Identification of a novel proline-directed serine/threonine protein kinase in rat pheochromocytoma

During investigations of the regulation of tyrosine hydroxylase (TH) by protein phosphorylation, a novel protein kinase activity has been discovered in rat pheochromocytoma. Originally detected as a trace contaminant in preparations of highly purified TH, this novel kinase activity phosphorylated TH at serine 8 in the proline-rich amino-terminal region of the enzyme. This particular site is not phosphorylated by, nor is the amino acid sequence surrounding this site selective for, any of the classical (i.e. well characterized) protein kinases. In this report, we describe the identification, characterization, and partial purification of this novel protein kinase. By utilizing a synthetic peptide corresponding to the amino-terminal region of TH, a selective assay for this protein kinase was developed. The kinase activity utilized ATP and magnesium, although GTP could also be utilized as a phosphate donor. The kinase activity was found to co-purify with TH activity through ammonium sulfate precipitation and DEAE-cellulose chromatography and could be only partially resolved from TH by heparin-agarose affinity chromatography. Substantial kinase activity could be resolved from TH by phosphocellulose chromatography. The novel kinase migrates as a protein with a molecular mass of approximately 45 kDa on gel permeation chromatography as well as sucrose density gradient centrifugation. Studies of site specificity indicate that this Ser/Thr kinase activity appears to be directed by an adjacent (carboxyl-terminal) proline residue, exhibiting a minimal recognition sequence of -X-Ser/Thr-Pro-X-. In addition to TH, this proline-directed protein kinase will also phosphorylate synapsin I, histone H1, and glycogen synthase, suggesting that this kinase may have multiple substrates in vivo. Additional findings indicate that the activity of proline-directed protein kinase is increased transiently in PC12 pheochromocytoma cells following treatment with nerve growth factor. Distinctions between this novel kinase and other well characterized protein kinases can be made on the basis of phosphorylation site specificity, chromatographic behavior, and physical characteristics.     

Identification of tyrosine 67 in bovine brain myelin basic protein as a specific phosphorylation site for thymus p56lck.

The protein-tyrosine kinase p56lck exhibits a restricted substrate specificity in vitro but can efficiently phosphorylate bovine myelin basic protein (MBP). Results obtained from both peptide mapping and fast atom bombardment mass spectrometry indicate that tyrosine 67 in the sequence -Thr-Thr-His-Tyr67-Gly-Ser-Leu-Pro-Gln-Lys- in bovine MBP is the specific phosphorylation site. p56lck does not phosphorylate the acidic, cytoplasmic domain of erythrocyte band 3. In contrast, p40, another protein-tyrosine kinase purified from bovine thymus that readily phosphorylates band 3, does not phosphorylate MBP. Therefore, MBP and band 3 may prove to be useful substrates for distinguishing between various tyrosine kinases on the basis of substrate specificity. In addition, identification of the recognition sequence in MBP for p56lck may contribute to an understanding of the structural features of physiological substrates for this kinase.     

Exceptional disfavor for proline at the P + 1 position among AGC and CAMK kinases establishes reciprocal specificity between them and the proline-directed kinases

To precisely regulate critical signaling pathways, two kinases that phosphorylate distinct sites on the same protein substrate must have mutually exclusive specificity. Evolution could assure this by designing families of kinase such as basophilic kinases and proline-directed kinase with distinct peptide specificity; their reciprocal peptide specificity would have to be very complete, since recruitment of substrate allows phosphorylation of even rather poor phosphorylation sites in a protein. Here we report a powerful evolutionary strategy that assures distinct substrates for basophilic kinases (PKA, PKG and PKC (AGC) and calmodulin-dependent protein kinase (CAMK)) and proline-directed kinase, namely by the presence or absence of proline at the P + 1 position in substrates. Analysis of degenerate and non-degenerate peptides by in vitro kinase assays reveals that proline at the P + 1 position in substrates functions as a "veto" residue in substrate recognition by AGC and CAMK kinases. Furthermore, analysis of reported substrates of two typical basophilic kinases, protein kinase C and protein kinase A, shows the lowest occurrence of proline at the P + 1 position. Analysis of crystal structures and sequence conservation provides a molecular basis for this disfavor and illustrate its generality.     

