CAMP-dependent protein kinase of sea urchin sperm phosphorylates sperm histone H1 on a single site

The phosphorylation of sperm specific histone H1 in the sea urchin Strongylocentrotus purpuratus occurs both in vivo and in vitro on a single serine site in the sequence Arg-Lys-Gly-Ser(P)-Ser-Asn-Ala-Arg. This is a preferred sequence for cAMP-dependent protein kinase. The in vitro phosphorylation is completely dependent on cAMP and is inhibited by the peptide protein kinase inhibitor. The protein kinase inhibitor H-8 blocks the in vivo phosphorylation of H1 without damaging motility, the acrosome reaction or the ability of sperm to fuse with and activate eggs.     

Phosphorylation of P0 Glycoprotein in Peripheral Nerve Myelin

The P0 protein in mammalian PNS myelin is known to undergo several posttranslational modifications, such as glycosylation, acylation, sulfation, and phosphorylation. Phosphorylation of purified P0 protein in vitro was studied comparatively using three enzymes, i.e., calcium/phospholipid-dependent protein kinase (protein kinase C), calcium/calmodulin-dependent protein kinase II (CaM kinase II), and the catalytic subunit of cyclic AMP-dependent protein kinase (A kinase). The phosphorylation of P0 protein by CaM kinase II was the greatest, followed by that by protein kinase C; phosphorylation by A kinase, however, was much lower. In order to identify phosphorylation sites, P0 protein was phosphorylated with [32P]ATP and each kinase and then digested with lysylendopeptidase. The resulting phosphopeptides were isolated by HPLC. Subsequent amino acid sequence analysis and comparison with the known sequence of P0 protein revealed that Ser181 and Ser204 were strongly phosphorylated by both protein kinase C and CaM kinase II. In addition, Ser214 was also phosphorylated by protein kinase C, but not by CaM kinase II. Because all of these sites are located in the cytoplasmic domain of P0 protein, phosphorylation may be important for maintenance of the major dense line of PNS myelin.     

Identification of the phosphoserine residue in histone H1 phosphorylated by protein kinase C

The site-specific phosphorylation of bovine histone H1 by protein kinase C was investigated in order to further elucidate the substrate specificity of protein kinase C. Protein kinase C was found to phosphorylate histone H1 to 1 mol per mol. Using N-bromosuccinimide and thrombin digestions, the phosphorylation site was localized to the globular region of the protein, containing residues 71-122. A tryptic peptide containing the phosphorylation site was purified. Modification of the phosphoserine followed by amino acid sequence analysis demonstrated that protein kinase C phosphorylated histone H1 on serine 103. This sequence, Gly97-Thr-Gly-Ala-Ser-Gly-Ser(PO4)-Phe-Lys105, supports the contention that basic amino acid residues C-terminal to the phosphorylation site are sufficient determinants for phosphorylation by protein kinase C.     

Cellular phosphorylation of an acidic proline-rich protein, PRP1, a secreted salivary phosphoprotein

Phosphorylation of many secreted salivary proteins is necessary for their biological functions. Identification of the kinase, which is responsible for in vivo phosphorylation, is complicated, because several of the protein phosphorylation sites conform both to the recognition sequence of casein kinase 2 (CK2) and Golgi kinase (G-CK), which both are found in the secretory pathway. This study was undertaken to determine the kinase recognition sequence in a secreted proline-rich salivary protein, PRP1, and thereby identify the responsible kinase. This was done by transfecting a human submandibular cell line, HSG, and a kidney cell line, HEK293, with expression vectors encoding wild-type or mutated PRP1. It was shown that phosphorylation occurred only at the same sites, Ser8 and 22, as in PRP1 purified from saliva. Phosphorylation at either site did not depend on the other site being phosphorylated. The sequence surrounding Ser8 has characteristics of both CK2 and G-CK recognition sequences, but destruction of the CK2 recognition site had no effect on phosphorylation, whereas no phosphorylation occurred if the G-CK recognition sequence was altered. The sequence surrounding Ser22 did not conform to any known kinase recognition sites. If Ser22 was mutated to Thr, no phosphorylation was seen, and a cluster of negatively charged residues at positions 27-29 was identified as part of the enzyme recognition site. Ser22 may be phosphorylated by a G-CK that recognizes an atypical substrate sequence or by a novel kinase. No difference in phosphorylation was seen between undifferentiated and differentiated HSG cells.     

