Role of enzyme-peptide substrate backbone hydrogen bonding in determining protein kinase substrate specificities

As part of a search for peptides that have specificity for selected protein kinases, the possibility that adenosine cyclic 3',5'-phosphate dependent protein kinase (A-kinase) recognizes the hydrogen-bonding potential of its peptide substrates was investigated. A-Kinase catalyzes the phosphorylation of five N alpha-methylated and four depsipeptide derivatives of Leu-Arg-Arg-Ala-Ser-Leu-Gly (peptide 1) at rates that differ by at least 7 orders of magnitude. These peptide 1 analogues each lack the ability to donate a hydrogen bond at selected positions in the peptide chain. If a particular amide hydrogen of a peptide amide is involved in hydrogen bonding, which is important for enzyme recognition, the prediction is that peptides which contain an ester or a N-methylated bond at that position in peptide 1 will be comparatively poor substrates. In contrast, if a depsipeptide has a reactivity comparable to that of peptide 1 but the analogous N-methylated peptide has a poor reactivity with A-kinase, the result might indicate that the N-methyl group causes unfavorable steric effects. The depsipeptide that lacks a Leu6 amide proton is a good substrate for A-kinase, but the corresponding N-methylated peptide is phosphorylated far less efficiently. This result and others presented in this paper suggest that although enzyme-substrate hydrogen bonding may play some role in A-kinase catalysis of phosphoryl group transfer, other explanations are necessary to account for the relative reactivities of N alpha-methylated and depsi-containing peptide 1 analogues.(ABSTRACT TRUNCATED AT 250 WORDS)     

A carboxy-terminal peptide from p47-phox is a substrate for phosphorylation by protein kinase C and by a neutrophil protein kinase.

Agonist-activated phosphorylation of neutrophil proteins including p47-phox, a cytosolic component of the respiratory burst oxidase, has been implicated in the signal transduction cascade which leads to activation of the superoxide generating respiratory burst. We have previously reported (J. Biol. Chem. 265, 17550-59) that in a cell-free activation system consisting of cytosol plus plasma membrane from human neutrophils, diacylglycerol acts synergistically with an anionic amphiphile such as sodium dodecyl sulfate (SDS) to augment superoxide generation and assembly of the oxidase, and that p47 phosphorylation can occur under these conditions. Herein, we show that a peptide corresponding to a carboxy terminal sequence of p47-phox is a substrate for phosphorylation both by purified protein kinase C (a mixture of alpha, beta, and gamma forms) and by a distinct kinase or kinases present in neutrophil cytosol. Based on its activator requirements, the neutrophil kinase differs from classical protein kinase C, but may be a protein kinase C variant, based on inhibition by a protein kinase C peptide. Although in the cell-free system phosphorylation occurs under conditions which are similar to those for activation of superoxide generation, phosphorylation is not required for activation (1). Rather, protein assembly or aggregation which occurs under activation conditions may also promote phosphorylation.     

Importance of protein kinase C targeting for the phosphorylation of its substrate, myristoylated alanine-rich C-kinase substrate

We visualized the translocation of myristoylated alanine-rich protein kinase C substrate (MARCKS) in living Chinese hamster ovary-K1 cells using MARCKS tagged to green fluorescent protein (MARCKS-GFP). MARCKS-GFP was rapidly translocated from the plasma membrane to the cytoplasm after the treatment with phorbol ester, which translocates protein kinase C (PKC) to the plasma membrane. In contrast, PKC activation by hydrogen peroxide, which was not accompanied by PKC translocation, did not alter the intracellular localization of MARCKS-GFP. Non-myristoylated mutant of MARCKS-GFP was distributed throughout the cytoplasm, including the nucleoplasm, and was not translocated by phorbol ester or by hydrogen peroxide. Phosphorylation of wild-type MARCKS-GFP was observed in cells treated with phorbol ester but not with hydrogen peroxide, whereas non-myristoylated mutant of MARCKS-GFP was phosphorylated in cells treated with hydrogen peroxide but not with phorbol ester. Phosphorylation of both MARCKS-GFPs reduced the amount of F-actin. These findings revealed that PKC targeting to the plasma membrane is required for the phosphorylation of membrane-associated MARCKS and that a mutant MARCKS existing in the cytoplasm can be phosphorylated by PKC activated in the cytoplasm without translocation but not by PKC targeted to the membrane.     