Identification of the ATP·Mg-dependent protein phosphatase activator (Fa ) as a synapsin I kinase that inhibits cross-linking of synapsin I with brain microtubules

The ATP·Mg-dependent protein phosphatase activating factor (Fa) has been identified and purified to near homogeneity from brain. In this report, as evidenced on SDS-polyacrylamide gel electrophoresis followed by autoradiography, factorFa has further been identified as a cAMP and Ca²⁺-independent brain kinase that could phosphorylate synapsin I, a neuronal protein that coats synaptic vesicles, binds to cytoskeleton, and is believed to be involved in the modulation of neurotransmission. Kinetic study further indicated that factorFa could phosphorylate synapsin I with a lowK _m value of about 2 µM and with a molar ratio of 1 mol of phosphate per mole of protein. Peptide mapping analysis revealed that factorFa specifically phosphorylated the tail region of synapsin I but on a unique site distinct from those phosphorylated by Ca²⁺/calmodulin-dependent protein kinase II and cAMP-dependent protein kinase, the two well-established synapsin I kinases. Functional study further revealed that factorFa could phosphorylate this unique specific site on the tail region of synapsin I and thereby inhibit cross-linking of synapsin I with microtubules. The results further suggest the possible involvement of factorFa as a synapsin I kinase in the regulation of axonal transport process of synaptic vesicles via the promotion of vesicles motility during neurotransmission.     

Structure and substrate specificity of the Pim-1 kinase

The Pim kinases are a family of three vertebrate protein serine/threonine kinases (Pim-1, -2, and -3) belonging to the CAMK (calmodulin-dependent protein kinase-related) group. Pim kinases are emerging as important mediators of cytokine signaling pathways in hematopoietic cells, and they contribute to the progression of certain leukemias and solid tumors. A number of cytoplasmic and nuclear proteins are phosphorylated by Pim kinases and may act as their effectors in normal physiology and in disease. Recent crystallographic studies of Pim-1 have identified unique structural features but have not provided insight into how the kinase recognizes its target substrates. Here, we have conducted peptide library screens to exhaustively determine the sequence specificity of active site-mediated phosphorylation by Pim-1 and Pim-3. We have identified the major site of Pim-1 autophosphorylation and find surprisingly that it maps to a novel site that diverges from its consensus phosphorylation motif. We have solved the crystal structure of Pim-1 bound to a high affinity peptide substrate in complexes with either the ATP analog AMP-PNP or the bisindolylmaleimide kinase inhibitor 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)maleimide HCl. These structures reveal an unanticipated mode of recognition for basic residues upstream of the phosphorylation site, distinct from that of other kinases with similar substrate specificity. The structures provide a rationale for the unusually high affinity of Pim kinases for peptide substrates and suggest a general mode for substrate binding to members of the CAMK group.     

An Arabidopsis protein phosphorylated in response to microbial elicitation, AtPHOS32, is a substrate of MAP kinases 3 and 6

Although mitogen-activated protein kinases (MAPKs) have been shown to be activated by a wide range of biotic and abiotic stimuli in diverse plant species, few in vivo substrates for these kinases have been identified. While studying proteins that are differentially phosphorylated upon treatment of Arabidopsis suspension cultures with the general bacterial elicitor peptide flagellin-22 (flg22), we identified two proteins with endogenous nickel binding properties that become phosphorylated after flg22 elicitation. These highly related proteins, AtPHOS32 and AtPHOS34, show similarity to bacterial universal stress protein A. We identified one of the phosphorylation sites on AtPHOS32 by nanoelectrospray ionization tandem mass spectrometry. Phosphorylation in a phosphoSer-Pro motif indicated that this protein may be a substrate of MAPKs. Using in vitro kinase assays, we confirmed that AtPHOS32 is a substrate of both AtMPK3 and AtMPK6. Specificity of phosphorylation was demonstrated by site-directed mutagenesis of the first phosphorylation site. In addition, immunosubtraction of both MAPKs from protein extracts removed detectable kinase activity toward AtPHOS32, indicating that the two MAPKs were the predominate kinases recognizing the motif in this protein. Finally, the target phosphorylation site in AtPHOS32 is conserved in AtPHOS34 and among apparent orthologues from many plant species, indicating that phosphorylation of these proteins by AtMPK3 and AtMPK6 orthologues has been conserved throughout evolution.     

