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PubMed1998

Biochemical characterization of HIV-1 Rev as a potent activator of casein kinase II in vitro.

The stimulatory effects of several DNA-binding basic proteins (histone and protamine) and HIV-1 Rev with arginine (Arg)-rich clusters on the activity of casein kinase II (CK-II) were investigated in vitro. It was found that recombinant Rev (rRev) and the synthetic oligo-fragments corresponding to the amino acid sequences of its Arg-rich cluster stimulate CK-II activity in a dose-dependent manner. The activated CK-II phosphorylates several cellular and viral proteins in HIV-1 infected human MOLT-4 cells, and also phosphorylates HIV-1 structural proteins, including recombinant reverse transcriptase (rRT). These phosphorylations are selectively inhibited by CK-II inhibitors, such as quercetin, oGA (a glycyrrhetinic acid derivative) and NCS-chrom (an enediyne containing antibiotic). The data presented here suggest that HIV-1 Rev acts as an effective potent activator of CK-II, which may be a cellular mediator promoting HIV-1 replication in virus-infected cells.

Amino Acid Sequence↗
PubMed1989

Characterization of a mitogen-activated, Ca2+-sensitive microtubule-associated protein-2 kinase.

We have previously found that treatment of quiescent mammalian fibroblast cells with several mitogenic factors activates in common a Ca2+-sensitive serine/threonine-specific protein kinase activity toward microtubule-associated protein 2 (MAP2) [Hoshi, M., Nishida, E. and Sakai, H. (1988) J. Biol. Chem. 263, 5396-5401]. Here, we characterized the mitogen-activated MAP2 kinase activity in rat 3Y1 cells. The activated kinase activity was detected in the cytosolic fraction but not in the membrane fraction. The inhibitory effect of Ca2+ on the kinase activity was reversible. Kinetic analyses revealed that the apparent Km values of the kinase activity for MAP2 and ATP were 1.6 microM and 30 microM, respectively. Free Ca2+ at 4 microM decreased apparent Vmax values for MAP2 and ATP without changing the apparent Km values. The MAP2 kinase had an apparent molecular mass of about 40 kDa as determined by gel filtration and by sucrose density gradient centrifugation. Myelin basic protein as well as MAP2 could serve as good substrates for this kinase, but 40S ribosomal protein S6, casein, histone, phosphorylase b, protamine, tubulin, actin and tau could not. These properties of the enzyme indicate that the Ca2+-sensitive MAP2 kinase may be a previously unidentified enzyme. Down-regulation of protein kinase C by prolonged phorbol ester treatment abolished the MAP2 kinase activation by phorbol ester, but did not prevent the MAP2 kinase activation by epidermal growth factor (EGF) or fresh serum. This suggests that the Ca2+-sensitive MAP2 kinase could be activated through protein-kinase-C-dependent and -independent pathways. Activation of the MAP2 kinase occurred shortly after the addition of EGF or phorbol ester even in the presence of protein synthesis inhibitors (cycloheximide, puromycin and emetin). Moreover, treatment of the EGF- or phorbol-ester-activated MAP2 kinase with acid phosphatase inactivated the kinase activity. Thus, the MAP2 kinase may be activated through phosphorylation.

Animals↗
PubMed1988

Characterization of four tyrosine protein kinases from the particulate fraction of rat spleen.

