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PubMed1994

Identification and characterization of delta B-CaM kinase and delta C-CaM kinase from rat heart, two new multifunctional Ca2+/calmodulin-dependent protein kinase isoforms.

We have identified, expressed and characterized two new isoforms of the multifunctional Ca2+/calmodulin-dependent kinase (CaM kinase) cloned from rat heart. Both isoforms are variants of the neuronal delta-CaM kinase (termed delta A-CaM kinase), and are designated as delta B-CaM kinase and delta C-CaM kinase. The new isoforms differ from delta A-CaM kinase in its isoform-specific insert region, between nucleotides 984 to 1087 of the delta A-CaM kinase cDNA. Replacing these 102 nucleotides, a sequence of 33 nucleotides which code for 11 amino acids (KRKSSSSQMM) are introduced in delta B-CaM kinase. The delta C-CaM kinase lacks all 102 nucleotides and the corresponding 34 amino acids which they encode. The predicted molecular masses of the delta B- and delta C-CaM kinase isoforms are 57,697 Da and 56,446 Da, respectively. Recombinant delta-CaM kinases purified from transfected COS-7 cells were found to associate into a larger holoenzyme estimated to contain 8 to 10 subunits. The relative subunit molecular masses on SDS-polyacrylamide gel electrophoresis are 59 kDa, 54 kDa and 52 kDa for delta A-, delta B- and delta C-CaM kinase, respectively. All three isoforms showed a strict dependence on Ca2+/calmodulin for activity and exhibited the Ca(2+)-dependent autophosphorylation and resultant conversion to Ca(2+)-independent kinase activity, characteristic features of multifunctional CaM kinase. Phosphopeptide analysis after partial CNBr digestion suggests that delta B-CaM kinase is the predominant soluble CaM kinase species purified from rat heart.

Amino Acid Sequence↗

PubMed1984

In vivo modulation of rat liver 3-hydroxy-3-methylglutaryl-coenzyme A reductase, reductase kinase, and reductase kinase kinase by mevalonolactone.

It has been previously demonstrated that the enzymic activity of rat liver 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase; EC 1.1.1.34) is modulated in vitro and in vivo by a bicyclic cascade system involving reversible phosphorylation of HMG-CoA reductase and reductase kinase. In the present study, administration of mevalonolactone to rats caused a rapid inhibition of HMG-CoA reductase activity. The initial short-term (20-min) reversible inhibition (38%) of enzyme activity was due to increased phosphorylation of HMG-CoA reductase. The inhibition of HMG-CoA reductase activity by increased phosphorylation was associated with an increased activity and phosphorylation (2- to 3-fold) of reductase kinase. The increased phosphorylation of reductase kinase was catalyzed by reductase kinase kinase, which was significantly elevated (3- to 4-fold) after the administration of mevalonolactone to rats. The mechanism for the in vivo activation of reductase kinase kinase is as yet unknown. Mevalonolactone administration was also associated with a significant inhibition of phosphoprotein phosphatase activity, which dephosphorylates both HMG-CoA reductase (activation) and reductase kinase (inactivation). These results indicate that mevalonolactone administration to rats in vivo was associated with an inhibition of HMG-CoA reductase activity by two mechanisms: (i) an increase in the degree of phosphorylation of both HMG-CoA reductase and reductase kinase due to increased activity of reductase kinase kinase; (ii) a decrease in the dephosphorylation of both HMG-CoA reductase and reductase kinase secondary to inhibition of phosphoprotein phosphatase activity. These combined effects favor an increase in the steady-state level of the phosphorylated forms of both HMG-CoA reductase and reductase kinase, resulting in a net reduction in the enzymic activity of HMG-CoA reductase and mevalonate formation. These results demonstrate that the activity of reductase kinase kinase is modulated in vivo, providing a mechanism for the regulation of the activities of both reductase kinase and HMG-CoA reductase.

AMP-Activated Protein Kinases↗

PubMed1994

Oligodeoxynucleotides antisense to mRNA encoding protein kinase A, protein kinase C, and beta-adrenergic receptor kinase reveal distinctive cell-type-specific roles in agonist-induced desensitization.

