Site-specific phosphorylation of L-form starch phosphorylase by the protein kinase activity from sweet potato roots

A 78-amino acid insertion (L78) is found in the low-affinity type (L-form) of starch phosphorylase (L-SP, EC 2.4.1.1). This insertion blocks the starch-binding site on the L-SP molecule, and it decreases the binding affinity of L-SP toward starch. The computational analysis of the amino acid sequence on L78 predicts several phosphorylation sites at its Ser residues. Indeed, from the immunoblotting results using antibodies against phosphoamino acids, we observed that the purified L-SP from mature sweet potato (Ipomoea batatas) roots is phosphorylated. This observation led us to the detection of a protein kinase activity in the protein fraction of the crude extract from the sweet potato roots. The kinase was partially purified by liquid chromatography, and its native molecular mass was estimated as 338 kDa. An expressed peptide (L78P) containing the essential part of L78 was intensively phosphorylated by the kinase. However, H-SP (the high-affinity isomer of SP lacking the L78 insertion) and the proteolytic modified L-SP, which lost its L78 fragment, could not be phosphorylated. Furthermore, using L78P mutants by site-directed mutagenesis at Ser residues on L78, we demonstrate that only one Ser residue on L78 is phosphorylated by the kinase. These results imply that this kinase is specific to L-SP, or more precisely, to the L78 insertion. The in vitro phosphorylated L-SP shows higher sensitivity to proteolytic modification, but has no change in its kinetic parameters. H.M. Chen and C.T. Lin contributed equally to this work.     

Vitamin E inhibits protein kinase C activity

Vitamin E (dl-alpha-tocopherol) has been found to inhibit in vitro brain protein kinase c with a half inhibitory concentration of 450 microM. The known plasma concentrations of vitamin E are one order of magnitude lower than the protein kinase c half-inhibitory concentration but it is also known that, at the membrane level where the active protein kinase c is located, the lipophilic vitamin E is more concentrated (Burton, G.W., Joyce, A. and Ingold, K.U. and Locke, S. (1983) Arch. Biochem. Biophys. 221, 281-290). It appears that vitamin E, in addition to its antioxidant function, may play a role in regulating the activity of protein kinase c.     

Purified protein kinase C phosphorylates microtubule-associated protein 2

We have investigated actions of purified protein kinase C on microtubule- and microfilament-related proteins. Among the cytoskeletal proteins examined, microtubule-associated protein 2 (MAP2) was found to serve as a good substrate. Other cytoskeletal proteins, tubulin, fodrin, cofilin, tropomyosin, and 53,000-Da protein, were very poorly phosphorylated. The amino acid residues of MAP2 that were phosphorylated by the protein kinase C were almost exclusively serine. The peptide mapping analysis indicated that protein kinase C and cAMP-dependent protein kinase phosphorylate MAP2 differently. The ability of MAP2 to interact with actin was markedly reduced by this protein kinase C-mediated phosphorylation. These data raise the possibility that phosphorylation of MAP2 by activated protein kinase C may be involved in cell-surface signal transduction.     

Growth factor-induced activation of a kinase activity which causes regulatory phosphorylation of p42/microtubule-associated protein kinase.

p42/microtubule-associated protein kinase (p42mapk) is activated by tyrosine and threonine phosphorylation, and its regulatory phosphorylation is likely to be important in signalling pathways involved in growth control, secretion, and differentiation. Here we show that treatment of quiescent 3T3 cells with diverse agonists results in the appearance of an activity capable of causing the in vitro phosphorylation of p42mapk on the regulatory tyrosine and to a lesser extent on the regulatory threonine, resulting in enzymatic activation of the p42mapk. This p42mapk-activating activity is capable of phosphorylating a kinase-defective p42mapk mutant, thus confirming its activity as a kinase.     