Specificity profiling of Pak kinases allows identification of novel phosphorylation sites

The p21-activated kinases (Paks) serve as effectors of the Rho family GTPases Rac and Cdc42. The six human Paks are divided into two groups based on sequence similarity. Group I Paks (Pak1 to -3) phosphorylate a number of substrates linking this group to regulation of the cytoskeleton and both proliferative and anti-apoptotic signaling. Group II Paks (Pak4 to -6) are thought to play distinct functional roles, yet their few known substrates are also targeted by Group I Paks. To determine if the two groups recognize distinct target sequences, we used a degenerate peptide library method to comprehensively characterize the consensus phosphorylation motifs of Group I and II Paks. We find that Pak1 and Pak2 exhibit virtually identical substrate specificity that is distinct from that of Pak4. Based on structural comparisons and mutagenesis, we identified two key amino acid residues that mediate the distinct specificities of Group I and II Paks and suggest a structural basis for these differences. These results implicate, for the first time, residues from the small lobe of a kinase in substrate selectivity. Finally, we utilized the Pak1 consensus motif to predict a novel Pak1 phosphorylation site in Pix (Pak-interactive exchange factor) and demonstrate that Pak1 phosphorylates this site both in vitro and in cultured cells. Collectively, these results elucidate the specificity of Pak kinases and illustrate a general method for the identification of novel sites phosphorylated by Paks.     

Utilization of oriented peptide libraries to identify substrate motifs selected by ATM

The ataxia telangiectasia mutated (ATM) gene encodes a serine/threonine protein kinase that plays a critical role in genomic surveillance and development. Here, we use a peptide library approach to define the in vitro substrate specificity of ATM kinase activity. The peptide library analysis identified an optimal sequence with a central core motif of LSQE that is preferentially phosphorylated by ATM. The contributions of the amino acids surrounding serine in the LSQE motif were assessed by utilizing specific peptide libraries or individual peptide substrates. All amino acids comprising the LSQE sequence were critical for maximum peptide substrate suitability for ATM. The DNA-dependent protein kinase (DNA-PK), a Ser/Thr kinase related to ATM and important in DNA repair, was compared with ATM in terms of peptide substrate selectivity. DNA-PK was found to be unique in its preference of neighboring amino acids to the phosphorylated serine. Peptide library analyses defined a preferred amino acid motif for ATM that permits clear distinctions between ATM and DNA-PK kinase activity. Data base searches using the library-derived ATM sequence identified previously characterized substrates of ATM, as well as novel candidate substrate targets that may function downstream in ATM-directed signaling pathways.     

Substrate specificity of phospholipid/Ca2+-dependent protein kinase as probed with synthetic peptide fragments of the bovine myelin basic protein

