.11)
-
3
1 -
-
-
-
E
-
4.12 (0.03)
1
0
-
-
-
E
C-4
(0.02)
5.93
3.39
C-3-B
C-3-A
C-2
-
-
-
-
2.12 (0.11) -
1
-
-
-
-
3.08
2 -
-
-
3
E
0
1.94 (0.27) (0.09) (0.05)
E
0
-
-
-
-
(0.01)
-
-
-
-
-
-
-
-
(0.03)
-
-
_
1 1
1.03 1.05
1 1
1
1 1 2
1.03
1.06 -
-
-
-
-
-
(0.07)
-
(0.06)
-
(0.31)
-
-
-
-
-
-
-
-
-
-
-
0.91
1
1.4
1
0.91
-
-
-
-
-
-
-
-
-
-
-
-
-
0.82
-
-
-
-
0.77
6 145
6 83.97
10 345.6
E
-
-
14 83.2
0
-
1 2
-
C-6
C-5
(0.13)
-
1
(0.06)
D 0.67
1
-1
-
0.87
-
1 -
5 151.4
4 274
Values given are moles of amino-acid residue per mole of peptide. Those in parentheses are presumed to be impurities. The quantities of amino acids expected from the primary sequence of BrCN-2 reported by Grosclaude et al. (18) are given as integral values; 0 and E refer to observed and expected values, respectively. * Corrected by 5% for destruction during hydrolysis. t Corrected by 10% for destruction during hydrolysis. Homoserine lactone, detected but not quantitated. § Tryptophan determination after aminopeptidase M digestion. t
3452
Biochemistry: Kemp et al.
BrCN-2 is shown with the segments corresponding. to the various chymotryptic peptides derived from it.
DISCUSSION It is clear from the comparison of the j3 casein genetic variants reported here that amino-acid substitutions may have profound effects on the phosphorylation of these variants. (3 Casein-B is the most effective substrate, differing from the common variant A2 by two amino-acid substitutions, namely, histidine instead of proline at position 67 and arginine instead of serine at position 122. The latter substitution accounts for the ability of variant B to serve as a substrate. This follows since variant A', which contains only the histidine substitution, is a poor substrate (see Table 2). Moreover, the results of studies with temperature-sensitive fragments of the casein variants indicated that the arginine substitution alone is responsible for the enhanced phosphorylation. Apparently, the presence of this particular amino acid residue causes a nearby serine, i.e., serine 124, to become a phosphorylation site for the protein kinase. Re-examination of the reported partial sequences around the phosphorylation sites of a number of the protein kinase substrates (see Table 1) indicates that an arginine residue occurs on the NH2-terminal side of most phosphorylation sites. In the two cases where an arginine residue has not been observed the sequences are incomplete, and since they are derived from tryptic phosphopeptides, it can be expected that either arginine or lysine will occupy the next position in the sequence towards the NH2-terminus. Initially, the apparent variability in the number of residues between the phosphorylated serine and the arginine was puzzling. However, preliminary consideration of the size of the guanidine side chain compared with the length of a peptide residue suggested that arginine could occupy a number of positions, perhaps between two and five residues from the serine. Although this discussion has emphasized the importance of the local primary sequence in determining substrate specificity, it is evident from studies done in this laboratory on chicken lysozyme that the tertiary structure of a protein may be important, at least in a negative sense, in that it can mask the specificity determinants present in the primary structure (9). A similar effect appears to be responsible for preventing glycogen phosphorylase from being phosphorylated by the cyclic AMP-dependent protein kinase even though the phosphorylation site sequence in phosphorylase contains an arginine in the appropriate position (24). However, D. J. Graves of the 'owa State University (personal communication) has found that a synthetic peptide [Ser-
Asp-Gln-Glu-Lys-Arg-Lys-Gln-Ile-Ser(P)-Val-Arg-Gly-Leul
corresponding to the phosphorylation site from glycogen phosphorylase can be phosphorylated by the cyclic AMPdependent protein kinase. Thus, even though the phosphorylase sequence contains the appropriate specificity determinants, these are not accessible in native phosphorylase. Not unexpectedly, the specificity determinants of the cyclic AMP-dependent protein kinase appear to be quite distinct from those of other protein kinases. Ribadeau-Dumas et al. (17) have proposed that the casein kinase thought to be responsible for the five naturally occurring phosphoserine residues in ,B casein-B (Ser-15, Ser-17, Ser-18, Ser-19, and Ser-35) recognizes either glutamate or phosphoserine residues as specificity determinants. On the other hand, phosphorylase kinase appears to have a requirement for basic residues on both sides of the phosphorylation site (24). This
Proc. Nat. Acad. Sci. USA 72 (1975)
observation is in accord with the greater substrate specificity of phosphorylase kinase as compared with that displayed by protein kinase in vitro. Further studies are in progress using synthetic peptides and chemical modification of protein substrates to examine the role of arginine and other residues in substrate specificity. A synthetic peptide with the sequence Arg-Gly-Tyr-Ser-Leu-Gly corresponding to the region around Ser-24 in chicken lysozyme is a substrate, whereas the nitroarginine derivative of this peptide is poorly phosphorylated, if.at all (Kemp et al., unpublished observations) by the protein kinase. This study could not have been undertaken without the generous gift of ( casein genetic variants from Mrs. E. W. Bingham and Dr. M. L. Groves of the USDA Eastern Regional Research Center. B.E.K. was a recipient of an Australian CSIRO Postdoctoral Studentship. This work was supported by grants from the National Institutes of Health (AM 16716, HL 14780, and AM 12842). 1.
