Phosphorylation of the yeast choline kinase by protein kinase C. Identification of Ser25 and Ser30 as major sites of phosphorylation

The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent and dependent on the concentrations of choline kinase (K(m) = 27 microg/ml) and ATP (K(m) = 15 microM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSSQRRHS (V5max/K(m) = 17.5 mm(-1) micromol min(-1) mg(-1)) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway, whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Although the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHSLTRQ) containing Ser30 was a substrate (V(max)/K(m) = 3.0 mm(-1) micromol min(-1) mg(-1)) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C.     

Differential phosphorylation of SNAP-25 in vivo by protein kinase C and protein kinase A

SNAP-25 is a key protein required for the fusion of synaptic vesicles with the plasma membrane during exocytosis. This study establishes that SNAP-25 is differentially phosphorylated by protein kinase C and protein kinase A in neuroendocrine PC12 cells. Using phosphopeptide mapping and site-directed mutagenesis we identified both Thr138 and Ser187 as the targets of SNAP-25 phosphorylation by protein kinase C and Thr138 as the exclusive site of SNAP-25 phosphorylation by protein kinase A in vivo. Finally, despite published data to the contrary, we demonstrate that stimulation of regulated exocytosis under physiological conditions is independent of a measurable increase in SNAP-25 phosphorylation in PC12 cells.     

Tau protein kinase I converts normal tau protein into A68-like component of paired helical filaments.

From bovine brain microtubules we purified tau protein kinase I (TPKI, Mr 45,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and tau protein kinase II (TPKII) whose activity was attributed to a 30-kDa protein on SDS-PAGE by affinity-labeling using an ATP analog. Both kinases were activated by tubulin. TPKII, but not TPKI, phosphorylated tau fragment peptides previously used for detection of a Ser/ThrPro kinase activity. Therefore, TPKII was considered to be the Ser/ThrPro kinase. TPKI was more effective than TPKII for producing the decrease of tau-1 immunoreactivity and mobility shift of tau on SDS-PAGE. Moreover, TPKI, but not TPKII nor other well-known protein kinases, generated an epitope present on paired helical filaments. These findings suggested that tau phosphorylated by TPKI resembled A-68, a component of paired helical filaments.     

Neuronal Cdc2-like protein kinase (Cdk5/p25) is associated with protein phosphatase 1 and phosphorylates inhibitor-2

Protein phosphatase 1 (PP1) is complexed with inhibitor 2 (I-2) in the cytosol. In rabbit muscle extract PP1.I-2 is activated upon preincubation with ATP/Mg. This activation is caused by phosphorylation of I-2 on Thr(72) by glycogen synthase kinase 3 (GSK3). We have found that PP1.I-2 in bovine brain extract is also activated upon preincubation with ATP/Mg. However, blocking GSK3 action by LiCl inhibited only approximately 29% of PP1 activity and indicated that GSK3 is not the sole PP1.I-2 activator in the brain. When bovine brain extract was analyzed by gel filtration PP1.I-2 and neuronal Cdc2-like protein kinase (NCLK), a heterodimer of Cdk5 and the regulatory p25 subunit, co-eluted as a approximately 450-kDa size species. The NCLK from the eluted column fractions bound to PP1-specific microcystin-Sepharose and glutathione S-transferase (GST)-I-2-coated glutathione-agarose beads. Similarly, PP1 from the eluted column fractions was pulled down with GST-Cdk5-coated glutathione-agarose beads. In vitro, NCLK phosphorylated I-2 on Thr(72) and activated PP1.I-2 in an ATP/Mg-dependent manner. NCLK bound to PP1 through its Cdk5 subunit and the PP1 binding region was localized to Cdk5 residues 28-41. Our data demonstrate that in brain extract PP1.I-2 and NCLK are associated within a complex of approximately 450 kDa and suggest that NCLK is one of the PP1.I-2-activating kinases in the mammalian brain.     