Characterization of the phosphorylation sites in the chicken and bovine myristoylated alanine-rich C kinase substrate protein, a prominent cellular substrate for protein kinase C

Little is known about the important cellular substrates for protein kinase C and their potential roles in mediating protein kinase C-dependent processes. We evaluated the protein kinase C phosphorylation sites in a major cellular substrate for the kinase, a protein of apparent Mr 80,000 in bovine and 60,000 in chicken tissues; we have recently determined the primary sequences of these proteins and tentatively named them the myristoylated alanine-rich C kinase substrates. The proteins were purified to apparent homogeneity from bovine and chicken brains, phosphorylated with protein kinase C, digested with trypsin, and the phosphopeptides purified and sequenced. Four distinct phosphopeptides were identified from both the bovine and chicken proteins. Two of the phosphorylated serines were contained in the repeated motif FSFKK, one in the sequence LSGF, and one in the sequence SFK. All four sites were contained within a basic domain of 25 amino acids which was identical in the chicken and bovine proteins. All of the sites phosphorylated in the cell-free system appeared to be phosphorylated in intact cells; an additional site may have been present in the proteins from intact cells. The identity of the phosphorylation site domains from two proteins of overall 65% amino acid sequence identity suggests a potential role for this domain in the physiological function of the myristoylated alanine-rich C kinase substrate proteins.     

Multiple specificities of brain Ca2+- and calmodulin-dependent protein kinase for substrate

The purified Ca2+- and calmodulin-dependent protein kinase from rat brain, which has a M.W. of 120,000 by gel filtration analysis, showed a broad substrate specificity. In addition to myosin light chain from chicken gizzard, the enzyme phosphorylated myelin basic protein, casein and two endogenous substrates in a Ca2+- and calmodulin-dependent manner. In contrast, chicken gizzard myosin light chain kinase exclusively phosphorylated myosin light chain.     

Effect of phospholipid on substrate phosphorylation by a catalytic fragment of protein kinase C

Limited tryptic digestion of protein kinase C purified from mouse brain generated a 36-kDa fragment which no longer required Ca2+ and phospholipid for activity or bound phorbol ester. Under appropriate conditions, the isolated fragment was stable for several months at 4 degrees C or upon freezing and storage at -70 degrees C. Kinetic characteristics of the fragment were similar to those for the intact protein kinase. Although the fragment did not require phospholipid for activity, anionic phospholipids affected the extent of its activity in a pH-, substrate-, and substrate concentration-dependent manner. This effect appeared to be due to complex formation between the phospholipid and substrate. The catalytic fragment thus permits detection of a second point of interaction of phospholipid with the protein kinase C system in addition to the already described phospholipid regulatory domain.     

Phosphatidylserine affects specificity of protein kinase C substrate phosphorylation and autophosphorylation

Protein kinase C substrate phosphorylation and autophosphorylation are differentially modulated by the phosphatidylserine concentration in model membranes. Both substrate phosphorylation and auto-phosphorylation display a cooperative dependence on phosphatidylserine in sonicated vesicles composed of diacylglycerol and either phosphatidylcholine or a mixture of cell lipids (cholesterol, sphingomyelin, phosphatidylethanolamine, and phosphatidylcholine). However, the concentration of phosphatidylserine required to support phosphorylation varies with individual substrates. In general, autophosphorylation is favored at intermediate phosphatidylserine concentrations, while substrate phosphorylation dominates at high phosphatidylserine concentrations. These different phosphatidylserine dependencies may reflect different affinities of particular substrates for negatively charged membranes. Increasing the negative surface charge of sonicated vesicles increases the rate of substrate phosphorylation. In contrast to the modulation exerted by phosphatidylserine, diacylglycerol activates protein kinase C equally toward substrate phosphorylation and autophosphorylation. These results indicate that both diacylglycerol and phosphatidylserine regulate protein kinase C activity in the membrane: diacylglycerol turns the enzyme on, while phosphatidylserine affects the specificity toward different substrates.     