The antioxidant butylated hydroxytoluene stimulates platelet protein kinase C and inhibits subsequent protein phosphorylation induced by thrombin

The phenolic antioxidant 2,6-bis(1,1-dimethyl ethyl)-4-methylphenol (BHT) evokes a transient phosphorylation of two platelet proteins of Mr 20,000 and 47,000 that are well-known substrates of protein kinase C (PKC) and, similarly to phorbol esters, a slight but persistent phosphorylation of a protein of Mr 26,000. These effects are observed both in the presence and in the absence of extracellular calcium, but are abolished in the presence of the protein kinase C inhibitor staurosporine. The phosphorylation of the 47 kDa protein takes place mostly at the serine and, to a lesser extent, at threonine residues. BHT induces an increased binding of tritiated phorbol dibutyrate to platelets indicating a PKC translocation from cytosol to plasma membrane. Addition of BHT (20 microM) a few min prior to thrombin causes inhibition of both agonist-evoked protein phosphorylation and increase in the Ca2+ concentration, the latter inhibition being counteracted by staurosporine. The inhibitory effect lasts for several minutes even after removal of BHT from the cellular suspending medium. Similar results are obtained with nordihydroguaiaretic acid, whereas 2- and 3-tert-butyl-4-methoxyphenol (BHA) produce only slight effects. BHT activates the protein kinase C purified from pig brain in a concentration-dependent manner (up to 200 microM), whereas it does not affect the activity of other purified protein kinases such as type 1 and 2 casein kinases, type II A, II B and III tyrosine protein kinases from rat spleen and the catalytic subunit of cyclic AMP-dependent protein kinase. It is concluded that, similarly to diacylglycerols and phorbol esters, these phenolic antioxidants activate the protein kinase C, which in turn desensitizes platelets towards subsequent phospholipase C activation.     

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

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

pp54 microtubule-associated protein 2 kinase. A novel serine/threonine protein kinase regulated by phosphorylation and stimulated by poly-L-lysine

An hepatic protein kinase that phosphorylates microtubule-associated protein 2 (MAP-2) on Ser/Thr residues is markedly activated after intraperitoneal injection of cycloheximide in the rat. The enzyme has been purified greater than 10,000-fold to near homogeneity and corresponds to a 54-kDa polypeptide, based on auto-phosphorylation, renaturation of activity from sodium dodecyl sulfate gels, and gel filtration. The protein kinase activity is unaffected by prior autophosphorylation, Ca2+, diacylglycerol and phospholipids, cyclic nucleotides, staurosporine, and protein kinase inhibitor, but can be totally and specifically deactivated by the Ser/Thr protein phosphatase 2A. The enzyme is inhibited completely but reversible by transition metals and p-chloromercuribenzoate, and is strongly stimulated by poly-L-lysine toward most, but not all protein substrates. The activity of the cycloheximide-stimulated MAP-2 kinase (pp54 MAP-2 kinase) toward potential polypeptide substrates was compared to that of an insulin-stimulated MAP-2 kinase (pp42 MAP-2 kinase). Although both MAP-2 kinases exhibited little or no ability to phosphorylate histones and casein, the two kinases had a distinguishable substrate specificity. At comparable MAP-2 phosphorylating activities, pp42 MAP-2 kinase, but not pp54 MAP-2 kinase, phosphorylated and activated the Xenopus S6 protein kinase II. Moreover, pp42 MAP-2 kinase phosphorylated myelin basic protein at 10-12-fold higher rates than did pp54 MAP-2 kinase. Cycloheximide-activated pp54 MAP-2 protein kinase appears to be a previously uncharacterized protein kinase that is itself regulated through Ser/Thr phosphorylation and, perhaps, polypeptide regulators with basic domains. The identity of the upstream regulatory elements and the native substrates remain to be established.     

Mitogen-activated-protein-kinase-catalyzed phosphorylation of microtubule-associated proteins, microtubule-associated protein 2 and microtubule-associated protein 4, induces an alteration in their function.

Mitogen-activated protein kinase (MAPK), a serine/threonine-specific protein kinase which is generally activated by stimulation with various growth factors and phorbol esters, utilizes microtubule-associated protein (MAP) 2 as a good substrate in vitro. We have found that MAPK-catalyzed phosphorylation of MAP2 resulted in a significant loss in its ability to induce tubulin polymerization. The chymotryptic fragments, containing a microtubule-binding domain of MAP2, were phosphorylated by MAPK and the ability of the fragments to induce tubulin polymerization was also greatly decreased by the phosphorylation, suggesting that phosphorylation of the microtubule-binding domain is important for functional alteration of MAP2. In addition to MAP2, a 190-kDa heat-stable MAP (MAP4) found in various tissues and cells, was a good substrate for MAPK in vitro. Phosphorylation of MAP4 inactivated tubulin polymerization. We examined the effect of phosphorylation of MAP2 and MAP4 on the dynamics of microtubules nucleated by purified centrosomes in vitro. The data showed that MAPK-catalyzed phosphorylation of MAP2 and MAP4 reduced their ability to increase the apparent elongation rate and the number of microtubules nucleated by the centrosome. Thus, MAPK is capable of phosphorylating MAPs and negatively regulating their microtubule-stabilizing function.     