Phosphorylation of protein phosphatase inhibitor-1 by protein kinase C

Inhibitor-1 becomes a potent inhibitor of protein phosphatase 1 when phosphorylated by cAMP-dependent protein kinase at Thr(35). Moreover, Ser(67) of inhibitor-1 serves as a substrate for cyclin-dependent kinase 5 in the brain. Here, we report that dephosphoinhibitor-1 but not phospho-Ser(67) inhibitor-1 was efficiently phosphorylated by protein kinase C at Ser(65) in vitro. In contrast, Ser(67) phosphorylation by cyclin-dependent kinase 5 was unaffected by phospho-Ser(65). Protein kinase C activation in striatal tissue resulted in the concomitant phosphorylation of inhibitor-1 at Ser(65) and Ser(67), but not Ser(65) alone. Selective pharmacological inhibition of protein phosphatase activity suggested that phospho-Ser(65) inhibitor-1 is dephosphorylated by protein phosphatase 1 in the striatum. In vitro studies confirmed these findings and suggested that phospho-Ser(67) protects phospho-Ser(65) inhibitor-1 from dephosphorylation by protein phosphatase 1 in vivo. Activation of group I metabotropic glutamate receptors resulted in the up-regulation of diphospho-Ser(65)/Ser(67) inhibitor-1 in this tissue. In contrast, the activation of N-methyl-d-aspartate-type ionotropic glutamate receptors opposed increases in striatal diphospho-Ser(65)/Ser(67) inhibitor-1 levels. Phosphomimetic mutation of Ser(65) and/or Ser(67) did not convert inhibitor-1 into a protein phosphatase 1 inhibitor. On the other hand, in vitro and in vivo studies suggested that diphospho-Ser(65)/Ser(67) inhibitor-1 is a poor substrate for cAMP-dependent protein kinase. These observations extend earlier studies regarding the function of phospho-Ser(67) and underscore the possibility that phosphorylation in this region of inhibitor-1 by multiple protein kinases may serve as an integrative signaling mechanism that governs the responsiveness of inhibitor-1 to cAMP-dependent protein kinase activation.     

Phosphorylation of human high mobility group N1 protein by protein kinase CK2

High mobility group (HMG) N1 protein, formerly known as HMG 14, is a member of the chromosomal HMG protein family. Protein kinase CK2 was previously reported to be able to phosphorylate bovine HMGN1 in vitro; Ser89 and Ser99, corresponding to Ser88 and Ser98 in human HMGN1, were shown to be major and minor recognition sites, respectively. In this report, we employed mass spectrometry and examined both the extent and the sites of phosphorylation in HMGN1 protein catalyzed by recombinant human protein kinase CK2. We found that five serine residues, i.e., Ser6, Ser7, Ser85, Ser88, and Ser98, in HMGN1 can be phosphorylated by the kinase in vitro. All five sites were previously shown to be phosphorylated in MCF-7 human breast cancer cells in vivo. Among these five sites, Ser6, Ser7, and Ser85 were new sites of phosphorylation induced by protein kinase CK2 in vitro.     

Sites of phosphorylation by protein kinase A in CDC25Mm/GRF1, a guanine nucleotide exchange factor for Ras

