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.     

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.     

Regulation of NDR protein kinase by hydrophobic motif phosphorylation mediated by the mammalian Ste20-like kinase MST3

NDR protein kinases are involved in the regulation of cell cycle progression and morphology. NDR1/NDR2 protein kinase is activated by phosphorylation on the activation loop phosphorylation site Ser281/Ser282 and the hydrophobic motif phosphorylation site Thr444/Thr442. Autophosphorylation of NDR is responsible for phosphorylation on Ser281/Ser282, whereas Thr444/Thr442 is targeted by an upstream kinase. Here we show that MST3, a mammalian Ste20-like protein kinase, is able to phosphorylate NDR protein kinase at Thr444/Thr442. In vitro, MST3 selectively phosphorylated Thr442 of NDR2, resulting in a 10-fold stimulation of NDR activity. MOB1A (Mps one binder 1A) protein further increased the activity, leading to a fully active kinase. In vivo, Thr442 phosphorylation after okadaic acid stimulation was potently inhibited by MST3KR, a kinase-dead mutant of MST3. Knockdown of MST3 using short hairpin constructs abolished Thr442 hydrophobic motif phosphorylation of NDR in HEK293F cells. We conclude that activation of NDR is a multistep process involving phosphorylation of the hydrophobic motif site Thr444/2 by MST3, autophosphorylation of Ser281/2, and binding of MOB1A.     

An array of insulin-activated, proline-directed serine/threonine protein kinases phosphorylate the p70 S6 kinase.

This study characterizes the insulin-activated serine/threonine protein kinases in H4 hepatoma cells active on a 37-residue synthetic peptide (called the SKAIPS peptide) corresponding to a putative autoinhibitory domain in the carboxyl-terminal tail of the p70 S6 kinase as well as on recombinant p70 S6 kinase. Three peaks of insulin-stimulated protein kinase active on both these substrates are identified as two (possibly three) isoforms of the 40-45-kDa erk/microtubule-associated protein (MAP)-2 kinase family and a 150-kDa form of cdc2. Although distinguishable in their substrate specificity, these protein kinases together with the p54 MAP-2 kinase share a major common specificity determinant reflected in the SKAIPS peptide: the requirement for a proline residue immediately carboxyl-terminal to the site of Ser/Thr phosphorylation. In addition, however, at least one peak of insulin-stimulated protein kinase active on recombinant p70, but not on the SKAIPS peptide, is present although not yet identified. MFP/cdc2 phosphorylates both rat liver p70 S6 kinase and recombinant p70 S6 kinase exclusively at a set of Ser/Thr residues within the putative autoinhibitory (SKAIPS peptide) domain. erk/MAP kinase does not phosphorylate rat liver p70 S6 kinase, but readily phosphorylates recombinant p70 S6 kinase at sites both within and in addition to those encompassed by the SKAIPS peptide sequences. Although the tryptic 32P-peptides bearing the cdc2 and erk/MAP kinase phosphorylation sites co-migrate with a subset of the sites phosphorylated in situ in insulin-stimulated cells, phosphorylation of the p70 S6 kinase by these proline-directed protein kinases in vitro does not reproducibly activate p70 S6 kinase activity. Thus, one or more erk/MAP kinases and cdc2 are likely to participate in the insulin-induced phosphorylation of the p70 S6 kinase. In addition to these kinases, however, phosphorylation of the p70 S6 kinase by other as yet unidentified protein kinases is necessary to recapitulate the multisite phosphorylation required for activation of the p70 S6 kinase.     

Recombinant rabbit muscle casein kinase I alpha is inhibited by heparin and activated by polylysine.