An isoform of microtubule-associated protein 4 inhibits kinesin-driven microtubule gliding

Recently, we revealed that microtubule-associated protein (MAP) 4 isoforms, which differ in the number of repeat sequences, alter the microtubule surface properties, and we proposed a hypothesis stating that the change in the surface properties may regulate the movements of microtubule motors [Tokuraku et al. (2003) J Biol Chem 278: 29609-29618]. In this study, we examined whether MAP4 isoforms affect the kinesin motor activity. When the MAP4 isoforms were present in an in vitro gliding assay, the five-repeat isoform but not the three- and four-repeat isoforms inhibited the movement of the microtubules in a concentration-dependent manner. The observation of individual microtubules revealed that in the presence of the five-repeat isoform, the microtubules completely stopped their movements or recurrently paused and resumed their movements, with no deceleration in the moving phase. The result can be explained by assuming that kinesin stops its movement when it encounters a microtubular region whose properties are altered by the MAPs. A sedimentation assay demonstrated that the MAP4 isoforms did not compete with kinesin for binding to microtubules, indicating that kinesin can bind to the MAP-bound microtubules, although it cannot move on them.     

Lidocaine inhibits stimulation-coupled responses of neutrophils and protein kinase C activity

The effects of lidocaine, a local anesthetic, on various stimulation-coupled responses of neutrophils were studied. Superoxide generation, generation of chemiluminescence, depolarization of membrane potential and transitional increase in intracellular Ca2+ were inhibited by lidocaine in a concentration dependent manner. Lidocaine also inhibited Ca(2+)-activated phospholipid-dependent protein kinase (PKC) in the presence of various concentrations of Ca2+, phosphatidylserine and dioleoylglycerol. For the inhibition of all these stimulation-coupled responses, a similar order of the lidocaine concentration was needed. As in the case of dibucaine (Mori, T., Takai, Y., Minakuchi, R., Yu, B. and Nishizuka, Y., J. Biol. Chem. 255:8378-8380, 1980), lidocaine inhibited PKC activity in a manner competitive with phosphatidylserine. Lidocaine also inhibited the phosphorylation of 47 kDa neutrophil cytosplasmic protein, a phosphorylated protein required for NADPH oxidase activation. Thus, the cellular membrane phospholipid may be one of the target sites of lidocaine for the inhibitory action on the various stimulation-coupled responses of neutrophils, and these effects of lidocaine may correlate with its inhibitory action on PKC activity.     

Suppression of Kir2.3 activity by protein kinase C phosphorylation of the channel protein at threonine 53

Kir2.3 plays an important part in the maintenance of membrane potential in neurons and myocardium. Identification of intracellular signaling molecules controlling this channel thus may lead to an understanding of the regulation of membrane excitability. To determine whether Kir2.3 is modulated by direct phosphorylation of its channel protein and identify the phosphorylation site of protein kinase C (PKC), we performed experiments using several recombinant and mutant Kir2.3 channels. Whole-cell Kir2.3 currents were inhibited by phorbol 12-myristate 13-acetate (PMA) in Xenopus oocytes. When the N-terminal region of Kir2.3 was replaced with that of Kir2.1, another member in the Kir2 family that is insensitive to PMA, the chimerical channel lost its PMA sensitivity. However, substitution of the C terminus was ineffective. Four potential PKC phosphorylation sites in the N terminus were studied by comparing mutations of serine or threonine with their counterpart residues in Kir2.1. Whereas substitutions of serine residues at positions 5, 36, and 39 had no effect on the channel sensitivity to PMA, mutation of threonine 53 completely eliminated the channel response to PMA. Interestingly, creation of this threonine residue at the corresponding position (I79T) in Kir2.1 lent the mutant channel a PMA sensitivity almost identical to the wild-type Kir2.3. These results therefore indicate that Kir2.3 is directly modulated by PKC phosphorylation of its channel protein and threonine 53 is the PKC phosphorylation site in Kir2.3.     