Activation of the neuronal Ras GDP/GTP exchange factor (GEF) CDC25Mm/GRF1 is known to be associated with phosphorylation of serine/threonine. To increase our knowledge of the mechanism involved, we have analyzed the ability of several serine/threonine kinases to phosphorylate CDC25Mm in vivo and in vitro. We could demonstrate the involvement of cAMP-dependent protein kinase (PKA) in the phosphorylation of CDC25Mm in fibroblasts overexpressing this RasGEF as well as in mouse brain synaptosomal membranes. In vitro, PKA was found to phosphorylate multiple sites on purified CDC25Mm, in contrast to protein kinase C, calmodulin kinase II, and casein kinase II, which were virtually inactive. Eight phosphorylated serines and one threonine were identified by mass spectrometry and Edman degradation. Most of them were clustered around the Ras exchanger motif/PEST motifs situated in the C-terminal moiety (residues 631-978) preceding the catalytic domain. Ser745 and Ser822 were the most heavily phosphorylated residues and the only ones coinciding with PKA consensus sequences. Substitutions S745D and S822D showed that the latter mutation strongly inhibited the exchange activity of CDC25Mm on Ha-Ras. The multiple PKA-dependent phosphorylation sites on CDC25Mm suggest a complex regulatory picture of this RasGEF. The results are discussed in the light of structural and/or functional similarities with other members of this RasGEF family.     

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

We previously reported that rat brain Ca(2+)/calmodulin-dependent protein kinase (CaM-kinase) IV is inactivated by cAMP-dependent protein kinase (PKA) [Kameshita, I. and Fujisawa, H. (1991) Biochem. Biophys. Res. Commun. 180, 191-196]. In the preceding paper, we demonstrated that changes in the activity of CaM-kinase IV by PKA results from the phosphorylation of CaM-kinase kinase alpha by PKA and identified six phosphorylation sites, Ser(24) for autophosphorylation, and Ser(52), Ser(74), Thr(108), Ser(458), and Ser(475) for phosphorylation by PKA. In the present study, a causal relationship between the phosphorylation and change in the activity toward PKIV peptide has been studied using mutant enzymes with amino acid substitutions at the six phosphorylation sites. The following conclusions can be drawn from the experimental results: (i) Phosphorylation of Ser74 and/or unidentified sites causes an increase in activity; (ii) phosphorylation of Thr(108) or Ser(458) causes a decrease in the activity; (iii) the inhibitory effect of the phosphorylation of Thr(108) is canceled by the stimulatory effect of the phosphorylation, but that of Ser(458) is not; and (iv) the inhibitory effects of Thr(108) and Ser(458) are synergistic. In contrast to the activity toward PKIV peptide, the activity toward CaM-kinase IV appears to be decreased by the phosphorylation of Thr(108), but not significantly affected by the phosphorylation of Ser(458).     

Mitogen activated protein (MAP) kinase transforms tau protein into an Alzheimer-like state.

The microtubule-associated protein tau is a major component of the paired helical filaments (PHFs) observed in Alzheimer's disease brains. The pathological tau is distinguished from normal tau by its state of phosphorylation, higher apparent M(r) and reaction with certain antibodies. However, the protein kinase(s) have not been characterized so far. Here we describe a protein kinase from brain which specifically induces the Alzheimer-like state in tau protein. The 42 kDa protein belongs to the family of mitogen activated protein kinases (MAPKs) and is activated by tyrosine phosphorylation. It is capable of phosphorylating Ser-Pro and Thr-Pro motifs in tau protein (approximately 14-16 P1 per tau molecule). By contrast, other proline directed Ser/Thr kinases such as p34(cdc2) combined with cyclin A or B have only minor effects on tau phosphorylation. We propose that MAP kinase is abnormally active in Alzheimer brain tissue, or that the corresponding phosphatases are abnormally passive, due to a breakdown of the normal regulatory mechanisms.     

Rabphilin-3A, a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotagmin.