The substrate specificity of phospholipid/Ca2+-dependent protein kinase (protein kinase C) was studied using synthetic peptides, in particular those corresponding to the amino acid sequence around serine 115 in bovine myelin basic protein (MBP). It was found that MBP (104-118) and MBP (104-123) were substrates for the enzyme, with apparent Km values of 14 and 10 microM, respectively. Neither MBP (111-118) nor MBP (111-123) were phosphorylated, indicating that an additional segment of sequence extending toward the N terminus, but not toward the C terminus, was essential for the substrate activity of the peptides. Of the alanine-substituted analogs examined, [Ala 105] MBP (104-118) was comparable to the parent peptide, whereas [Ala 107] MBP (104-118) and [Ala 113] MBP-(104-118) were much poorer substrates. These findings indicated that lysine 105 was not essential, but both arginine 107 and arginine 113 were important specificity determinants. Initial studies revealed that [Ala 113] MBP (104-118) inhibited phosphorylation by the enzyme of the parent peptide and, to a lesser extent, the intact MBP(1-170). Serine 115 was the only site phosphorylated in the analog peptides [Ala 105] MBP (104-118) and [Ala 107]MBP (104-118). In the parent peptide, serine 115 was the initial site of phosphorylation but after prolonged phosphorylation other sites became phosphorylated (serine 110 and/or serine 112), further supporting the concept that arginine residues act as essential substrate specificity determinants for phospholipid/Ca2+-dependent protein kinase.     

Cyclin B and Cyclin A Confer Different Substrate Recognition Properties on CDK2

The transitions of the cell cycle are regulated by the cyclin dependent protein kinases(CDKs). The cyclins activate their respective CDKs and confer substrate recognitionproperties. We report the structure of phospho-CDK2/cyclin B and show that cyclin Bconfers M phase-like properties on CDK2, the kinase that is usually associated with S phase.Cyclin B produces an almost identical activated conformation of CDK2 as that produced bycyclin A. There are differences between cyclin A and cyclin B at the recruitment site, whichin cyclin A is used to recruit substrates containing an RXL motif. Because of sequencedifferences this site in cyclin B binds RXL motifs more weakly than in cyclin A. Despitesimilarity in kinase structures, phospho-CDK2/cyclin B phosphorylates substrates, such asnuclear lamin and a model peptide derived from p107, at sequences SPXX that differ fromthe canonical CDK2/cyclin A substrate recognition motif, SPXK. CDK2/cyclin Bphosphorylation at these non-canonical sites is not dependent on the presence of a RXLrecruitment motif. The p107 peptide contained two SP motifs each followed by a noncanonicalsequence of which only one site (Ser640) is phosphorylated by pCDK2/cyclin Awhile two sites are phosphorylated by pCDK2/cyclin B. The second site is too close to theRXL motif to allow the cyclin A recruitment site to be effective, as previous work has shownthat there must be at least 16 residues between the catalytic site serine and the RXL motif.Thus the cyclins A and B in addition to their role in promoting the activatory conformationalswitch in CDK2, also provide differential substrate specificity.     

Ca(2+)-calmodulin-dependent protein kinases Ia and Ib from rat brain I. Identification, purification, and structural comparisons.

Two Ca(2+)-calmodulin (CaM)-dependent protein kinases were purified from rat brain using as substrate a synthetic peptide based on site 1 (site 1 peptide) of the synaptic vesicle-associated protein, synapsin I. One of the purified enzymes was an approximately 89% pure protein of M(r) = 43,000 which bound CaM in a Ca(2+)-dependent fashion. The other purified enzyme was an apparently homogenous protein of M(r) = 39,000 accompanied by a small amount of a M(r) = 37,000 form which may represent a proteolytic product of the 39-kDa enzyme. The 39-kDa protein bound CaM in a Ca(2+)-dependent fashion. Gel filtration analysis indicated that both enzymes are monomers. The 43- and 39-kDa enzymes are named Ca(2+)-CaM-dependent protein kinases Ia and Ib (CaM kinases Ia, Ib), respectively. The specific activities of CaM kinases Ia and Ib were similar (5-8 mumol/min/mg protein). CaM kinase Ia (but not CaM kinase Ib) activity was enhanced by addition of a CaM-Sepharose column wash (non-binding) fraction suggesting the existence of an "activator" of CaM kinase Ia. Both kinases phosphorylated exogenous substrates (site 1 peptide and synapsin I) in a Ca(2+)-CaM-dependent fashion and both kinases underwent autophosphorylation. CaM kinase Ia autophosphorylation was Ca(2+)-CaM-dependent and occurred exclusively on threonine while CaM kinase Ib autophosphorylation showed Ca(2+)-CaM independence and occurred on both serine and threonine. Proteolytic digestion of autophosphorylated CaM kinases Ia and Ib yielded phosphopeptides of differing M(r). These characteristics, as well as enzymatic and regulatory properties (DeRemer, M. F., Saeli, R. J. Brautigen, D. L., and Edelman, A. M. (1992) J. Biol. Chem. 267, 13466-13471), indicate that CaM kinases Ia and Ib are distinct and possibly previously unrecognized enzymes.     