2. 3.
4. 5. 6. 7. 8. 9.
10. 11. 12. 13.
Soderling, T. R. & Park, C. R. (1974) in Advances in Cyplic
Nucleotide Research, eds. Greengard, P. & Robison, G. A. (Raven Press, New York), Vol. 4, pp. 283-333. Langan, T. A. (1973) in Advances in Cyclic Nucleotide Research, eds. Greengard, P. & Robison, G. A. (Raven Press, New York), Vol. 3, pp. 99-153. Cohen, P., Watson, D. C. & Dixon, G. H. (1975) Eur. J. Biochem. 51,79-92. Cardinale, C. J. & Udenfriend, S. (1974) Adv. Enzymol. 41, 245-300. Heide, K. & Schwick, H. G. (1973) Angew. Chem. Int. Ed. Engl. 12, 721-733. Langan, T. A. (1971) Ann. N.Y. Acad. Sci. 185, 166-180. Carnegie, P. R., Kemp, B. E., Dunkley, P. R. & Murray, A. W. (1973) Biochem. J. 135,569-572. Carnegie, P. R., Dunkley, P. R., Kemp, B. E. & Murray, A. W. (1974) Nature 249, 147-150. Bylund, D. B. & Krebs, E. G. (1975) J. Biol. Chem., in press. Daile, P. & Carnegie, P. R. (1974) Biochem. Bwphys. Res. Commun. 61, 852-858. Beavo, J. A., Bechtel, P. J. & Krebs, E. G. (1974) in Methods in Enzymology, eds. O'Malley, B. W. & Hardman, J. G. (Academic Press, New York), Vol. 38, Part C, pp. 299-308. Steers, E., Jr., Graven, G. R., Anfinsen, C. B. & Bethune, J. L. (1965) J. Biol. Chem. 240,2478-2484. Huang, T. S., Bylund, D. B., Stull, J. T. & Krebs, E. G. (1974)
FEBS Lett. 42,249-252. 14. Weigele, M., De Bernardo, S. L., Tengi, J. A. & Leimgruber, W. (1972) J. Am. Chem. Soc. 94,5927-5928. 15. Grosclaude, F., Mahe, M. F., Mercier, J. C. & RibadeauDumas, B. (1972) Eur. J. Biochem. 26,328-337. 16. Groves, M. L., Gordon, W. G., Kalan, E. B. & Jones, S. B. (1973) J. Dairy Sci. 56,558-568. 17. Ribadeau-Dumas, B., Grosclaude, F. & Mercier, J. C. (1970) Eur. J. Biochem. 14,451-459. 18. Hjelmquist, G., Andersson, J., Edlund, B. & Engstrom, L. (1974) Biochem. Biophys. Res. Commun. 61,559-563. 19. Larner, J. & Sanger, F. (1965) J. Mol. Biol. 11,491-500. 20. Moir, A. J. G., Wilkinson, J. M. & Perry, S. V. (1974) FEBS Lett. 42, 253-256. 21. DeLange, R. J. & Smith, E. L. (1971) Annu. Rev. Biochem. 40,279-314. 22. Farago, A., Romhanyi, T., Antoni, F., Takats, A. & Fabian, F. (1975) Nature 254,88. 23. Groves, M. L. & Gordon, W. G. (1969) Biochim. Biophys. Acta 194, 421-432. 24. Graves, D. J., Martensen, T. M., Tu, J. I. & Tessmer, G. M. (i974) in Metabolic Interconversion of Enzymes, eds. Fischer, E. H., Krebs, E. G., Neurath, H. & Stadtman, E. R. (Springer-Verlag, New York), pp. 53-61.