In a previous study (H. Shirataki, K. Kaibuchi, T. Yamaguchi, K. Wada, H. Horiuchi, and Y. Takai, J. Biol. Chem. 267:10946-10949, 1992), we highly purified from bovine brain crude membranes the putative target protein for smg p25A/rab3A p25, a ras p21-related small GTP-binding protein implicated in neurotransmitter release. In this study, we have isolated and sequenced the cDNA of this protein from a bovine brain cDNA library. The cDNA had an open reading frame encoding a protein of 704 amino acids with a calculated M(r) of 77,976. We tentatively refer to this protein as rabphilin-3A. Structural analysis of rabphilin-3A revealed the existence of two copies of an internal repeat that were homologous to the C2 domain of protein kinase C as described for synaptotagmin, which is known to be localized in the membrane of the synaptic vesicle and to bind to membrane phospholipid in a Ca(2+)-dependent manner. The isolated cDNA was expressed in COS7 cells, and the encoded protein was recognized with an anti-rabphilin-3A polyclonal antibody and was identical in size with rabphilin-3A purified from bovine brain by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Moreover, both rabphilin-3A purified from bovine brain and recombinant rabphilin-3A made a complex with the GTP gamma S-bound form of rab3A p25 but not with the GDP-bound form of rab3A p25. Immunoblot and Northern (RNA) blot analyses showed that rabphilin-3A was highly expressed in bovine and rat brains. These results indicate that rabphilin-3A is a novel protein that has C2 domains and selectively interacts with the GTP-bound form of rab3A p25.     

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.     

Non-requirement of calcium on protamine phosphorylation by calcium-activated, phospholipid-dependent protein kinase

Phosphorylation of clupeine sulfate by purified rat brain calcium-activated, phospholipid-dependent protein kinase (protein kinase C) was studied. In the absence of Ca2+, phosphatidylserine and diolein markedly stimulated its phosphorylation. However Ca2+ did not stimulate but inhibit this phosphorylation about 30% in the presence of phospholipids. Random polymer (Arg, Ser) 3:1 and (Lys, Ser) 3:1 could be phosphorylated by protein kinase C. In the presence of phospholipids Ca2+ is not needed for the phosphorylation of polymer (Arg, Ser) 3:1, while Ca2+ is necessary for polymer (Lys, Ser) 3:1. Non-requirement of Ca2+ on clupeine phosphorylation by protein kinase C is briefly discussed.     

Comparison of phosphorylation sites in protamines between protein kinase C and cAMP-dependent protein kinase

Phosphorylation sites of protamines by protein kinase C and cAMP-dependent protein kinase (protein kinase A) were studied. Using clupeine Y1 as a substrate, protein kinase C phosphorylates both Ser and Thr residues, whereas protein kinase A phosphorylates only Ser residue(s). Protein kinase C phosphorylates all Ser and Thr residues of clupeine Y2 and Z, however protein kinase A phosphorylates mainly Ser9 and slightly Thr5 in clupeine Y2 and Ser6 and Ser10 in clupeine Z. These results suggest that protein kinase C recognizes more sites than those of protein kinase A and may participate in protamine phosphorylation in vivo.     

Synthetic myelin basic protein peptide analogs are specific inhibitors of phospholipid/calcium-dependent protein kinase (protein kinase C)

Synthetic peptide analogs of the bovine myelin basic protein (MBP) corresponding to residues 104-118 were found to specifically inhibit phospholipid/ Ca2+-dependent protein kinase (protein kinase C). The peptides [Ala107]MBP (104-118) and [Ala113]MBP (104-118) inhibited protein phosphorylation of intact MBP, histone H1 and peptide phosphorylation with MBP(104-123), MBP(104-118) or [Ala105]MBP (104-118) as substrates. The inhibitor peptides [Ala107]MBP(104-118) and [Ala113]MBP (104-118), containing alanine in place of the arginine recognition sites, apparently inhibited the enzyme noncompetitively with respect to substrates, with IC50 values ranging from 46-145 and 28-62 microM, respectively. These peptide analogs did not inhibit cyclic AMP-dependent protein kinase or myosin light chain kinase but inhibited phospholipid/Ca2+-dependent phosphorylation of endogenous proteins in the total, solubilized fraction of rat brain.     