The casein kinase I (CKI) family consists of widely distributed monomeric Ser/Thr protein kinases that have a preference for acidic substrates. Four mammalian isoforms are known. A full length cDNA encoding the CKI alpha isoform was cloned from a rabbit skeletal muscle cDNA library and was utilized to construct a bacterial expression vector. Active CKI alpha was expressed in Escherichia coli as a polypeptide of Mr 36,000. The protein kinase phosphorylated casein, phosvitin and a specific peptide substrate (D4). The enzyme was inhibited by the isoquinolinesulfonamide CKI-7, half-maximally at 70 microM. Heparin inhibited phosphorylation of the D4 peptide or phosvitin by CKI alpha. Polylysine activated when the D4 peptide was the substrate but had no effect on phosvitin phosphorylation. It is becoming clear that the individual CKI isoforms have different kinetic properties and hence could have quite distinct cellular functions.     

Mechanism of activation of protein kinase B by insulin and IGF-1.

Insulin activated endogenous protein kinase B alpha (also known as RAC/Akt kinase) activity 12-fold in L6 myotubes, while after transfection into 293 cells PKBalpha was activated 20- and 50-fold in response to insulin and IGF-1 respectively. In both cells, the activation of PKBalpha was accompanied by its phosphorylation at Thr308 and Ser473 and, like activation, phosphorylation of both of these residues was prevented by the phosphatidylinositol 3-kinase inhibitor wortmannin. Thr308 and/or Ser473 were mutated to Ala or Asp and activities of mutant PKBalpha molecules were analysed after transfection into 293 cells. The activity of wild-type and mutant PKBalpha was also measured in vitro after stoichiometric phosphorylation of Ser473 by MAPKAP kinase-2. These experiments demonstrated that activation of PKBalpha by insulin or insulin-like growth factor-1 (IGF-1) results from phosphorylation of both Thr308 and Ser473, that phosphorylation of both residues is critical to generate a high level of PKBalpha activity and that the phosphorylation of Thr308 in vivo is not dependent on phosphorylation of Ser473 or vice versa. We propose a model whereby PKBalpha becomes phosphorylated and activated in insulin/IGF-1-stimulated cells by an upstream kinase(s).     

Regulation of zipper-interacting protein kinase activity in vitro and in vivo by multisite phosphorylation

Zipper-interacting protein kinase (ZIPK) is a widely expressed serine/threonine kinase implicated in cell death and smooth muscle contractility, but its mechanism of regulation is unknown. We have identified six phosphorylation sites in ZIPK that regulate both its enzyme activity and localization, including Thr180, Thr225, Thr265, Thr299, Thr306, and Ser311. Mutational analysis showed that phosphorylation of Thr180 in the kinase activation T-loop, Thr225 in the substrate-binding groove, and Thr265 in kinase subdomain X is essential for full ZIPK autophosphorylation and activity toward exogenous substrates. Abrogation of phosphorylation of Thr299, Thr306, and Ser311 had little effect on enzyme activity, but mutation of Thr299 and Thr300 to alanine resulted in redistribution of ZIPK from the cytosol to the nucleus. Mutation of Thr299 alone to alanine caused ZIPK to assume a diffuse cellular localization, whereas T299D redistributed the enzyme to the cytoplasm. C-terminal truncations of ZIPK at amino acid 273 or 342 or mutation of the leucine zipper motif increased ZIPK activity toward exogenous substrates by severalfold, suggesting a phosphorylation-independent autoinhibitory role for the C-terminal domain. Additionally, mutation of the leucine zipper reduced the ability of ZIPK to oligomerize and also caused ZIPK to relocalize from the cytoplasm to the nucleus in vivo. Together, our findings show that ZIPK is positively regulated by phosphorylation within its kinase domain and that it contains an inhibitory C-terminal domain that controls enzyme activity, localization, and oligomerization.     