The roles of three protein kinases, cyclic AMP-dependent protein kinase (protein kinase A), protein kinase C, and beta-adrenergic receptor kinase (beta ARK), implicated in agonist-induced desensitization of guanine nucleotide-binding protein (G-protein)-coupled receptors were explored in four different cell lines after 48 hr of incubation with oligodeoxynucleotides antisense to the mRNA encoding each kinase. Desensitization of beta 2-adrenergic receptors was analyzed in cell types in which the activities of the endogenous complement of protein kinases A and C and beta ARK were distinctly different. Protein kinase A was necessary for desensitization of rat osteosarcoma cells (ROS 17/2.8), whereas the contribution of beta ARK to desensitization was insignificant. In Chinese hamster ovary cells that stably express beta 2-adrenergic receptors and in smooth muscle cells (DDT1MF-2), oligodeoxynucleotides antisense to beta ARK mRNA nearly abolished desensitization, whereas oligodeoxynucleotides antisense to protein kinase A mRNA attenuated desensitization to a lesser extent. In human epidermoid carcinoma cells (A-431), oligodeoxynucleotides antisense to either protein kinase A mRNA or beta ARK mRNA attenuated agonist-induced desensitization, providing a third scenario in which two kinases constitute the basis for agonist-induced desensitization. In sharp contrast, oligodeoxynucleotides antisense to protein kinase C mRNA were found to enhance rather than attenuate desensitization in DDT1MF-2 and A-431 cell lines, demonstrating counterregulation between prominent protein kinases in desensitization. Using antisense oligodeoxynucleotides to "knock out" target protein kinases in vivo, we reveal distinctive cell-type-specific roles of protein kinase A, protein kinase C, and beta ARK in agonist-induced desensitization.

Animals↗

PubMed1998

Activation of phosphatidylinositol 3-kinase through glycoprotein 130 induces protein kinase B and p70 S6 kinase phosphorylation in cardiac myocytes.

Phosphatidylinositol (PI) 3-kinase is known to be activated by cytokine stimulation through different types of receptors to transduce intracellular responses. We have previously reported that leukemia inhibitory factor (LIF) induces the activation of Janus kinase signal transducer and activator of transcription (JAK-STAT) and mitogen-activated protein (MAP) kinase pathways through glycoprotein (gp) 130 in cardiac myocytes. However, whether PI 3-kinase is involved in regulation of gp130 signaling and the activation mechanisms by which it associates with other tyrosine-phosphorylated proteins remain unknown. We found that LIF induced the activation of PI 3-kinase in cardiac myocytes. Moreover, JAK1 binds to PI 3-kinase, and LIF stimulation increases the PI 3-kinase activity in JAK1 immunoprecipitates. Activation of MAP kinase and protein kinase B by LIF was attenuated by wortmannin. LIF-induced p70 S6 kinase activation, protein synthesis, and c-fos mRNA expression were inhibited by wortmannin and rapamycin. Both inhibitors failed to appreciably affect the phosphorylation of STAT3. In conclusion, PI 3-kinase is activated with LIF in cardiac myocytes, and JAK1 is found to associate with this enzyme. PI 3-kinase provides a crucial link between gp130, MAP kinase, protein kinase B, and p70 S6 kinase in cardiac myocytes.

Androstadienes↗

PubMed2000

Src family kinase activity is required for Kit-mediated mitogen-activated protein (MAP) kinase activation, however loss of functional retinoblastoma protein makes MAP kinase activation unnecessary for growth of small cell lung cancer cells.