Identification of epidermal growth factor Thr-669 phosphorylation site peptide kinases as distinct MAP kinases and p34cdc2.

A synthetic peptide modeled after the major threonine (T669) phosphorylation site of the epidermal growth factor (EGF) receptor was an efficient substrate (apparent Km approximately 0.45 mM) for phosphorylation by purified p44mpk, a MAP kinase from sea star oocytes. The peptide was also phosphorylated by a related human MAP kinase, which was identified by immunological criteria as p42mapk. Within 5 min of treatment of human cervical carcinoma A431 cells with EGF or phorbol myristate acetate (PMA), a greater than 3-fold activation of p42mapk was measured. However, Mono Q chromatography of A431 cells extracts afforded the resolution of at least three additional T669 peptide kinases, some of which may be new members of the MAP kinase family. One of these (peak I), which weakly adsorbed to Mono Q, phosphorylated myelin basic protein (MBP) and other MAP kinase substrates, immunoreacted as a 42 kDa protein on Western blots with four different MAP kinase antibodies, and behaved as a approximately 45 kDa protein upon Superose 6 gel filtration. Another T669 peptide kinase (peak IV), which bound more tightly to Mono Q than p42mapk (peak II), exhibited a nearly identical substrate specificity profile to that of p42mapk, but it immunoreacted as a 40 kDa protein only with anti-p44mpk antibody on Western blots, and eluted from Superose 6 in a high molecular mass complex of greater than 400 kDa. By immunological criteria, the T669 peptide kinase in Mono Q peak III was tentatively identified as an active form of p34cdc2 associated with cyclin A. The Mono Q peaks III and IV kinases were modestly stimulated following either EGF or PMA treatments of A431 cells, and they exhibited a greater T669 peptide/MBP ratio than p42mapk. These findings indicated that multiple proline-directed kinases may mediate phosphorylation of the EGF receptor.     

Substrate discrimination among mitogen-activated protein kinases through distinct docking sequence motifs

Mitogen-activated protein kinases (MAPKs) mediate cellular responses to a wide variety of extracellular stimuli. MAPK signal transduction cascades are tightly regulated, and individual MAPKs display exquisite specificity in recognition of their target substrates. All MAPK family members share a common phosphorylation site motif, raising questions as to how substrate specificity is achieved. Here we describe a peptide library screen to identify sequence requirements of the DEF site (docking site for ERK FXF), a docking motif separate from the phosphorylation site. We show that MAPK isoforms recognize DEF sites with unique sequences and identify two key residues on the MAPK that largely dictate sequence specificity. Based on these observations and computational docking studies, we propose a revised model for MAPK interaction with substrates containing DEF sites. Variations in DEF site sequence requirements provide one possible mechanism for encoding complex target specificity among MAPK isoforms.     

Ca2+/calmodulin-dependent microtubule-associated protein 2 kinase: broad substrate specificity and multifunctional potential in diverse tissues