Phosphorylation of high-mobility-group proteins by the calcium-phospholipid-dependent protein kinase and the cyclic AMP-dependent protein kinase

Purified lamb thymus high-mobility-group (HMG) proteins 1, 2, and 17 have been investigated as potential substrates for the Ca2+-phospholipid-dependent protein kinase and the cAMP-dependent protein kinase. HMG proteins 1, 2, and 17 are phosphorylated by the Ca2+-phospholipid-dependent protein kinase; the reactions are totally Ca2+ and lipid dependent and are not inhibited by the inhibitor protein of the cAMP-dependent protein kinase. HMG 17 is phosphorylated predominantly in a single seryl residue, Ser 24 in the sequence Gln-Arg-Arg-Ser 24-Ala-Arg-Leu-Ser 28-Ala-Lys, with the second seryl moiety, Ser 28, modified to a markedly lesser degree. HMGs 1 and 2 are also phosphorylated in only seryl residues but with each there are multiple phosphorylation sites. HMG 17, but not HMG 1 or 2, is also phosphorylated by the cAMP-dependent protein kinase with the site phosphorylated being the minor of the two phosphorylated by the Ca2+-phospholipid-dependent protein kinase; the Km for phosphorylation by the cAMP-dependent enzyme is 50-fold higher than that by the Ca2+-phospholipid-dependent enzyme. HMG 17 is an equally effective substrate for the Ca2+-phospholipid-dependent protein kinase either as the pure protein or bound to nucleosomes. Preliminary evidence has indicated that lamb thymus HMG 14 is also a substrate for the Ca2+-phospholipid-dependent enzyme. It is phosphorylated with a Km similar to that of HMG 17 (4-6 microM), and a comparison of tryptic peptides suggests that it is phosphorylated in a site that is homologous with Ser 24 of HMG 17 and distinct from the sites phosphorylated by the cAMP-dependent protein kinase.     

Substrates enhance autophosphorylation and activation of p21-activated protein kinase gamma-PAK in the absence of activation loop phosphorylation.

The p21-activated protein kinase gamma-PAK from rabbit, expressed in insect cells, is activated following binding of Cdc42(GTPgammaS). The rate of autophosphorylation is increased fivefold and the protein kinase activity 13-fold, as measured with the synthetic heptapeptide (AKRESAA). The mutant K278R, where the invariant lysine in the catalytic site is replaced by arginine, shows neither autophosphorylation nor activity. Replacement of the conserved threonine in the catalytic domain with alanine (T402A) reduces autophosphorylation and protein kinase activity to 1% that of the wild-type gamma-PAK, indicating autophosphorylation of Thr402 in the activation loop is essential for protein kinase activity. In contrast, certain protein substrates such as histone 2B, histone 4 and myelin basic protein, stimulate both autophosphorylation and protein kinase activity to levels similar to those observed with Cdc42(GTPgammaS). This substrate-level activation does not require autophosphorylation of Thr402 in the activation loop. As shown with T402A, the protein kinase activity with histone 4 is similar to that observed with recombinant wild-type gamma-PAK. Basic proteins or peptides which are not substrates of gamma-PAK, such as histone 1 and polylysine, do not stimulate autophosphorylation or activity. Other substrates such as the Rous sarcoma virus protein NC are phosphorylated by gamma-PAK following activation by Cdc42(GTPgammaS), but are not phosphorylated by T402A. The data suggest that some substrates can override the requirement for Cdc42(GTPgammaS), by activating gamma-PAK directly.     

A casein kinase II-related activity is involved in phosphorylation of microtubule-associated protein MAP-1B during neuroblastoma cell differentiation