Small cell lung cancer (SCLC) is characterized by multiple genetic alterations that include inactivation of the retinoblastoma protein (Rb), the establishment of several autocrine loops including that induced by coexpression of stem cell factor (SCF) and Kit, and the ectopic expression and activation of Src family kinases. Previous studies have shown that Lck associates with, and becomes activated by, Kit after SCF stimulation of SCLC cells. In the present study, we have demonstrated that PP1, a pharmacological inhibitor of Src kinases, blocked SCF-mediated activation of mitogen-activated protein (MAP) kinase, but it also inhibited Kit activation. However, MAP kinase activation was more sensitive than Kit activation to the effects of PP1. Overexpression of Lck reduced the sensitivity of MAP kinase activation to PP1 without altering the sensitivity of Kit activation, which suggested a role for Lck in SCF-mediated MAP kinase activation. Inducible expression of a dominant negative Lck inhibited MAP kinase activation in a dose-dependent manner, which confirmed that Src family kinase activity is required for SCF-induced MAP kinase activation. The growth of cells that expressed dominant negative Lck was unaffected, however, despite the inhibition of MAP kinase. Growth was also unaffected by the inhibition of the MAP kinase pathway using PD 98059, but sensitivity to the MAP/extracellular signal-regulated kinase kinase inhibitor could be partially restored by expression of wild-type Rb. Therefore, MAP kinase activation seems to be dispensable for the growth of SCLC only in the absence of Rb expression. These data suggest that the SCF/Kit autocrine loop, through activation of Lck and subsequently MAP kinase, and the mutational inactivation of Rb contribute to the loss of G1-S phase checkpoint regulation during the pathogenesis of SCLC. Furthermore, the data demonstrate that, in established SCLC cell lines, proliferative signal transduction initiated by Kit is mediated by pathways other than the classic MAP kinase pathway.

Blotting, Western↗

PubMed2003

Mitogen-activated protein kinase kinase inhibitor suppresses cyclin B1 synthesis and reactivation of p34cdc2 kinase, which improves pronuclear formation rate in matured porcine oocytes activated by Ca2+ ionophore.

To investigate the role of mitogen-activated protein (MAP) kinase kinase (MEK)/MAP kinase cascade on p34cdc2 kinase activity and cyclin B1 levels during parthenogenetic activation of porcine oocytes, MEK activity, MAP kinase activity, p34cdc2 kinase activity, and cyclin B1 levels were assayed in mature porcine oocytes after treatment with different concentrations of Ca2+ ionophore. A high concentration of Ca2+ ionophore (50 microM) rapidly reduced MEK activity in oocytes for up to 8 h of culture. MEK activity in the 10-microM treatment group was significantly higher. The low concentration treatment transiently decreased p34cdc2 kinase activity but did not affect MAP kinase activity and ultimately induced reactivation of p34cdc2 kinase via the synthesis of cyclin B1. On the other hand, treatments of a high concentration of Ca2+ ionophore or a low concentration of Ca2+ ionophore plus MEK inhibitor, U0126, linearly decreased MAP kinase activity following the decrease of p34cdc2 kinase activity; most of these oocytes formed pronuclei. These results suggest that decreasing MAP kinase activity is essential to maintaining low p34cdc2 kinase activity resulting from the degradation of cyclin B via a Ca(2+)-dependent pathway; lower activities of both MAP kinase and p34cdc2 kinase induce normal meiotic completion and pronuclear formation of parthenogenetically activated porcine oocytes.

Animals↗

PubMed1995

Phosphorylation and activation of Ca(2+)-calmodulin-dependent protein kinase IV by Ca(2+)-calmodulin-dependent protein kinase Ia kinase. Phosphorylation of threonine 196 is essential for activation.

Purified pig brain Ca(2+)-calmodulin (CaM)-dependent protein kinase Ia kinase (Lee, J. C., and Edelman, A. M. (1994) J. Biol. Chem. 269, 2158-2164) enhances, by up to 24-fold, the activity of recombinant CaM kinase IV in a reaction also requiring Ca(2+)-CaM and MgATP. The addition of brain extract, although capable of activating CaM kinase IV by itself, provides no further activation beyond that induced by purified CaM kinase Ia kinase, consistent with the lack of a requirement of additional components for activation. Activation is accompanied by the development of significant (38%) Ca(2+)-CaM-independent CaM kinase IV activity. In parallel fashion to its activation, CaM kinase IV is phosphorylated in a CaM kinase Ia kinase-, Ca(2+)-CaM-, and MgATP-dependent manner. Phosphorylation occurs on multiple serine and threonine residues with a Ser-P:Thr-P ratio of approximately 3:1. The identical requirements for phosphorylation and activation and a linear relationship between extent of phosphorylation of CaM kinase IV and its activation state indicate that CaM kinase IV activation is induced by its phosphorylation. Replacement of Thr-196 of CaM kinase IV with a nonphosphorylatable alanine by site-directed mutagenesis abolishes both the phosphorylation and activation of CaM kinase IV, demonstrating that Thr-196 phosphorylation is essential for activation.