Chemoattractant-induced tyrosine phosphorylation and activation of microtubule-associated protein kinase in human neutrophils.

Activation of human neutrophils by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) induces tyrosine phosphorylation of several polypeptides, including a prominent band of approximately 41 kDa. A polypeptide of identical electrophoretic mobility was recognized by a monoclonal antibody raised against a sequence corresponding to amino acids 325-345 of ERK-1, one of a family of mitogen-activated protein (MAP) kinases. To establish the possible identity of these polypeptides, extracts from control and fMLP-treated cells were immunoprecipitated with immobilized antiphosphotyrosine antibodies. Reactivity with anti-ERK-1 antibodies was observed only in the precipitate of chemoattractant-stimulated cells. These data imply that a MAP kinase constitutes at least part of the tyrosine-phosphorylated 41-kDa polypeptide. By using an in vitro renaturation assay, treatment of intact cells with fMLP was found to stimulate several protein kinases, including one of approximately 41 kDa. Renaturation of samples immunoprecipitated with antiphosphotyrosine antibodies revealed the presence of an active protein kinase in chemoattractant-stimulated, but not in control cells. The immunoprecipitated kinase comigrated with the 41-kDa tyrosine phosphorylated polypeptide and the anti-ERK-1 reactive band. We conclude that a MAP kinase closely related or identical to ERK-1 is tyrosine phosphorylated and activated when human neutrophils are stimulated by chemotactic peptides. The rapid phosphorylation of this kinase, which is apparent within seconds, is compatible with a role in the activation of the respiratory burst and/or other neutrophil responses.     

Caldesmon phosphorylation in intact human platelets by cAMP-dependent protein kinase and protein kinase C

Caldesmon is a calmodulin- and actin-binding protein present in both smooth and non-muscle tissue. The present study demonstrates that platelet caldesmon is a substrate for cAMP-dependent protein kinase (protein kinase A). Purified platelet caldesmon has an apparent molecular mass of 82 kDa on sodium dodecyl sulfate-polyacrylamide gels and can be phosphorylated in vitro by the catalytic subunit of protein kinase A to a level of 2 mol of phosphate/mol of caldesmon. Phosphorylation of caldesmon by protein kinase A results in a shift in the apparent molecular mass of the protein to 86 kDa. When caldesmon was immunoprecipitated from intact platelets treated with prostacyclin (PGI2) the same shift in apparent molecular mass of caldesmon was observed. Comparison of two-dimensional tryptic phosphopeptide maps of caldesmon phosphorylated in vitro by protein kinase A with caldesmon immunoprecipitated from intact platelets verified that protein kinase A was responsible for the observed increase in caldesmon phosphorylation in PGI2-treated platelets. The present study demonstrates that although caldesmon is basally phosphorylated in the intact platelet, activation of protein kinase A by PGI2 results in the significant incorporation of phosphate into two new sites. In addition, the effects of phorbol ester, collagen, and thrombin on caldesmon phosphorylation were also examined. Although phorbol ester treatment results in a significant increase in caldesmon phosphorylation apparently by protein kinase C, treatment of intact platelets with thrombin or collagen does not result in an increase in caldesmon phosphorylation.     

Site-specific phosphorylation of tau accompanied by activation of mitogen-activated protein kinase (MAPK) in brains of Niemann-Pick type C mice

Niemann-Pick type C (NPC) disease is characterized by an accumulation of cholesterol in most tissues and progressive neurodegeneration with the formation of neurofibrillary tangles. Neurofibrillary tangles are composed of paired helical filaments (PHF), a major component of which is the hyperphosphorylated tau. In this study we used NPC heterozygous and NPC homozygous mouse brains to investigate the molecular mechanism responsible for tauopathy in NPC. Immunoblot analysis using anti-tau antibodies (Tau-1, PHF-1, AT-180, and AT-100) revealed site-specific phosphorylation of tau at Ser-396 and Ser-404 in the brains of NPC homozygous mice. Mitogen-activated protein kinase, a potential serine kinase known to phosphorylate tau, was activated, whereas other serine kinases such as glycogen synthase kinase-3beta and cyclin-dependent kinase 5 were inactive. Morphological examination demonstrated that a number of neurons, the perikarya of which strongly immunostained with PHF-1, exhibited polymorphorous cytoplasmic inclusion bodies and multi-concentric lamellar-like bodies. Importantly, the accumulation of intracellular cholesterol in NPC mouse brains was determined to be a function of age. From these results we conclude that abnormal cholesterol metabolism due to the genetic mutation in NPC1 may be responsible for activation of the mitogen-activated protein kinase-signaling pathway and site-specific phosphorylation of tau in vivo, leading to tauopathy in NPC.     