Regulation of calcineurin by phosphorylation. Identification of the regulatory site phosphorylated by Ca2+/calmodulin-dependent protein kinase II and protein kinase C

The site in calcineurin, the Ca2+/calmodulin (CaM)-dependent protein phosphatase, which is phosphorylated by Ca2+/CaM-dependent protein kinase II (CaM-kinase II) has been identified. Analyses of 32P release from tryptic and cyanogen bromide peptides derived from [32P]calcineurin plus direct sequence determination established the site as -Arg-Val-Phe-Ser(PO4)-Val-Leu-Arg-, which conformed to the consensus phosphorylation sequence for CaM-kinase II (Arg-X-X-Ser/Thr-). This phosphorylation site is located at the C-terminal boundary of the putative CaM-binding domain in calcinerin (Kincaid, R. L., Nightingale, M. S., and Martin, B. M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8983-8987), thereby accounting for the observed inhibition of this phosphorylation when Ca2+/CaM is bound to calcineurin. Since the phosphorylation site sequence also contains elements of the specificity determinants for Ca2+/phospholipid-dependent protein kinase (protein kinase C) (basic residues both N-terminal and C-terminal to Ser/Thr), we tested calcineurin as a substrate for protein kinase C. Protein kinase C catalyzed rapid stoichiometric phosphorylation, and the characteristics of the reaction were the same as with CaM-kinase II: 1) the phosphorylation was blocked by binding of Ca2+/CaM to calcineurin; 2) phosphorylation partially inactivated calcineurin by increasing the Km (from 9.9 +/- 1.1 to 17.5 +/- 1.1 microM 32P-labeled myosin light chain); and 3) [32P]calcineurin exhibited very slow autodephosphorylation but was rapidly dephosphorylated by protein phosphatase IIA. Tryptic and thermolytic 32P-peptide mapping and sequential phosphoamino acid sequence analysis confirmed that protein kinase C and CaM-kinase II phosphorylated the same site.     

The localization of the cAMP-dependent protein kinase phosphorylation site in the platelet rat protein, rap 1B

Rap 1B is a low molecular weight G protein which is phosphorylated by cAMP-dependent protein kinase. In order to identify the site of phosphorylation by cAMP-dependent protein kinase, purified rap 1B from human platelets was phosphorylated and subjected to limited proteolysis with trypsin. Single digestion fragment containing the phosphorylation site was obtained and purified by reversed-phase HPLC. Sequence analysis of the phosphorylated digestion fragment demonstrated that the sequence of the phosphorylation site was -Lys-Lys-Ser-Ser-. This sequence is near the carboxy terminus and is adjacent to the site of membrane attachment of the protein.     

Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase).

Mitogen-activated protein kinase (MAP kinase) is a 42 kd serine/threonine protein kinase whose enzymatic activity requires phosphorylation of both tyrosyl and threonyl residues. As a step in elucidating the mechanism(s) for activation of this enzyme, we have determined the sites of regulatory phosphorylation. Following proteolytic digestion of 32P-labeled pp42/MAP kinase with trypsin, only a single phosphopeptide was detected by two-dimensional peptide mapping, and this peptide contained both phosphotyrosine and phosphothreonine. The amino acid sequence of the peptide, including the phosphorylation sites, was determined using a combination of Fourier transform mass spectrometry and collision-activated dissociation tandem mass spectrometry with electrospray ionization. The sequence for the pp42/MAP kinase tryptic phosphopeptide is similar (but not identical) to a sequence present in the ERK1- and KSS1-encoded kinases. The two phosphorylation sites are separated by only a single residue. The regulation of activity by dual phosphorylations at closely spaced threonyl and tyrosyl residues has a functional correlate in p34cdc2, and may be characteristic of a family of protein kinases regulating cell cycle transitions.     