A neuroblastoma protein related to the brain microtubule-associated protein, MAP-1B, as determined by immunoprecipitation and coassembly with brain microtubules, becomes phosphorylated when N2A mouse neuroblastoma cells are induced to generate microtubule-containing neurites. To characterize the protein kinases that may be involved in this in vivo phosphorylation of MAP-1B, we have studied its in vitro phosphorylation. In brain microtubule protein, MAP-1B appears to be phosphorylated in vitro by an endogenous casein kinase II-like activity which also phosphorylates the related protein MAP-1A but scarcely phosphorylates MAP-2. A similar kinase activity has been detected in cell-free extracts of differentiating N2A cells. Using brain MAP preparations devoid of endogenous kinase activities and different purified protein kinases, we have found that MAP-1B is barely phosphorylated by cAMP-dependent protein kinase, Ca/calmodulin-dependent protein kinase, or Ca/phospholipid-dependent protein kinase whereas MAP-1B is one of the preferred substrates, together with MAP-1A, for casein kinase II. Brain MAP-1B phosphorylated in vitro by casein kinase II efficiently coassembles with microtubule proteins in the same way as in vivo phosphorylated MAP-1B from neuroblastoma cells. Furthermore, the phosphopeptide patterns of brain MAP-1B phosphorylated in vitro by either purified casein kinase II or an extract obtained from differentiating neuroblastoma cells are identical to each other and similar to that of in vivo phosphorylated neuroblastoma MAP-1B. Thus, we suggest that the observed phosphorylation of a protein identified as MAP-1B during neurite outgrowth is mainly due to the activation of a casein kinase II-related activity in differentiating neuroblastoma cells. This kinase activity, previously implicated in beta-tubulin phosphorylation (Serrano, L., J. Díaz-Nido, F. Wandosell, and J. Avila, 1987. J. Cell Biol. 105: 1731-1739), may consequently have an important role in posttranslational modifications of microtubule proteins required for neuronal differentiation.     

pp54 microtubule-associated protein-2 kinase requires both tyrosine and serine/threonine phosphorylation for activity.

pp54 microtubule-associated protein-2 (MAP-2) kinase, a recently discovered protein serine/threonine kinase (Kyriakis, J., and Avruch, J. (1990) J. Biol. Chem. 265, 17355-17363), is shown to contain immunoreactive phosphotyrosine residues. Treatment with recombinant rat brain protein tyrosine phosphatase-1 deactivates pp54 MAP-2 kinase, concomitant with the removal of phosphotyrosine residues. Protein (serine/threonine) phosphatase-1 also deactivates pp54 MAP-2 kinase in a specific fashion. pp54 MAP-2 kinase joins pp42 MAP-2 kinase and cdc2/maturation-promoting factor as one of only three serine/threonine protein kinases known to be regulated by phosphorylation at both tyrosine and, independently, at serine/threonine residues. In view of these shared regulatory properties, a role for pp54 MAP-2 kinase in the control of cell division is likely.     

Defective regulation of mitogen-activated protein kinase activity in a 3T3 cell variant mitogenically nonresponsive to tetradecanoyl phorbol acetate.

Mitogen-activated protein (MAP) kinase is a serine/threonine-specific protein kinase which is activated in response to various mitogenic agonists (e.g., epidermal growth factor, insulin, and the tumor promoter tetradecanoyl phorbol acetate [TPA]) and requires both threonine and tyrosine phosphorylation for activity. This enzyme has recently been shown to be identical or closely related to pp42, a protein which becomes tyrosine phosphorylated in response to mitogenic stimulation. Neither the kinases which regulate MAP kinase/pp42 nor the in vivo substrates for this enzyme are known. Because MAP MAP kinase is activated and phosphorylated in response both to agents which stimulate tyrosine kinase receptors and to agents which stimulate protein kinase C, a serine/threonine kinase, we have examined the regulation and phosphorylation of this enzyme in 3T3-TNR9 cells, a variant cell line partially defective in protein kinase C-mediated signalling. In this communication, we show that in the 3T3-TNR9 variant cell line, TPA does not cause the characteristically rapid phosphorylation of pp42 or the activation and phosphorylation of MAP kinase. This defective response is not due to the absence of the MAP kinase/pp42 protein itself because both tyrosine phosphorylation of MAP kinase/pp42 and its enzymatic activation could be induced by platelet-derived growth factor in the 3T3-TNR9 cells. Thus, the defect in these variant cells apparently resides in some aspect of the regulation of MAP kinase phosphorylation. Since the 3T3-TNR9 cells are also defective with respect to the TPA-induced increase in ribosomal protein S6 kinase, these in vivo results reinforce the earlier in vitro finding that MAP kinase can regulate S6 kinase activity. These findings suggest a key role for MAP kinase in a kinase cascade cascade involved in the control of cell proliferation.     