Four distinct tyrosine protein kinases active on poly(Glu4,Tyr1) and angiotensin II, and operationally termed TPK-I, TPK-IIA, TPK-IIB and TPK-III have been resolved and partially purified from rat spleen particulate fraction by combining DEAE-Sepharose, heparin-Sepharose, phosphocellulose and polylysine-agarose chromatographies. Once partially purified all of them are free of Ser/Thr-specific protein kinase activity as judged using casein, histones, protamine and the peptide Arg-Arg-Ala-Ser-Val-Ala as substrates. TPK-I (apparent molecular mass 64 kDa, by gel filtration) and TPK-IIA (54 kDa) share several properties, including substrate specificity and stimulation by heparin; the latter however is much more responsive to polylysine then the former (10- and 3-fold maximum stimulation, respectively). Conversely TPK-IIB (51 kDa) is markedly inhibited by heparin and it is also characterized by its unique substrate specificity: unlike the other three tyrosine protein kinases it by far prefers the tetrapeptide Glu-Tyr-Ala-Ala over the decapeptide Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly and readily phosphorylates band-3 protein of red cell membrane. The unusual preference for Mg2+ over Mn2+ as activator and the capability to phosphorylate calmodulin distinguish TPK-III (61 kDa) from the other isoenzymes. Moreover TPK-III is insensitive to heparin and polylysine and is inhibited by quercetin much more efficiently than the other enzymes (I50 = 10 microM). Upon incubation with [gamma-32P]ATP, TPK-I, TPK-IIA and TPK-III give rise to alkali-stable radiolabeled components of 61, 55 and 52 kDa respectively, as evaluated by PAGE/SDS. In every case such a radiolabeling takes place also in the presence of a large excess of phosphorylatable substrate (angiotensin II) while it is readily reversed by isotopic dilution with 10-fold excess unlabeled ATP, supporting the view that it represents an autophosphorylation process. No (auto)phosphorylation product(s) could be detected in TPK-IIB even if its amount, in terms of catalytic activity, was 10-fold higher than that of the others.

Amino Acids↗
PubMed1983

Purification and characterization of the brain calmodulin-dependent protein kinase (kinase II), which is involved in the activation of tryptophan 5-monooxygenase.

Calmodulin-dependent protein kinase (kinase II) [Yamauchi, T. and Fujisawa, H. (1980) FEBS Lett. 116, 141-144], which is involved in the activation of tryptophan 5-monooxygenase, was purified 720-fold with a 36% yield from rat cerebral cortex using ammonium sulfate fractionation and chromatography on Sepharose CL-4B, calmodulin-Sepharose 4B and phosphocellulose. The purified enzyme showed one major protein band corresponding in mobility to a molecular weight of 55000 and a faint band upon sodium dodecyl sulfate/polyacrylamide disc gel electrophoresis, whereas it gave a single protein band upon polyacrylamide gel electrophoresis without sodium dodecyl sulfate. The molecular weight and the sedimentation coefficient of the kinase were determined to be 540000 by sedimentation equilibrium and 16.5 S by sucrose density gradient centrifugation. The kinase required absolutely calmodulin and Ca2+ for its activity and the apparent Ka values for calmodulin and Ca2+ were 10 nM and 1.6 microM respectively. The Km values for ATP and Mg2+ were calculated to be 0.06 mM and 1 mM, respectively. The concentration of tryptophan 5-monooxygenase required to produce half-maximal effects on its activation by the kinase was estimated to be as low as 0.3 nM, on the basis of the finding that the molecular weight and the specific activity of tryptophan 5-monooxygenase were 245000 and 374 nmol min-1 mg protein-1 respectively [Nakata, H. and Fujisawa, H. (1982) Eur. J. Biochem. 122, 41-47]. The kinase phosphorylated casein, smooth muscle myosin light chain as well as some endogenous proteins of brain cytosol. The enzyme did not phosphorylate significantly histone, protamine, and phosphorylase b. Some other properties of the kinase were examined.

Amino Acids↗
PubMed1987

The protein phosphatases of Drosophila melanogaster and their inhibitors.