Phosphorylation regulates the activity of the eEF-2-specific Ca(2+)- and calmodulin-dependent protein kinase III

The activity of the eukaryotic elongation factor 2 (eEF-2)-specific Ca(2+)- and calmodulin-dependent protein kinase III (CaM PK III) is regulated by phosphorylation. The kinase can be inactivated by treatment with alkaline phosphatase and subsequently reactivated by endogenous protein kinase. This kinase can be substituted for by the catalytic subunit of cAMP-dependent protein kinase but not by casein kinase II. The purified kinase preparation contains only one protein as judged by gel electrophoresis. This protein has a molecular mass of approximately 90 kDa and an isoelectric point of 5.2. Reactivation of the eEF-2 kinase is associated with the phosphorylation of this protein. The amino acid sequence obtained from the 90-kDa protein reveals substantial homology with that of murine heat shock protein 86 (HSP 86) a member of the HSP 90-family. Conventional preparations of HSP 90 contain an inactive eEF-2 kinase that could be activated after dephosphorylation and phosphorylation by the catalytic subunit of cAMP-dependent protein kinase.     

Amino acid sequence of rat thymus histone H2B and identification of the in vitro phosphorylation sites.

The amino acid sequence of rat thymus histone obtained in highly purified form by preparative electrophoresis, was determined. This sequence is identical to the sequence of calf thymus histone H2B. The in vitro phosphorylation of the rat histone with a cyclic AMP-dependent protein kinase isolated from rat pancreas led to the identification of four sites of phosphorylation: two major ones, at serine residues 32 and 36, and two minor ones, specific of the rat protein kinase, at serine residues 87 and 91.     

Characterization of the Phosphorylation Site of PP59, a Substrate for Cyclic AMP-Dependent Protein Kinase That Is Enriched in the Synaptic Membrane Fraction of Rat Cerebellum

Abstract: We have identified previously a synaptic membrane-associated protein, PP59, that serves as a substrate for cyclic AMP-dependent protein kinase and is enriched in rat cerebellum. We show here that PP59 can be extracted from synaptic plasma membranes with a combination of 2% Triton X-100 plus 1 M KCl. A 290-fold purification of PP59 was achieved by selective solubilization, followed by continuous-elution preparative gel electrophoresis. To determine the amino acid sequence surrounding the cyclic AMP-dependent protein kinase phosphorylation site within PP59, the partially purified ³²P-phosphorylated protein was digested with chymotrypsin, and radiolabeled peptides were purified by sequential reversed-phase HPLC in two different solvent systems. Automated Edman degradation revealed a single phosphorylation site contained within the sequence Ala-Arg-Glu-Arg-Ser-Asp-Ser(P)-Thr-Gly-Ser-Ser-Ser-Val-Tyr. No strong sequence homology to this peptide fragment with other known peptides or proteins in the SwissProt, PIR, or GenPept databases could be found. A synthetic peptide containing this unique 14-amino acid sequence was used to develop polyclonal anti-peptide antibodies that were affinity-purified and shown to recognize intact PP59 as determined by western blotting. These antibodies specifically inhibited the phosphorylation of PP59 by cyclic AMP-dependent protein kinase in an in vitro phosphorylation assay containing synaptic plasma membranes.     

Regulation of Ca(2+)/calmodulin-dependent protein kinase kinase alpha by cAMP-dependent protein kinase: I. Biochemical analysis