Biochemical characterization of the tobacco 42-kD protein kinase activated by osmotic stress

In tobacco (Nicotiana tabacum), hyperosmotic stress induces rapid activation of a 42-kD protein kinase, referred to as Nicotiana tabacum osmotic stress-activated protein kinase (NtOSAK). cDNA encoding the kinase was cloned and, based on the predicted amino acid sequence, the enzyme was assigned to the SNF1-related protein kinase type 2 (SnRK2) family. The identity of the enzyme was confirmed by immunoprecipitation of the active kinase from tobacco cells subjected to osmotic stress using antibodies raised against a peptide corresponding to the C-terminal sequence of the kinase predicted from the cloned cDNA. A detailed biochemical characterization of NtOSAK purified from stressed tobacco cells was performed. Our results show that NtOSAK is a calcium-independent Ser/Thr protein kinase. The sequence of putative phosphorylation sites recognized by NtOSAK, predicted by the computer program PREDIKIN, resembled the substrate consensus sequence defined for animal and yeast (Saccharomyces cerevisiae) AMPK/SNF1 kinases. Our experimental data confirmed these results, as various targets for AMPK/SNF1 kinases were also efficiently phosphorylated by NtOSAK. A range of protein kinase inhibitors was tested as potential modulators of NtOSAK, but only staurosporine, a rather nonspecific protein kinase inhibitor, was found to abolish the enzyme activity. In phosphorylation reactions, NtOSAK exhibited a preference for Mg(2+) over Mn(2+) ions and an inability to use GTP instead of ATP as a phosphate donor. The enzyme activity was not modulated by 5'-AMP. To our knowledge, these results represent the first detailed biochemical characterization of a kinase of the SnRK2 family.     

Phosphorylation of human CTP synthetase 1 by protein kinase C: identification of Ser(462) and Thr(455) as major sites of phosphorylation

Phosphorylation of human CTP synthetase 1 by mammalian protein kinase C was examined. Using purified Escherichia coli-expressed CTP synthetase 1 as a substrate, protein kinase C activity was time- and dose-dependent and dependent on the concentrations of ATP and CTP synthetase 1. The protein kinase C phosphorylation of the recombinant enzyme was accompanied by a 95-fold increase in CTP synthetase 1 activity. Phosphopeptide mapping and phosphoamino acid analyses showed that CTP synthetase 1 was phosphorylated on multiple serine and threonine residues. The induction of PKC1(R398A)-encoded protein kinase C resulted in a 50% increase for human CTP synthetase 1 phosphorylation in the Saccharomyces cerevisiae ura7Delta ura8Delta mutant lacking yeast CTP synthetase activity. Synthetic peptides that contain the protein kinase C motif for Ser(462) and Thr(455) were substrates for mammalian protein kinase C, and S462A and T455A mutations resulted in decreases in the extent of CTP synthetase 1 phosphorylation that occurred in vivo. Phosphopeptide mapping analysis of S. cerevisiae-expressed CTP synthetase 1 mutant enzymes phosphorylated with mammalian protein kinase C confirmed that Ser(462) and Thr(455) were phosphorylation sites. The S. cerevisiae-expressed and purified S462A mutant enzyme exhibited a 2-fold reduction in CTP synthetase 1 activity, whereas the purified T455A mutant enzyme exhibited a 2-fold elevation in CTP synthetase 1 activity (Choi, M.-G., and Carman, G.M. (2006) J. Biol. Chem. 282, 5367-5377). These data indicated that protein kinase C phosphorylation at Ser(462) stimulates human CTP synthetase 1 activity, whereas phosphorylation at Thr(455) inhibits activity.     

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.     