Protein phosphatases-1, 2A and 2B have been identified in membrane and soluble fractions of Drosophila melanogaster heads. Similarities between Drosophila and mammalian protein phosphatase-1 included specificity for the beta subunit of phosphorylase kinase, sensitivity to inhibitor-1 and inhibitor-2, inhibition by protamine, retention by heparin-Sepharose and selective interaction with membranes. In addition, an inactive form of protein phosphatase-1, termed protein phosphatase-1I, was detected in the soluble fraction that could be activated by preincubation with MgATP and mammalian glycogen synthase kinase-3. Inhibitor-2 partially purified from Drosophila had an identical molecular mass to its mammalian counterpart, and recombined with mammalian protein phosphatase-1 to form a hybrid protein phosphatase-1I. Similarities between Drosophila and mammalian protein phosphatase-2A included preferential dephosphorylation of the alpha subunit of phosphorylase kinase, insensitivity to inhibitors-1 and -2, activation by protamine, exclusion from heparin-Sepharose and apparent molecular mass. A Ca2+-dependent calmodulin-stimulated protein phosphatase (protein phosphatase-2B) that was inhibited by trifluoperazine was identified in the soluble fraction. The remarkable similarities between Drosophila protein phosphatases and their mammalian counterparts are indicative of strict phylogenetic conservation and demonstrate that the procedures used to classify mammalian protein phosphatases have a wider application. Characterisation of the Drosophila phosphatases will facilitate genetic analysis of dephosphorylation systems and their possible roles in neuronal and behavioural plasticity in Drosophila.

Adenosine Triphosphate↗
PubMedDate not supplied

The apparent inhibitor of the protein kinase in the human seminal plasma: an explanation.

The protein kinase in the human seminal plasma, as assayed by its ability to catalyze the transfer of phosphate from [gama-32P]ATP to the protamine, was found to linearly increase with dilution of the seminal plasma. Extrapolation of the enzyme activity to the undiluted seminal plasma indicated that te protein kinase existed in the ejaculate as the inactive enzyme. An inhibitor of the protein kinase was postulated and the results suggested that the inhibitor was a heat-labile macromolecule. However, the linear increase of the protein kinase activity could also be obtained by increasing ATP concentration. In addition, a high Mg+2-dependent ATPase activity was found in the seminal plasma, its activity exhibited inverse relationship with the activity of the protein kinase. The results suggested that the apparent inhibitor of the protein kinase in the human seminal plasma was the destruction of ATP by the ATPase.

Adenosine Triphosphatases↗
PubMed2000

An unusual form of purifying selection in a sperm protein.

Protamines are small, highly basic DNA-binding proteins found in the sperm of animals. Interestingly, the proportion of arginine residues in one type of protamine, protamine P1, is about 50% in mammals. Upon closer examination, it was found that both the total number of amino acids and the positions of arginine residues have changed considerably during the course of mammalian evolution. This evolutionary pattern suggests that protamine P1 is under an unusual form of purifying selection, in which the high proportion of arginine residues is maintained but the positions may vary. In this case, we would expect that the rate of nonsynonymous substitution is not particularly low compared with that of synonymous substitution, despite purifying selection. We would also expect that the selection for a high arginine content results in a high frequency of the nucleotide G in the coding region of this gene, because all six arginine codons contain at least one G. These expectations were confirmed in our study of mammalian protamine genes. Analysis of nonmammalian vertebrate genes also showed essentially the same patterns of evolutionary changes, suggesting that this unusual form of purifying selection has been active since the origin of bony vertebrates. The protamine gene of an insect species shows similar patterns, although its purifying selection is less intense. These observations suggest that arginine-rich selection is a general feature of protamine evolution. The driving force for arginine-rich selection appears to be the DNA-binding function of protamine P1 and an interaction with a protein kinase in the fertilized egg.

Amino Acid Sequence↗
PubMed1994

Partial purification of a type eta protein kinase C from murine brain: separation from other protein kinase C isoenzymes and characterization.

Various murine tissues were tested, by using a protein kinase C-eta-specific antiserum, for the expression of type eta protein kinase C. Brain was found to be the richest source of a type eta isoenzyme. Native protein kinase C-eta was partially purified from the cytosol of murine brain by chromatography on DEAE-Sepharose, hydroxyapatite and protamine-agarose. This procedure resulted in a separation of protein kinase C-eta from the other phorbol 12-myristate 13-acetate (PMA)-responsive isoenzymes (alpha, beta, gamma, delta, epsilon) and allowed, for the first time, characterization of the native enzyme. The protein kinase C of type eta from mouse brain is a phospholipid-dependent Ca(2+)-unresponsive protein kinase. Both PMA and bryostatin activate the kinase for phosphorylation of a substrate as well as for autophosphorylation. Various pseudo-substrate-related peptides are suitable as substrates for the eta-type kinase, peptide delta being the best and peptides eta and epsilon the poorest substrates. The enzyme is inhibited by staurosporine and staurosporine-related compounds, such as K252a and Gö 6976. However, protein kinase C-eta, like protein kinase C-delta, is around two orders of magnitude less sensitive towards Gö 6976 than are the Ca(2+)-responsive isoenzymes (alpha, beta, gamma). The eta-type protein kinase C exhibits an extreme tendency to lose its PMA-responsiveness. Consequently, purification of the enzyme to homogeneity has not yet been successful.