Cyclic AMP-independent regulation of protein kinase A substrate phosphorylation by Kelch repeat proteins

Pseudohyphal and invasive growth in the yeast Saccharomyces cerevisiae is regulated by the kelch repeat-containing proteins Gpb1p and Gpb2p, which act downstream of the G protein alpha-subunit Gpa2p. Here we show that deletion of GPB1 and GPB2 causes increased haploid invasive growth in cells containing any one of the three protein kinase A (PKA) catalytic subunits, suggesting that Gpb1p and Gpb2p are able to inhibit each of these kinases. Cells containing gpb1Delta gpb2Delta mutations also display increased phosphorylation of the PKA substrates Sfl1p and Msn2p, indicating that Gpb1p and Gpb2p are negative regulators of PKA substrate phosphorylation. Stimulation of PKA-dependent signaling by gpb1Delta gpb2Delta mutations occurs in cells that lack both adenylyl cyclase and the high-affinity cyclic AMP (cAMP) phosphodiesterase. This effect is also seen in cells that lack the low-affinity cAMP phosphodiesterase. Given that these three enzymes control the synthesis and degradation of cAMP, these results indicate that the effect of Gpb1p and Gpb2p on PKA substrate phosphorylation does not occur by regulating the intracellular cAMP concentration. These findings suggest that Gpb1p and Gpb2p mediate their effects on the cAMP/PKA signaling pathway either by inhibiting the activity of PKA in a cAMP-independent manner or by activating phosphatases that act on PKA substrates.     

A synthetic peptide substrate for selective assay of protein kinase C.

Among various phosphate acceptor proteins and peptides so far tested, a synthetic peptide having the sequence surrounding Ser(8) of myelin basic protein, Gln-Lys-Arg-Pro-Ser(8)-Gln-Arg-Ser-Lys-Tyr-Leu, (MBP4-14), is the most specific and convenient substrate which can be used for selective assay of protein kinase C. This peptide is not phosphorylated by cyclic AMP-dependent protein kinase, casein kinases I and II, Ca2+/calmodulin-dependent protein kinase II, or phosphorylase kinase, and can be routinely used for the assay of protein kinase C with low background in the crude tissue extracts. The Km value is considerably low (7 microM) with a Vmax value of twice as much as that for H1 histone.     

Resveratrol preferentially inhibits protein kinase C-catalyzed phosphorylation of a cofactor-independent, arginine-rich protein substrate by a novel mechanism.

Resveratrol, a polyphenolic natural product abundantly present in grape skins, is a candidate cancer chemopreventive agent that antagonizes each stage of carcinogenesis and inhibits protein kinase C (PKC), a key mediator of tumor promotion. While resveratrol has been shown to antagonize both isolated and cellular forms of PKC, the weak inhibitory potency observed against isolated PKC cannot account for the reported efficacy of the polyphenol against PKC in cells. In this report, we analyze the mechanism of PKC inhibition by resveratrol. Our results indicate that resveratrol has a broad range of inhibitory potencies against purified PKC that depend on the nature of the substrate and the cofactor dependence of the phosphotransferase reaction. Resveratrol weakly inhibited the Ca2+/phosphatidylserine-stimulated activity of a purified rat brain PKC isozyme mixture (IC(50) = 90 microM) by competition with ATP (K(i) = 55 microM). Consistent with the kinetic evidence for a catalytic domain-directed mechanism, resveratrol inhibited the lipid-dependent activity of PKC isozymes with divergent regulatory domains similarly, and it was even more effective in inhibiting a cofactor-independent catalytic domain fragment (CDF) of PKC generated by limited proteolysis. This suggested that regulatory features of PKC might impede resveratrol inhibition of the enzyme. To explore this, we examined the effects of resveratrol on PKC-catalyzed phosphorylation of the cofactor-independent substrate protamine sulfate, which is a polybasic protein that activates PKC by a novel mechanism. Resveratrol potently inhibited protamine sulfate phosphorylation (IC(50) = 10 microM) by a mechanism that entailed antagonism of the activation of PKC by protamine sulfate and did not involve competition with either substrate. On the basis of the presence of PKC isozymes at subcellular sites rich in polybasic proteins, it has been proposed that certain endogenous polybasic PKC substrates may activate PKC in cells by the same mechanism as protamine sulfate. Our results suggest that antagonism by resveratrol of the phosphorylation of cellular PKC substrates that resemble protamine sulfate in their interactions with PKC may contribute to the efficacy of resveratrol against PKC in cells.     

Definition of a consensus sequence for peptide substrate recognition by p44mpk, the meiosis-activated myelin basic protein kinase