In previous studies, we described a soluble Ca2+/calmodulin-dependent protein kinase which is the major Ca2+/calmodulin-dependent microtubule-associated protein 2 (MAP-2) kinase in rat brain [Schulman, H. (1984) J. Cell Biol. 99, 11-19; Kuret, J. A., & Schulman, H. (1984) Biochemistry 23, 5495-5504]. We now demonstrate that this protein kinase has broad substrate specificity. Consistent with a multifunctional role in cellular physiology, we show that in vitro the enzyme can phosphorylate numerous substrates of both neuronal and nonneuronal origin including vimentin, ribosomal protein S6, synapsin I, glycogen synthase, and myosin light chains. We have used MAP-2 to purify the enzyme from rat lung and show that the brain and lung kinases have nearly indistinguishable physical and biochemical properties. A Ca2+/calmodulin-dependent protein kinase was also detected in rat heart, rat spleen, and in the ring ganglia of the marine mollusk Aplysia californica. Partially purified MAP-2 kinase from each of these three sources displayed endogenous phosphorylation of a 54 000-dalton protein. Phosphopeptide analysis reveals a striking homology between this phosphoprotein and the 53 000-dalton autophosphorylated subunit of the major rat brain Ca2+/calmodulin-dependent protein kinase. The enzymes phosphorylated MAP-2, synapsin I, and vimentin at peptides that are identical with those phosphorylated by the rat brain kinase. This enzyme may be a multifunctional Ca2+/calmodulin-dependent protein kinase with a widespread distribution in nature which mediates some of the effects of Ca2+ on microtubules, intermediate filaments, and other cellular constituents in brain and other tissues.     

The structural basis for specificity of substrate and recruitment peptides for cyclin-dependent kinases

Progression through the eukaryotic cell cycle is driven by the orderly activation of cyclin-dependent kinases (CDKs). For activity, CDKs require association with a cyclin and phosphorylation by a separate protein kinase at a conserved threonine residue (T160 in CDK2). Here we present the structure of a complex consisting of phosphorylated CDK2 and cyclin A together with an optimal peptide substrate, HHASPRK. This structure provides an explanation for the specificity of CDK2 towards the proline that follows the phosphorylatable serine of the substrate peptide, and the requirement for the basic residue in the P+3 position of the substrate. We also present the structure of phosphorylated CDK2 plus cyclin A3 in complex with residues 658-668 from the CDK2 substrate p107. These residues include the RXL motif required to target p107 to cyclins. This structure explains the specificity of the RXL motif for cyclins.     

Protein kinases display minimal interpositional dependence on substrate sequence: potential implications for the evolution of signalling networks

Characterization of in vitro substrates of protein kinases by peptide library screening provides a wealth of information on the substrate specificity of kinases for amino acids at particular positions relative to the site of phosphorylation, but provides no information concerning interdependence among positions. High-throughput techniques have recently made it feasible to identify large numbers of in vivo kinase substrates. We used data from experiments on the kinases ATM/ATR and CDK1, and curated CK2 substrates to evaluate the prevalence of interactions between substrate positions within a motif and the utility of these interactions in predicting kinase substrates. Among these data, evidence of interpositional sequence dependencies is strikingly rare, and what dependency exists does little to aid in the prediction of novel kinase substrates. Significant increases in the ability of models to predict kinase-substrate specificity beyond position-independent models must come largely from inclusion of elements of biological and cellular context, rather than further analysis of substrate sequences alone. Our results suggest that, evolutionarily, kinase substrate fitness exists in a smooth energetic landscape. Taken with results from others indicating that phosphopeptide-binding domains do exhibit interpositional dependence, our data suggest that incorporation of new substrate molecules into phospho-signalling networks may be rate-limited by the evolution of suitability for binding by phosphopeptide-binding domains.     

Identification of the ATP·Mg-dependent protein phosphatase activator (Fa) as a synapsin I kinase that inhibits cross-linking of synapsin I with brain microtubules