Ca(2+)/calmodulin-dependent protein kinases (CaM-kinases) I and IV are activated upon phosphorylation of their Thr(177) and Thr(196), respectively, by the upstream Ca(2+)/calmodulin-dependent protein kinases CaM-kinase kinase alpha and beta, and deactivated upon dephosphorylation by protein phosphatases such as CaM-kinase phosphatase. Recent studies demonstrated that the activity of CaM-kinase kinase alpha is decreased upon phosphorylation by cAMP-dependent protein kinase (PKA), and the relationship between the inhibition and phosphorylation of CaM-kinase kinase alpha by PKA has been studied. In the present study, we demonstrate that the activity of CaM-kinase kinase alpha toward PKIV peptide, which contains the sequence surrounding Thr(196) of CaM-kinase IV, is increased by incubation with PKA in the presence of Ca(2+)/calmodulin but decreased in its absence, while the activity toward CaM-kinase IV is decreased by incubation with PKA in both the presence and absence of Ca(2+)/calmodulin. Six phosphorylation sites on CaM-kinase kinase alpha, Ser(24) for autophosphorylation, and Ser(52), Ser(74), Thr(108), Ser(458), and Ser(475) for phosphorylation by PKA, were identified by amino acid sequence analysis of the phosphopeptides purified from the tryptic digest of the phosphorylated enzymes. The presence of Ca(2+)/calmodulin suppresses phosphorylation on Ser(52), Ser(74), Thr(108), and Ser(458) by PKA, but accelerates phosphorylation on Ser(475). The changes in the activity of the enzyme upon phosphorylation appear to occur as a result of conformational changes induced by phosphorylation on several sites.     

Location and function of three sites phosphorylated on rat acetyl-CoA carboxylase by the AMP-activated protein kinase

1. We have sequenced two tryptic/chymotryptic peptides (TC3 and TC3a) containing a third site phosphorylated on rat acetyl-CoA carboxylase by the AMP-activated protein kinase. Comparison with the complete sequence of rat acetyl-CoA carboxylase predicted from the cDNA sequence [López-Casillas et al. (1988) Proc. Natl Acad. Sci. USA 85, 5784-5788] shows that this site corresponds to Ser1215. 2. Comparison of the cDNA sequence with previous amino acid sequence data identifies the other two sites for the AMP-activated protein kinase as Ser79 and Ser1200. A total of eight serine residues phosphorylated in vitro by six protein kinases can now be identified: six of these (Ser23, Ser25, Ser29, Ser77, Ser79 and Ser95) are clustered in the amino terminal region, while two (Ser1200 and Ser1215) are located in the central region. 3. Prior phosphorylation of Ser77 and Ser1200 by cyclic-AMP-dependent protein kinase prevents subsequent phosphorylation of Ser79 and Ser1200, but not Ser1215, by the AMP-activated protein kinase. Phosphorylation of Ser1215 under these conditions is not associated with a change in enzyme activity. 4. Limited trypsin treatment of native acetyl-CoA carboxylase selectively cleaves off the highly phosphorylated amino-terminal region containing Ser79. 5. Phosphorylation at Ser79 and Ser1200 by the AMP-activated protein kinase dramatically decreases Vmax and increases the A0.5 for citrate. Phosphorylation at Ser77 and Ser1200 by cyclic-AMP-dependent protein kinase causes more modest changes in the A0.5 for citrate and the Vmax. Dephosphorylation, or removal of the amino-terminal region containing Ser77/79 using trypsin, reverses all of these effects. 6. These results suggest that the effects of the AMP-activated protein kinase on acetyl-CoA carboxylase activity are mediated entirely by phosphorylation of Ser79, and not Ser1200 and Ser1215. The smaller effects of cyclic-AMP-dependent protein kinase are mediated by phosphorylation of Ser77.     

Insulin-activated protein kinases phosphorylate a pseudosubstrate synthetic peptide inhibitor of the p70 S6 kinase

p70 S6 kinase, a major insulin-mitogen-activated ribosomal S6 protein kinase in mammalian cells, is activated by phosphorylation of multiple Ser/Thr residues on the enzyme polypeptide. A synthetic peptide, corresponding to a 37-residue segment from the carboxyl-terminal tail of the kinase which resembles the sequence phosphorylated in S6, acts as a competitive inhibitor of p70 S6 kinase without itself being phosphorylated by the enzyme. This synthetic peptide is phosphorylated by an array of protein kinases which are rapidly activated by insulin. Thus, these sequences of p70 S6 kinase constitute a potential autoinhibitory pseudosubstrate site, whose phosphorylation is catalyzed by candidate upstream-activating protein kinases.     