Substrate specificity of Ca(2+)/calmodulin-dependent protein kinase phosphatase: kinetic studies using synthetic phosphopeptides as model substrates

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKPase) dephosphorylates and regulates multifunctional Ca(2+)/calmodulin-dependent protein kinases. In order to elucidate the mechanism of substrate recognition by CaMKPase, we chemically synthesized a variety of phosphopeptide analogs and carried out kinetic analysis using them as CaMKPase substrates. This is the first report using systematically synthesized phosphopeptides as substrates for kinetic studies on substrate specificities of protein Ser/Thr phosphatases. CaMKPase was shown to be a protein Ser/Thr phosphatase having a strong preference for a phospho-Thr residue. A Pro residue adjacent to the dephosphorylation site on the C-terminal side and acidic clusters around the dephosphorylation site had detrimental effects on dephosphorylation by CaMKPase. Deletion analysis of a model substrate peptide revealed that the minimal length of the substrate peptide was only 2 to 3 amino acid residues including the dephosphorylation site. The residues on the C-terminal side of the dephosphorylation site were not essential for dephosphorylation, whereas the residue adjacent to the dephosphorylation site on the N-terminal side was essential. Ala-scanning analysis suggested that CaMKPase did not recognize a specific motif around the dephosphorylation site. Myosin light chain phosphorylated by protein kinase C and Erk2 phosphorylated by MEK1 were poor substrates for CaMKPase, while a synthetic phosphopeptide corresponding to the sequence around the phosphorylation site of the former was not dephosphorylated by CaMKPase but that of the latter was fairly good substrate. These data suggest that substrate specificity of CaMKPase is determined by higher-order structure of the substrate protein rather than by the primary structure around its dephosphorylation site. Use of phosphopeptide substrates also revealed that poly-L-lysine, an activator for CaMKPase, activated the enzyme mainly through increase in the V(max) values.     

Phosphorylation sites on tau identified by nanoelectrospray mass spectrometry: differences in vitro between the mitogen-activated protein kinases ERK2, c-Jun N-terminal kinase and P38, and glycogen synthase kinase-3beta

The stress-activated kinases c-Jun N-terminal kinase (JNK) and p38 are members of the mitogen-activated protein (MAP) kinase family and take part in signalling cascades initiated by various forms of stress. Their targets include the microtubule-associated protein tau, which becomes hyperphosphorylated in Alzheimer's disease. It is necessary, as a forerunner for in vivo studies, to identify the protein kinases and phosphatases that are responsible for phosphate turnover at individual sites. Using nanoelectrospray mass spectrometry, we have undertaken an extensive comparison of phosphorylation in vitro by several candidate tau kinases, namely, JNK, p38, ERK2, and glycogen synthase kinase 3beta (GSK3beta). Between 10 and 15 sites were identified for each kinase. The three MAP kinases phosphorylated Ser202 and Thr205 but not detectably Ser199, whereas conversely GSK3beta phosphorylated Ser199 but not detectably Ser202 or Thr205. Phosphorylated Ser404 was found with all of these kinases except JNK. The MAP kinases may not be strictly proline specific: p38 phosphorylated the nonproline sites Ser185, Thr245, Ser305, and Ser356, whereas ERK2 was the most strict. All of the sites detected except Thr245 and Ser305 are known or suspected phosphorylation sites in paired helical filament-tau extracted from Alzheimer brains. Thus, the three MAP kinases and GSK3beta are importantly all strong candidates as tau kinases that may be involved in the pathogenic hyperphosphorylation of tau in Alzheimer's disease.     

Tyrosine kinase activity of a Ca2+/calmodulin-dependent protein kinase II catalytic fragment

A 30-kDa fragment of Ca²⁺/calmodulin-dependent protein kinase II (30K-CaMKII) is a constitutively active protein Ser/Thr kinase devoid of autophosphorylation activity. We have produced a chimeric enzyme of 30K-CaMKII (designated CX₄₀-30K-CaMKII), in which the N-terminal 40 amino acids of Xenopus Ca²⁺/calmodulin-dependent protein kinase I (CX₄₀) were fused to the N-terminal end of 30K-CaMKII. Although CX₄₀-30K-CaMKII exhibited essentially the same substrate specificity as 30K-CaMKII, it underwent significant autophosphorylation. Surprisingly, its autophosphorylation site was found to be Tyr-18 within the N-terminal CX₄₀ region of the fusion protein, although it did not show any Tyr kinase activity toward exogenous substrates. Several lines of evidence suggested that the autophosphorylation occurred via an intramolecular mechanism. These data suggest that even typical Ser/Thr kinases such as 30K-CaMKII can phosphorylate Tyr residues under certain conditions. The possible mechanism of the Tyr residue autophosphorylation is discussed.     