The ATP·Mg-dependent protein phosphatase activating factor (Fa) has been identified and purified to near homogeneity from brain. In this report, as evidenced on SDS-polyacrylamide gel electrophoresis followed by autoradiography, factorFa has further been identified as a cAMP and Ca²⁺-independent brain kinase that could phosphorylate synapsin I, a neuronal protein that coats synaptic vesicles, binds to cytoskeleton, and is believed to be involved in the modulation of neurotransmission. Kinetic study further indicated that factorFa could phosphorylate synapsin I with a lowK _m value of about 2 µM and with a molar ratio of 1 mol of phosphate per mole of protein. Peptide mapping analysis revealed that factorFa specifically phosphorylated the tail region of synapsin I but on a unique site distinct from those phosphorylated by Ca²⁺/calmodulin-dependent protein kinase II and cAMP-dependent protein kinase, the two well-established synapsin I kinases. Functional study further revealed that factorFa could phosphorylate this unique specific site on the tail region of synapsin I and thereby inhibit cross-linking of synapsin I with microtubules. The results further suggest the possible involvement of factorFa as a synapsin I kinase in the regulation of axonal transport process of synaptic vesicles via the promotion of vesicles motility during neurotransmission.     

Identification by mass spectrometry of threonine 97 in bovine myelin basic protein as a specific phosphorylation site for mitogen-activated protein kinase.

Bovine myelin basic protein (MBP) was found to be an excellent in vitro substrate (apparent Km = 50 microM) for MAP (mitogen-activated protein) kinase and can be used in lieu of microtubule-associated protein 2 for purification and functional studies of the enzyme. MBP phosphotransferase activity co-purified with MAP kinase during sequential DE52, phenyl-Superose, and gel filtration chromatography, and kinase activities for the two substrates were co-regulated by mitogen stimulation. MAP kinase phosphorylated MBP exclusively on threonine, and only one major phosphopeptide was generated by digestion with trypsin or endoproteinase Lys-C. Using mass spectrometry, we determined that the phosphorylation site is threonine 97, present in the conserved triproline loop of MBP, with (partial) sequence -Thr-Pro-Arg-Thr97-Pro-Pro-Pro-. Thr97 is a known in vivo phosphorylation site in MBP although enzymes capable of phosphorylating this site have not been identified previously. MAP kinase phosphorylated peptide 88-109 from rabbit MBP and a synthetic peptide 91-109 from human MBP but did not phosphorylate either the histone H1 peptide, utilized by p34cdc2, or the peptide substrate for the recently described proline-directed kinase. Thus, the sequence surrounding threonine 97 in bovine MBP may contain essential features of a recognition sequence for MAP kinase.     

Purification and characterization of Ca2+/calmodulin-dependent protein kinase I from bovine brain

Ca2+/calmodulin-dependent protein kinase (Ca2+/CaM kinase I), which phosphorylates site I of synapsin I, has been highly purified from bovine brain. The physical properties and substrate specificity of Ca2+/CaM kinase I were distinct from those of all other known Ca2+/CaM kinases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified enzyme preparation consisted of two major polypeptides of Mr 37,000 and 39,000 and a minor polypeptide of Mr 42,000. In the presence of Ca2+ and calmodulin (CaM), all three polypeptides bound CaM, were autophosphorylated on threonine residues, and were labeled by the photoaffinity label 8-azido-ATP. Peptide maps of the three autophosphorylated polypeptides were very similar. The Stokes radius and the sedimentation coefficient of the enzyme were, respectively, 31.8 A and 3.25 s. A molecular weight of 42,400 and a frictional ratio of 1.38 were calculated from the above values, suggesting that Ca2+/CaM kinase I is a monomer. It is possible that the polypeptides of lower molecular weight are derived from the polypeptide of Mr 42,000 by proteolysis; alternatively, the polypeptides may represent isozymes of Ca2+/CaM kinase I. Synapsin I (site I) was the best substrate tested (Km, 2-4 microM) for Ca2+/CaM kinase I. Of many additional proteins tested, only protein III (a phosphoprotein related to synapsin I) and smooth muscle myosin light chain were phosphorylated. Ca2+/CaM kinase I was found in highest concentration in brain, where it showed widespread regional and subcellular distributions. In addition, the enzyme had a widespread and predominantly cytosolic tissue distribution. The widespread neuronal and tissue distribution of Ca2+/CaM kinase I suggests that other substrates might exist for this enzyme in both neuronal and non-neuronal tissues.