Identification of type III protein kinase C in bovine aortic tissue

We identified a subtype of protein kinase C in bovine aortic tissue. In Western blots, both the soluble and the particulate fractions from the aorta reacted only with MC-3a. In the case of hydroxylapatite column chromatography, a single activity peak of protein kinase C from the soluble and the particulate fractions was obtained with about 140 mM of potassium phosphate, a finding similar to that with the Type III protein kinase C from rabbit brain. The sandwich-type enzyme immunoassay for protein kinase C, with which the contents of each protein kinase C isozyme can be determined in the crude extracts, revealed that the Type III bovine aortic protein kinase C included 25.9 ng/mg protein. These results strongly suggest that it is the Type III protein kinase C which is mainly expressed in aortic tissue. Kinetic parameters of the Type III protein kinase C of the soluble and the particulate fractions, with respect to the Km for ATP, were 33 and 15 microM and the Km values for myosin light chain from chicken gizzard were 6.3 and 4.6 microM, respectively.     

A novel method to identify protein kinase substrates: eEF2 kinase is phosphorylated and inhibited by SAPK4/p38delta

We have developed a method of general application for identifying putative substrates of protein kinases in cell extracts. Using this procedure, we identified the physiological substrates of several mitogen-activated protein kinase kinases and an authentic substrate of stress-activated protein kinase (SAPK) 2a/p38. A 120 kDa protein was detected in skeletal muscle extracts that was phosphorylated rapidly by SAPK4/p38delta, but poorly by SAPK2/p38, SAPK3/p38gamma, SAPK1/JNK or extracellular signal-regulated kinase 2 (ERK2). It was purified and identified as eukaryotic elongation factor 2 kinase (eEF2K). SAPK4/p38delta phosphorylated eEF2K at Ser359 in vitro, causing its inactivation. eEF2K became phosphorylated at Ser359 and its substrate eEF2 became dephosphorylated (activated) when KB cells were exposed to anisomycin, an agonist that activates all SAPKs, including SAPK4/p38delta. The anisomycin-induced phosphorylation of Ser359 was unaffected by SB 203580, U0126 or rapamycin, and was prevented by overexpression of a catalytically inactive SAPK4/p38delta mutant, suggesting that SAPK4/p38delta may mediate the inhibition of eEF2K by this stress. The phosphorylation of eEF2K at Ser359 was also induced by insulin-like growth factor-1. However, this was blocked by rapamycin, indicating that Ser359 is targeted by at least two signalling pathways.     

A single amino acid mutation in SNAP-25 induces anxiety-related behavior in mouse

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a presynaptic protein essential for neurotransmitter release. Previously, we demonstrate that protein kinase C (PKC) phosphorylates Ser(187) of SNAP-25, and enhances neurotransmitter release by recruiting secretory vesicles near to the plasma membrane. As PKC is abundant in the brain and SNAP-25 is essential for synaptic transmission, SNAP-25 phosphorylation is likely to play a crucial role in the central nervous system. We therefore generated a mutant mouse, substituting Ser(187) of SNAP-25 with Ala using "knock-in" technology. The most striking effect of the mutation was observed in their behavior. The homozygous mutant mice froze readily in response to environmental change, and showed strong anxiety-related behavior in general activity and light and dark preference tests. In addition, the mutant mice sometimes exhibited spontaneously occurring convulsive seizures. Microdialysis measurements revealed that serotonin and dopamine release were markedly reduced in amygdala. These results clearly indicate that PKC-dependent SNAP-25 phosphorylation plays a critical role in the regulation of emotional behavior as well as the suppression of epileptic seizures, and the lack of enhancement of monoamine release is one of the possible mechanisms underlying these defects.     