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.     

Separation of the different microtubule-associated tau protein species from bovine brain and their mode II phosphorylation by Ca2+/phospholipid-dependent protein kinase C

Bovine brain tau protein (tau) consists of four closely related phosphoproteins named tau 1, tau 2, tau 3 and tau 4, that range in size from 55 to 68 kDa (as determined by gel electrophoresis). Here we report an improved large-scale purification method for tau protein and the separation of the four individual tau protein species. The separation of the individual tau protein was accomplished by two chromatographic techniques: hydroxyapatite chromatography allowed the separation of two pairs of tau protein (tau 1 and tau 3) and (tau 2 and tau 4); fast protein liquid chromatography on a Mono Q column at basic pH achieved the resolution of the individual tau protein species in each pair derived from hydroxyapatite columns. Chromatography on the Mono Q column revealed that tau protein possesses previously unrecognized, highly reactive sulfhydryl groups that may oxidize to form intermolecular disulfide bridges. The isolation of individual species of tau in substantial quantities permitted an improved amino acid analysis that demonstrated the occurrence of cysteine and tryptophan in the protein. The availability of individual tau protein species greatly simplified the analysis for mode II phosphorylation of tau, which was found to be catalyzed by the calcium/phospholipid-dependent protein kinase C. The mode II phosphorylation of tau by protein kinase C was not associated with a mobility shift for tau protein in SDS-polyacrylamide gel electrophoresis, in contrast to mode I phosphorylation of tau by the Ca2+/calmodulin-dependent kinase, which produces a substantial shift in mobility.     

Selective phosphorylation of human DNA methyltransferase by protein kinase C

Human DNA methyltransferase, the enzyme thought to be responsible for the somatic inheritance of patterns of DNA methylation, is an effective substrate for phosphorylation by protein kinase C. This provides a plausible mechanistic link between the action of tumor promoting phorbol esters, which stimulate protein kinase C, and abnormal patterns of DNA methylation often observed in transformed cells.     

The phosphorylation of eukaryotic ribosomal protein S6 by protein kinase C

Purified Ca2+-dependent and phospholipid-dependent protein kinase (protein kinase C) from bovine brain catalysed the phosphorylation of ribosomal protein S6 when incubated with 40S ribosomal subunits from rat liver or from hamster fibroblasts. The phosphorylation was dependent on Ca2+ and phospholipid, and occurred under ionic conditions similar to those which support protein biosynthesis in vitro. Protein kinase C phosphorylated at least three sites on ribosomal protein S6 when incubated with unphosphorylated ribosomes, and increased the extent of phosphorylation of ribosomes previously phosphorylated predominantly on two sites by cyclic-AMP-dependent protein kinase, converting some molecules to the tetraphosphorylated or pentaphosphorylated form. This indicates that protein kinase C can phosphorylate sites on ribosomal protein S6 other than those phosphorylated by the cyclic-AMP-dependent protein kinase, and this conclusion was confirmed by analysis of tryptic phosphopeptides. These results strengthen the possibility that protein kinase C might be involved in catalysing the multisite phosphorylation of ribosomal protein S6 in certain circumstances in vivo.     

Altered erythrocyte protein kinase C activity and membrane protein phosphorylation in chronic myelogenous leukemia

The membrane protein kinase C (PKC) content was found to be higher in erythrocytes form patients suffering from chronic myelogenous leukemia (CML) compared to normal erythrocytes. PKC activity was also higher in the cytosol and after translocation to the membrane, as assessed by histone phosphorylation. The increased PKC activity in CML erythrocytes was associated with abnormal phosphorylation of protein 4.1. Since phosphorylation-dephosphorylation mechanisms are likely candidates for controlling membrane protein associations, the altered PKC activity may be one of the factors responsible for altered thermal sensitivity and mechanical stability of CML erythrocytes.