Substrate specificity of protein kinase C. Use of synthetic peptides corresponding to physiological sites as probes for substrate recognition requirements

Although the Ca2+/phospholipid-dependent protein kinase, protein kinase C, has a broad substrate specificity in vitro, the enzyme appears considerably less promiscuous in vivo. To date only a handful of proteins have been identified as physiological substrates for this protein kinase. In order to determine the basis for this selectivity for substrates in intact cells, we have probed the substrate primary sequence requirements of protein kinase C using synthetic peptides corresponding to sites of phosphorylation from four of the known physiological substrates. We have also identified the acetylated N-terminal serine of chick muscle lactate dehydrogenase as an in vitro site of phosphorylation for this protein kinase. These comparative studies have demonstrated that, in vivo, the enzyme exhibits a preference for one basic residue C-terminal to the phosphorylatable residue, as in the sequence: Ser/Thr-Xaa-Lys/Arg, where Xaa is usually an uncharged residue. Additional basic residues, both N and C-terminal to the target amino acid, enhance the Vmax and Km parameters of phosphorylation. None of the peptides based on physiological phosphorylation sites of protein kinase C was an efficient substrate of cAMP-dependent protein kinase, emphasizing the distinct site-recognition selectivities of these two pleiotropic protein kinases. The favorable kinetic parameters of several of the synthetic peptides, coupled with their selectivity for phosphorylation by protein kinase C, will facilitate the assay of this enzyme in the presence of other protein kinases in tissue and cell extracts.     

Ca2+/Calmodulin-Dependent Protein Kinase II and Protein Kinase C Phosphorylate a Synthetic Peptide Corresponding to a Sequence That Is Specific for the γ2L Subunit of the GABAA Receptor

Abstract: The γ₂ subunit of the GABA_A receptor (GABA_A-R) is alternatively spliced. The long variant (γ_2L) contains eight additional amino acids that possess a consensus sequence site for protein phosphorylation. Previous studies have demonstrated that a peptide or fusion protein containing these eight amino acids is a substrate for protein kinase C (PKC), but not cyclic AMP-dependent protein kinase A (PKA)-stimulated phosphorylation. We have examined the ability of PKA, PKC, and Ca²⁺/calmodulin-dependent protein kinase (CAM kinase II) to phosphorylate a synthetic peptide corresponding to residues 336–351 of the intracellular loop of the γ_2L subunit and inclusive of the alternatively spliced phosphorylation consensus sequence site. PKC and CAM kinase II produced significant phosphorylation of this peptide, but PKA was ineffective. The K _m values for PKC-and CAM kinase II-stimulated phosphorylation of this peptide were 102 and 35 μM , respectively. Maximal velocities of 678 and 278 nmol of phosphate/min/mg were achieved by PKC and CAM kinase II, respectively. The phosphorylation site in the eight-amino-acid insert of the γ_2L subunit has been shown to be necessary for ethanol potentiation of the GABA_A-R. Thus, our results suggest that PKC, CAM kinase II, or both may play a role in the effects of ethanol on GABAergic function.     

Phosphorylation of nitric oxide synthase by protein kinase A.

Nitric oxide synthase was purified to apparent homogeneity from the cytosolic fractions obtained from rat and porcine cerebellum. Enzyme activity--measured as [3H]citrulline formation after incubation with [3H]arginine--was dependent on Ca2+/calmodulin, NADPH, and tetrahydro-L-biopterin. Specific activity varied between 450 to 780 nmol/min/mg protein. Purified nitric oxide synthases showed a single band on 8% SDS/PAGE gels and had an apparent molecular mass of 150,000 Da. The purified proteins were used as substrate for phosphorylation with different protein kinases. In the assays using two Ca2+/calmodulin-dependent protein kinases, CaM kinase II and CaM kinase-Gr, protein kinase C, and the catalytic subunit of protein kinase A, nitric oxide synthase was exclusively phosphorylated by protein kinase A. Such phosphorylation was linear over time for at least 60 min and resulted in nearly stoichiometric phosphate/protein incorporation. The serine in the protein kinase A-consensus sequence KRFGS is probably the site of phosphorylation in nitric oxide synthase. Kemptide, a known protein kinase A substrate, inhibited phosphorylation of nitric oxide synthase in a dose-dependent manner. No changes in nitric oxide synthase activity were observed upon phosphorylation by protein kinase A.     