Calpastatin, an endogenous calpain-inhibitor protein, regulates the cleavage of the Cdk5 activator p35 to p25

Cyclin-dependent kinase 5 (Cdk5) is a Ser/Thr kinase that is activated by binding to its regulatory subunit, p35. The calpain-mediated cleavage of p35 to p25 and the resulting aberrant activity and neurotoxicity of Cdk5 have been implicated in neurological disorders, such as Alzheimer's disease. To gain further insight into the molecular mechanisms underlying the pathological function of Cdk5, we investigated the role of the calpain inhibitor protein calpastatin (CAST), in controlling the aberrant production of p25. For this purpose, brain tissue from wild-type, CAST-over-expressing (transgenic), and CAST knockout mice were analyzed. Cleavage of p35 to p25 was increased in extracts from CAST knockout mice, compared with wild-type. Conversely, generation of p25 was not detected in brain lysates from CAST-over-expressing mice. CAST expression was 5-fold higher in mouse cerebellum than cerebral cortex. Accordingly, p25 production was lower in the cerebellum than the cerebral cortex. Furthermore, the Ca(2+) -dependent degradation of p35 by proteasome was evident when calpain was inhibited. Taken together, these results suggest that CAST is a crucial regulator of calpain activity, the production of p25, and, hence, the deregulation of Cdk5. Therefore, impairment of CAST expression and its associated mechanisms may contribute to the pathogenesis of neurodegenerative disorders.     

Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins.

MAP kinase-activated protein kinase-2 (MAPKAP kinase-2) phosphorylates the serine residues in murine heat shock protein 25 (hsp25) and human heat shock protein 27 (hsp27) which are phosphorylated in vivo in response to growth factors and heat shock, namely Ser15 and Ser86 (hsp25) and Ser15, Ser78 and Ser82 (hsp27). Ser86 of hsp25 and the equivalent residue in hsp27 (Ser82) are phosphorylated preferentially in vitro. The small heat shock protein is present in rabbit skeletal muscle and hsp25 kinase activity in skeletal muscle extracts co-purifies with MAPKAP kinase-2 activity throughout the purification of the latter enzyme. These results suggest that MAPKAP kinase-2 is the enzyme responsible for the phosphorylation of these small heat shock proteins in mammalian cells.     

Phosphorylation blocks the activity of tubulin polymerization-promoting protein (TPPP): identification of sites targeted by different kinases

Tubulin polymerization-promoting protein (TPPP), an unfolded brain-specific protein interacts with the tubulin/microtubule system in vitro and in vivo, and is enriched in human pathological brain inclusions. Here we show that TPPP induces tubulin self-assembly into intact frequently bundled microtubules, and that the phosphorylation of specific sites distinctly affects the function of TPPP. In vitro phosphorylation of wild type and the truncated form (Delta3-43TPPP) of human recombinant TPPP was performed by kinases involved in brain-specific processes. A stoichiometry of 2.9 +/- 0.3, 2.2 +/- 0.3, and 0.9 +/- 0.1 mol P/mol protein with ERK2, cyclin-dependent kinase 5 (Cdk5), and cAMP-dependent protein kinase (PKA), respectively, was revealed for the full-length protein, and 0.4-0.5 mol P/mol protein was detected with all three kinases when the N-terminal tail was deleted. The phosphorylation sites Thr(14), Ser(18), Ser(160) for Cdk5; Ser(18), Ser(160) for ERK2, and Ser(32) for PKA were identified by mass spectrometry. These sites were consistent with the bioinformatic predictions. The three N-terminal sites were also found to be phosphorylated in vivo in TPPP isolated from bovine brain. Affinity binding experiments provided evidence for the direct interaction between TPPP and ERK2. The phosphorylation of TPPP by ERK2 or Cdk5, but not by PKA, perturbed the structural alterations induced by the interaction between TPPP and tubulin without affecting the binding affinity (K(d) = 2.5-2.7 microM) or the stoichiometry (1 mol TPPP/mol tubulin) of the complex. The phosphorylation by ERK2 or Cdk5 resulted in the loss of microtubule-assembling activity of TPPP. The combination of our in vitro and in vivo data suggests that ERK2 can regulate TPPP activity via the phosphorylation of Thr(14) and/or Ser(18) in its unfolded N-terminal tail.