Synthetic peptides have been used to define the consensus amino acid sequence for substrate recognition by the meiosis-activated myelin basic protein (MBP) kinase (p44mpk), which was purified from maturing sea star oocytes. This protein kinase shares many properties with the mitogen-activated microtubule-associated protein-2 kinase (p42mapk) in vertebrates. Recently, Thr-97 in the tryptic fragment KNIVTPRTPPPSQGK of bovine MBP was identified as the major site of phosphorylation by p44mpk (Sanghera, J. S., Aebersold, R., Morrison, H. D., Bures, E. J., and Pelech, S. L. (1990) FEBS Lett. 273, 223-226). Synthetic peptides modeled after this sequence revealed that the presence of a proline residue C-terminal (+1 position) to the phosphorylatable threonine (or serine) residue was critical for recognition by p44mpk. Although not essential, a proline residue located at the -2 position enhanced the Vmax of peptide phosphorylation. Basic, acidic, and non-polar residues were equally tolerated at the -1 position. The presence of an amino acid residue at position -3 also increased peptide phosphorylation. Thus, the optimum consensus sequence for phosphorylation by p44mpk was defined as Pro-X-(Ser/Thr)-Pro, where X is a variable amino acid residue, but ideally not a Pro. Peptides that included this sequence were phosphorylated by p44mpk with Vmax values approaching 1 mumol.min-1.mg-1 and with apparent Km values of approximately 1 mM). Pseudosubstrate peptides in which the phosphorylatable residue was replaced by valine or alanine were weak inhibitors of p44mpk (apparent Ki values of approximately 3 mM). Over 40 distinct protein kinases contain Pro-X-(Ser/Thr)-Pro sequences including the human receptors for insulin and epidermal growth factor, and kinases encoded by the human proto-oncogenes abl, neu, and raf-1, and Schizosaccharomyces pombe cell cycle control genes ran-1 and wee-1. Multiple putative sites were also identified in rat microtubule-associated protein-2, human retinoblastoma protein, human tau protein, and Drosophila myb protein and RNA polymerase II.     

Role of substrate in determining the phospholipid specificity of protein kinase C activation

The phospholipid selectivity of protein kinase C (PKC) activation was examined by using two substrates, histone and a random copolymer of lysine and serine [poly(lysine, serine)] (PLS), plus phospholipids provided as vesicles or as Triton-mixed micelle preparations. The results indicated that substrate-phospholipid interaction was an essential component of PKC activation and that many in vitro properties of PKC activation are attributable to this interaction. The substrate histone interacted with phospholipid-Triton mixed micelles containing phosphatidylserine (PS), but not with those containing phosphatidylinositol (PI) or phosphatidylglycerol (PG). In direct correlation, only PS-Triton mixed micelles were effective in supporting PKC activity. Also, the minimum PS composition (4 mol % in Triton) required to induce significant histone-PS interaction coincided with the minimum composition required for phosphorylation of histones. Moreover, the PS composition required for maximum activity varied with the histone concentration of the reaction. In contrast to histone, PLS interacted with phospholipid-Triton mixed micelles containing either PS, PI, or PG, and all these mixed micelles supported the phosphorylation of PLS. In fact, by selection of appropriate experimental conditions (e.g., concentration of substrate and phospholipid), any of the three mixed micelles could appear the most effective in supporting PKC activity. Phospholipid vesicles containing PS, PG, or PI were found to interact with both histone and PLS and to support the activity of PKC. Physical properties of the solution and conditions used for preparation of phospholipid vesicles had considerable influence on PKC activation. At high phospholipid concentrations, vesicles containing PS, PI, or PG supported the activity of PKC to essentially the same level, provided that the physical differences among the phospholipid vesicles were minimized.(ABSTRACT TRUNCATED AT 250 WORDS)     

Importance of substrate conformation in the phosphorylation of chromatin-associated proteins by exogenous protein kinase C.

Protein kinase C (PKC)-mediated phosphorylation of chromatin-associated proteins was studied in vitro. HL-60 and HeLa nuclear proteins were notably unresponsive to exogenously added brain PKC. In contrast, 3T3 fibroblasts and lymphocytes from primary cultures exhibited PKC-dependent phosphorylation of chromatin-associated proteins when chromatin was induced to expand. Unexpanded nuclei in all cell lines were unresponsive. Responsiveness was particularly obvious in the decondensed chromatin of primary lymphocytes, where a large number of proteins were phosphorylated in response to exogenous PKC. DNAase-I and micrococcal nuclease strongly modulated these phosphorylation patterns indicating that the substrates were DNA-associated. It was concluded that although substrate conformation, i.e. condensation state, was the primary determining factor in control of PKC-dependent nuclear protein phosphorylation, different cell lines greatly differ in their overall responsiveness to exogenous PKC.     