Identification of casein kinase 1, casein kinase 2, and cAMP-dependent protein kinase-like activities in Trypanosoma evansi

Trypanosoma evansi contains protein kinases capable of phosphorylating endogenous substrates with apparent molecular masses in the range between 20 and 205 kDa. The major phosphopolypeptide band, pp55, was predominantly localized in the particulate fraction. Anti-alpha and anti-beta tubulin monoclonal antibodies recognized pp55 by Western blot analyses, suggesting that this band corresponds to phosphorylated tubulin. Inhibition experiments in the presence of emodin, heparin, and 2,3-bisphosphoglycerate indicated that the parasite tubulin kinase was a casein kinase 2 (CK2)-like activity. GTP, which can be utilized instead of ATP by CK2, stimulated rather than inactivated the phosphorylation of tubulin in the parasite homogenate and particulate fraction. However, GTP inhibited the cytosolic CK2 responsible for phosphorylating soluble tubulin and other soluble substrates. Casein and two selective peptide substrates, P1 (RRKDLHDDEEDEAMSITA) for casein kinase (CK1) and P2 (RRRADDSDDDDD) for CK2, were recognized as substrates in T. evansi. While the enzymes present in the soluble fraction predominantly phosphorylated P1, P2 was preferentially labeled in the particulate fractions. These results demonstrated the existence of CK1-like and CK2-like activities primarily located in the parasite cytosolic and membranous fractions, respectively. Histone II-A and kemptide (LRRASVA) also behaved as suitable substrates, implying the existence of other Ser/Thr kinases in T. evansi. Cyclic AMP only increased the phosphorylation of histone II-A and kemptide in the cytosol, demonstrating the existence of soluble cAMP-dependent protein kinase-like activities in T. evansi. However, no endogenous substrates for this enzyme were identified in this fraction. Further evidences were obtained by using PKI (6-22), a reported inhibitor of the catalytic subunit of mammalian cAMP-dependent protein kinases, which specifically hindered the cAMP-dependent phosphorylation of histone II-A and kemptide in the parasite soluble fraction. Since the sum of the values obtained in the parasite cytosolic and particulate fractions were always higher than the values observed in the total T. evansi lysate, the kinase activities examined here appeared to be inhibited in the original extract.     

Activation of Dictyostelium myosin light chain kinase A by phosphorylation of Thr166.

Phosphorylation of the regulatory light chain is an important mechanism for the activation of myosin in non-muscle cells. Unlike most myosin light chain kinases (MLCKs), MLCK-A from Dictyostelium is not activated by Ca2+/calmodulin. Autophosphorylation increases activity, but only to a low level, suggesting that there is an additional activation mechanism. Here, we show that MLCK-A is autophosphorylated on Thr289, which is C-terminal to the catalytic domain. Phosphorylation of MLCK-A increases in response to concanavalin A (conA) treatment of cells, which was previously shown to activate MLCK-A. However, a mutant kinase with an alanine at position 289 (T289A) is also phosphorylated in vivo, indicating that there is an additional phosphorylated residue. Based on comparisons with other protein kinases, we tested whether phosphorylation of Thr166 drives activation of MLCK-A. Our data indicate that phosphorylation of Thr289 occurs in vivo, but is not associated with conA-induced activation, whereas phosphorylation of Thr166 by some as yet unidentified kinase is associated with activation. Replacement of Thrl66 with glutamate results in a 12-fold increase in activity as compared with the wild-type enzyme, supporting the idea that phosphorylation of Thr166 increases MLCK-A activity.