Prediction of kinase-specific phosphorylation sites with sequence features by a log-odds ratio approach

Protein phosphorylation plays important roles in a variety of cellular processes. Detecting possible phosphorylation sites and their corresponding protein kinases is crucial for studying the function of many proteins. This article presents a new prediction system, called PhoScan, to predict phosphorylation sites in a kinase-family-specific way. Common phosphorylation features and kinase-specific features are extracted from substrate sequences of different protein kinases based on the analysis of published experiments, and a scoring system is developed for evaluating the possibility that a peptide can be phosphorylated by the protein kinase at the specific site in its sequence context. PhoScan can achieve a specificity of above 90% with sensitivity around 90% at kinase-family level on the data experimented. The system is applied on a set of human proteins collected from Swiss-Prot and sets of putative phosphorylation sites are predicted for protein kinase A, cyclin-dependent kinase, and casein kinase 2 families. PhoScan is available at http://bioinfo.au.tsinghua.edu.cn/phoscan/.     

Identification of the phosphorylation sites of the murine small heat shock protein hsp25.

Native phosphorylated mouse small heat shock protein hsp25 from Ehrlich ascites tumor cells was isolated and the in vivo phosphorylation sites of the protein were determined. Furthermore, native hsp25 was phosphorylated by the endogenous kinase(s) in a cell-free system as well as recombinant hsp25 was phosphorylated in vitro by protein kinase C and catalytic subunit of cAMP-dependent protein kinase. The two major phosphorylation sites of native and recombinant hsp25 were determined as Ser-15 and Ser-86. There are no differences in the hsp25 phosphorylation sites phosphorylated by the protein kinase C, the catalytic subunit of cAMP-dependent protein kinase and the unknown intracellular kinase(s). The serine residues identified exist in all known small mammalian stress proteins and are located in the conserved kinase recognition sequence Arg-X-X-Ser.     

Cross-talk between the two divergent insulin signaling pathways is revealed by the protein kinase B (Akt)-mediated phosphorylation of adapter protein APS on serine 588

The APS adapter protein is recruited to the autophosphorylated kinase domain of the insulin receptor and initiates the phosphatidylinositol 3-kinase (PI3K)-independent pathway of insulin-stimulated glucose transport by recruiting CAP and c-Cbl. In this study, we have identified APS as a novel substrate for protein kinase B/Akt using an antibody that exhibits insulin-dependent immunoreactivity with a phosphospecific antibody raised against the protein kinase B substrate consensus sequence RXRXX(pS/pT) and a phosphospecific antibody that recognizes serine 21/9 of glycogen synthase kinase-3alpha/beta. This phosphorylation of APS is observed in both 3T3-L1 adipocytes and transfected cells. The insulin-stimulated serine phosphorylation of APS was inhibited by a PI3-kinase inhibitor, LY290004, a specific protein kinase B (PKB) inhibitor, deguelin, and knockdown of Akt. Serine 588 of APS is contained in a protein kinase B consensus sequence for phosphorylation conserved in APS across multiple species but not found in other members of this family, including SH2-B and Lnk. Mutation of serine 588 to alanine abolished the insulin-stimulated serine phosphorylation of APS and prevented the localization of APS to membrane ruffles. A glutathione S-transferase fusion protein containing amino acids 534-621 of APS was phosphorylated by purified PKB in vitro, and mutation of serine 588 abolished the PKB-mediated phosphorylation of APS in vitro. Taken together, this study identifies APS as a novel physiological substrate for PKB and the first serine phosphorylation site on APS. These data therefore reveal the molecular cross-talk between the insulin-activated PI3-kinase-dependent and -independent pathways previously thought to be distinct and divergent.