Characterization of yeast pyruvate kinase 1 as a protein kinase A substrate, and specificity of the phosphorylation site sequence in the whole protein

Pyk1 (pyruvate kinase 1) from Saccharomyces cerevisiae was characterized as a substrate for PKA (protein kinase A) from bovine heart and yeast. By designing Pyk1 synthetic peptides containing potential PKA sequence targets (Ser22, Thr94 and Thr478) we determined that the peptide S22 was a substrate for PKA in vitro, with a K(sp)* (specificity constant) 10-fold and 3-fold higher than Kemptide for bovine heart and yeast PKA respectively. In vitro phosphorylation of the Pyk1 S22A mutant protein was decreased by as much as 90% when compared with wild-type Pyk1 and the Pyk1 T94A mutant. The K(sp)* values for Pyk1 and Pyk1 T94A were the same, indicating that both proteins are phosphorylated at the same site by PKA. Two-dimensional PAGE of Pyk1 and Pyk1 S22A indicates that in vivo the S22A mutation prevented the formation of one of the Pyk1 isoforms. We conclude that in yeast the major PKA phosphorylation site of Pyk1 is Ser22. Phosphorylation of Ser22 leads to a Pyk1 enzyme that is more active in the absence of FBP (fructose 1,6-bisphosphate). The specificity of yeast and mammalian PKA towards the S22 peptide and towards whole Pyk1 protein was measured and compared. The K(sp)* for the S22 peptide is higher than that for Pyk1, indicating that the peptide modelled on Pyk1 is a much better substrate than Pyk1, regardless of which tissue was used as the source of PKA. However, the K(m) of Pyk1 protein is lower than that of the better substrate, the S22 peptide, indicating that ground-state substrate binding is not the major determinant of substrate specificity for PKA.     

Ca2+ phospholipid-dependent and independent phosphorylation of synthetic peptide substrates by protein kinase C

Several synthetic peptides reproducing fragments of protamines have been used as model substrates for Ca2+/phospholipid-dependent protein kinase C, tested both in the absence of any effector (basal conditions) and upon activation by either Ca2+ and phosphatidylserine (or diacylglycerol) or limited proteolysis. Only the peptide Arg4-Tyr-Gly-Ser-Arg6-Tyr [Ga(52-65)] shares the unique property of protamines of being readily phosphorylated even under basal conditions. Optimal activity in the absence of effectors is observed with Tris/HCl buffer pH 7.5; Pipes and Hepes are less effective at pH 7.5, and at pH 6.5 basal phosphorylation is reduced. Under the best conditions for basal phosphorylation of Ga(52-65), its derivative with ornithine replaced for arginine and those corresponding to its C-terminal fragments Gly-Ser-Arg6-Tyr [Ga(57-65)] and Gly-Ser-Arg3 [Ga(57-61)], as well as the peptides Pro-Arg5-Ser2-Arg-Pro-Val-Arg [Th(1-12)], Arg4-Tyr-Arg2-Ser-Thr-Val-Ala [Th(13-23)] and Arg2-Leu-Ser2-Leu-Arg-Ala are not significantly affected though all of them, like histones, are more or less readily phosphorylated upon activation of protein kinase C by Ca2+/phosphatidylserine. The peptide Ser2-Arg-Pro-Val-Arg [Th(7-12)] however, corresponding to the C-terminal part of Th(1-12), is not phosphorylated even in the presence of activators. Limited proteolysis can roughly mimic the Ca2+/phosphatidylserine effect inducing however different extents of activation depending on the nature of the peptide substrates. Our results support the following two conclusions. Basal phosphorylation by protein kinase C in the absence of any effector requires peptide substrates whose target residue(s) are included between two extended arginyl blocks and is also dependent on pH and nature of the buffer. Peptides having extended clusters of either arginyl or ornithyl residues on the C-terminal side of serine are also readily phosphorylated, but they need activation of protein kinase by either Ca2+/phosphatidylserine or limited proteolysis. The same is true of peptides having basic residues only on the N-terminal side, or even on both sides but in limited number.     

Inhibition of cyclic AMP-dependent protein kinase by analogues of a synthetic peptide substrate.

Analogues of the synthetic substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly in which the serine is replaced by other amino acids inhibited the activity of the catalytic subunit of cyclic AMP-dependent protein kinase from beef skeletal muscle (Peak I). All of the analogues were competitive with respect to peptide substrate but apparent Ki values varied depending on the particular amino acid that was substituted for serine. Inhibition was also competitive with respect to mixed histone as determined in experiments utilizing one of the analogues. Acetylation of the terminal amino group of Leu-Arg-Arg-Ala-Ser-Leu-Gly lowered the Km for this substrate from 16 micrometer to 3 micrometer, but a similar modification of the inhibitory analogue Leu-Arg-Arg-Ala-Ala-Leu-Gly resulted in no major change in the Ki value. An amount of inhibitory peptide sufficient to inhibit the cyclic AMP-dependent protein kinase by 90% caused less than 10% inhibition of several cyclic AMP-independent protein kinases indicating a high degree of specificity of inhibition by the peptide analogues. The experiments show that synthetic peptide analogues could be useful in identifying phosphorylation reactions catalyzed by cyclic AMP-dependent protein kinase as distinguished from other protein kinase reactions.     