Alkaloids↗
PubMed1988

Characterization of a polypeptide-dependent membrane protein kinase that specifically phosphorylates NS protein of vesicular stomatitis virus in vitro.

Phosphorylation of membrane-associated proteins by protein kinases in the membrane fraction from HeLa S3 cells was rapidly increased when the cells were infected with vesicular stomatitis virus (VSV). SDS-PAGE followed by autoradiography revealed polypeptides with molecular sizes of Mr. 53,000, 44,000, 42,000, 35,000, 30,000 and 27,000 in the kinase fraction from uninfected cells to be highly phosphorylated. Virus-coding NS protein (Mr. 40,000) was phosphorylated when the membrane fraction from virus-infected cells was incubated with [gamma-32P]ATP in the presence of histone H1 and Mg2+. Under these conditions, histone H1 functioned as a stimulator for NS protein phosphorylation by the kinases. One (kinase III) of the membrane-associated kinases was partially purified from HeLa S3 cells using FPLC (type Mono Q) after DEAE-cellulose column chromatography. The enzymatic properties of kinase III were similar to those reported for a polypeptide-dependent protein kinase (protein kinase P), because (a) both kinases highly phosphorylated beta-casein, although no phosphorylation was observed with histones; (b) several endogenous substrates from HeLa S3 cell membrane were phosphorylated by the kinases in the presence of basic proteins, such as histones, protamine and poly-Lys; (c) their activity was insensitive to a low concentration (19 micrograms/ml) of heparin, which highly inhibited casein kinase II activity; and (d) the kinases were extractable from the plasma membrane using Triton X-100. In addition, provided evidence suggests that kinase III may play an important role in an early stage of VSV replication through its specific phosphorylation of NS protein and membrane proteins in virus infected cells.

Animals↗
PubMed1990

Protein kinase C activation by platelet-activating factor is independent of enzyme translocation.

The subcellular distribution and activation state of protein kinase C (PKC) was studied after short-term exposure of rabbit platelets to platelet-activating factor (PAF). Cytosolic and nonidet P-40-solubilized particulate extracts prepared from treated platelets were subjected to analytical column chromatography on MonoQ, hydroxylapatite and Superose 6/12. PKC activity was assayed by the ability of the enzyme to phosphorylate the following substrates: (i) histone H1 in the presence of the activators calcium, diacylglycerol and phosphatidylserine; (ii) histone H1 following proteolytic activation of PKC with 0.5 micrograms trypsin/ml; and (iii) protamine in the absence of calcium and lipid. PAF treatment for 1-20 min elicited a rapid 2-4-fold activation of both cytosolic and particulate-derived PKC as assessed by all three methods. On the other hand, there were no significant PAF-induced changes in the level of [3H]phorbol-12,13-dibutyrate binding by soluble and particulate-associated PKC. Hydroxyapatite column chromatography revealed that in non-treated rabbit platelets the type II (beta) form of PKC predominated, but PAF appeared to induce a shift in the elution profile from this resin. The stability of the PAF activation of PKC to column chromatography and the altered binding affinity to hydroxylapatite indicated that the stimulation might be a consequence of covalent modification, albeit minor, since PKC still eluted as an 80 kDa protein from Superose 6/12. As the PAF-induced increases in the kinase activity of PKC were preserved even after proteolytic activation with trypsin, but were without effect on the phorbol ester binding activity, such a putative modification may have occurred within or near the catalytic domain of PKC. These findings imply that PAF may directly modulate the activity of preexisting membrane-associated PKC by a novel mechanism, rather than by eliciting its recruitment from the cytoplasm.