Transforming properties and substrate specificities of the protein tyrosine kinase oncogenes ros and src and their recombinants.

To determine the sequences of the oncogenes src (encoded by Rous sarcoma virus [RSV]) and ros (encoded by UR2) that are responsible for causing different transformation phenotypes and to correlate those sequences with differences in substrate recognition, we constructed recombinants of the two transforming protein tyrosine kinases (PTKs) and studied their biological and biochemical properties. A recombinant with a 5' end from src and a 3' end from ros, called SRC x ROS, transformed chicken embryo fibroblasts (CEF) to a spindle shape morphology, mimicking that of UR2. Neither of the two reverse constructs, ROS x SRC I and ROS x SRC II, could transform CEF. However, a transforming variant of ROS x SRC II appeared during passages of the transfected cells and was called ROS x SRC (R). ROS x SRC (R) contains a 16-amino-acid deletion that includes the 3' half of the transmembrane domain of ros. Unlike RSV, ROS x SRC (R) also transformed CEF to an elongated shape similar to that of UR2. We conclude that distinct phenotypic changes of RSV- and UR2-infected cells do not depend solely on the kinase domains of their oncogenes. We next examined cellular proteins phosphorylated by the tyrosine kinases of UR2, RSV, and their recombinants as well as a number of other avian sarcoma viruses including Fujinami sarcoma virus Y73, and some ros-derived variants. Our results indicate that the UR2-encoded receptorlike PTK P68gag-ros and its derivatives have a very restricted substrate specificity in comparison with the nonreceptor PTKs encoded by the rest of the avian sarcoma viruses. Data from ros and src recombinants indicate that sequences both inside and outside the catalytic domains of ros and src exert a significant effect on the substrate specificity of the two recombinant proteins. Phosphorylation of most of the proteins in the 100- to 200-kDa range correlated with the presence of the 5' src domain, including the SH2 region, but not with the kinase domain in the recombinants. This corroborates the conclusion given above that the kinase domain of src or ros per se is not sufficient to dictate the transforming morphology of these two oncogenes. High-level tyrosyl phosphorylation of most of the prominent substrates of src is not sufficient to cause a round-shape transformation morphology.     

Site-specific phosphorylation of the purified receptor for calcium-channel blockers by cAMP- and cGMP-dependent protein kinases, protein kinase C, calmodulin-dependent protein kinase II and casein kinase II

Five protein kinases were used to study the phosphorylation pattern of the purified skeletal muscle receptor for calcium-channel blockers (CaCB). cAMP kinase, cGMP kinase, protein kinase C, calmodulin kinase II and casein kinase II phosphorylated the 165-kDa and the 55-kDa proteins of the purified CaCB receptor. The 130/28-kDa and the 32-kDa protein of the receptor are not phosphorylated by these protein kinases. Among these protein kinases only cAMP kinase phosphorylated the 165-kDa subunit with 2-3-fold higher initial rate than the 55-kDa subunit. Casein kinase II phosphorylated the 165-kDa and the 55-kDa protein of the receptor with comparable rates. cGMP kinase, protein kinase C and calmodulin kinase II phosphorylated preferentially the 55-kDa protein. The 55-kDa protein is phosphorylated 50 times faster by cGMP kinase and protein kinase C than by calmodulin kinase II or casein kinase II and about 10 times faster by these enzymes than by cAMP kinase. Two-dimensional peptide maps of the 165-kDa subunit yielded a total of 11 phosphopeptides. Four or five peptides are phosphorylated specifically by cAMP kinase, cGMP kinase, casein kinase II and protein kinase C, whereas the other peptides are modified by several kinases. The same kinases phosphorylate 11 peptides in the 55-kDa subunit. Again, some of these peptides are modified specifically by each kinase. These results suggest that the 165-kDa and the 55-kDa subunit contain specific phosphorylation sites for cAMP kinase, cGMP kinase, casein kinase II and protein kinase C. Phosphorylation of these sites may be relevant for the in vivo function of the CaCB receptor.