Amino Acid Sequence↗
PubMed1993

Activation of protein kinase C by selective binding of arginine-rich polypeptides.

Protein substrates for protein kinase C (PKC) have phosphorylation domains that are typically rich in the basic amino acids arginine and lysine. However, arginine-rich proteins may interact with PKC differently than lysine-rich proteins, i.e. lysine-rich histone requires phospholipid and Ca2+ to be phosphorylated, whereas the arginine-rich protein, protamine, can bypass these effector requirements. We have studied the interaction of PKC with protamine, histone, poly-L-arginine, and poly-L-lysine to better understand the role of basic protein domains in PKC activation, effector dependence, and substrate specificity. Using a microtiter binding assay, PKC was found to bind tightly to protamine and poly-L-arginine, but not to histone or poly-L-lysine, in the absence of phospholipid, Ca2+, and MgATP. Furthermore, poly-L-arginine was much more potent than poly-L-lysine at inhibiting protamine phosphorylation; i.e. 1-2 nM poly-L-arginine was sufficient to cause 50% inhibition of protamine phosphorylation, whereas over 300 microM poly-L-lysine was needed to reach 50% inhibition. Autophosphorylation of PKC in the absence of activators was potently stimulated by protamine and poly-L-arginine, but not by histone or poly-L-lysine, suggesting selective stimulation of PKC by arginine-rich polypeptides. Double-reciprocal plots of protamine phosphorylation using either a mixture of isozymes (alpha/beta/gamma) or isolated PKC-beta were parabolic, and analysis of the kinetic data on velocity/[protamine] versus velocity plots indicated positive cooperativity with respect to protamine. These findings are consistent with those from autophosphorylation experiments in that PKC appears to be selectively stimulated by arginine-rich polypeptides. These results suggest that PKC can preferentially bind arginine-rich proteins in the absence of phospholipid and Ca2+. This interaction appears to be distal to the catalytic site and thus binding of arginine-rich proteins may allosterically activate PKC. Selective stimulation of PKC by arginine-rich proteins may be a mechanism by which protamine can bypass activator requirements. Furthermore, control of PKC activity by activator-independent binding of arginine-rich polypeptides suggests that altering access to certain cellular proteins may be a mechanism for PKC regulation in vivo.

Adenosine Triphosphate↗
PubMed1995

Expression and characterization of PKD, a phorbol ester and diacylglycerol-stimulated serine protein kinase.

A novel protein kinase (named PKD) with an NH2-terminal region containing two cysteine-rich motifs has been expressed in COS-7 cells and identified as a receptor for phorbol esters. COS-7 cells transfected with a PKD cDNA construct (pcDNA3-PKD) exhibit a marked (4.8-fold) increase in [3H]phorbol 12,13-dibutyrate binding. An antiserum raised against the COOH-terminal 15 amino acids of PKD specifically recognized a single 110-kDa band in PKD-transfected cells. PKD prepared by elution from immunoprecipitates with the immunizing peptide efficiently phosphorylated the synthetic peptide syntide-2. The enzyme only poorly phosphorylated a variant syntide-2 where arginine 4 has been replaced by an alanine. The addition of [3H]phorbol 12,13-dibutyrate, 1-oleoyl-2-acetylglycerol, or 1,2-dioctanoyl-sn-glycerol in the presence of dioleoylphosphatidylserine stimulated the syntide-2 kinase activity of PKD in a synergistic fashion (4-6-fold). Furthermore, the autophosphorylation of PKD was strikingly stimulated by the same lipid activators (14-24-fold). Similar properties were found with PKD isolated from mouse lung. The substrate specificity of PKD is different from that of previously identified members of the protein kinase C family since it does not efficiently phosphorylate histone III-S, protamine sulfate, or a synthetic peptide based upon the conserved pseudosubstrate region of the protein kinase C family. Taken together, these data unambiguously establish PKD as a phorbol ester receptor and as a novel phospholipid/diacylglycerol-stimulated protein kinase.

Amino Acid Sequence↗