Amino Acid Sequence↗

PubMed2002

IgA Fc receptor (FcalphaR) cross-linking recruits tyrosine kinases, phosphoinositide kinases and serine/threonine kinases to glycolipid rafts.

The human IgA Fc receptor (FcalphaR, CD89) triggers several important physiological functions, including phagocytosis, NADPH oxidase activation and antigen presentation. Efforts are underway to delineate FcalphaR signal-transduction pathways that control these functions. In a previous study, we demonstrated that cross-linking of FcalphaR increased its partitioning into membrane glycolipid rafts and was accompanied by gamma-chain-dependent recruitment and phosphorylation of the tyrosine kinases Lck/Yes-related novel protein tyrosine kinase (Lyn) and Bruton's tyrosine kinase (Btk). Here we have performed a more extensive characterization of signalling effectors recruited to rafts on FcalphaR cross-linking. We demonstrate that in addition to tyrosine kinases Lyn and Btk, FcalphaR cross-linking also recruits B-lymphocyte kinase (Blk) and spleen tyrosine kinase (Syk) to rafts. We show recruitment of phosphoinositide kinases, including 3-phosphoinositide 3-kinase and phospholipase Cgamma2, and serine/threonine kinases such as protein kinase C (PKC) alpha, PKCepsilon, and protein kinase B (PKB) alpha. This suggests that lipid rafts serve as sites for FcalphaR-triggered recruitment of multiple classes of signalling effectors. We further demonstrate that tyrosine kinases and PKCalpha have a sustained association with rafts, whereas phosphoinositide 3-kinase and its downstream effectors have a transient association with rafts. This is consistent with temporally regulated divergence of FcalphaR signalling pathways in rafts. Furthermore, we suggest the spatial separation of signalling effectors by transport of phosphoinositide 3-kinase, phosphoinositide-dependent kinase 1, PKBalpha and PKCepsilon to endocytic compartments containing internalized FcalphaR.

1-Phosphatidylinositol 4-Kinase↗

PubMed1997

Phosphatidylinositol 3-kinase links the interleukin-2 receptor to protein kinase B and p70 S6 kinase.

Phosphatidylinositol 3-kinase (PI 3-kinase) is activated by the cytokine interleukin-2 (IL-2). We have used a constitutively active PI 3-kinase to identify IL-2-mediated signal transduction pathways directly regulated by PI 3-kinase in lymphoid cells. The serine/threonine protein kinase B (PKB)/Akt can act as a powerful oncogene in T cells, but its positioning in normal T cell responses has not been explored. Herein, we demonstrate that PKB is activated by IL-2 in a PI 3-kinase-dependent fashion. Importantly, PI 3-kinase signals are sufficient for PKB activation in IL-2-dependent T cells, and PKB is a target for PI 3-kinase signals in IL-2 activation pathways. The present study establishes also that PI 3-kinase signals or PKB signals are sufficient for activation of p70 S6 kinase in T cells. PI 3-kinase can contribute to, but is not sufficient for, activation of extracellular signal-regulated kinases (Erks) and Erk effector pathways. Therefore, PI 3-kinase is a selective regulator of serine/threonine kinase signal transduction pathways in T lymphocytes, and this enzyme provides a crucial link between the interleukin-2 receptor, the protooncogene PKB, and p70 S6 kinase.

Enzyme Activation↗

PubMed1992

A purified S6 kinase kinase from Xenopus eggs activates S6 kinase II and autophosphorylates on serine, threonine, and tyrosine residues.

S6 kinases I and II have been purified previously from Xenopus eggs and shown to be activated by phosphorylation on serine and threonine residues. An S6 kinase clone, closely related to S6 kinase II, was subsequently identified and the protein product was expressed in a baculovirus system. Using this protein, termed "rsk" for Ribosomal Protein S6 Kinase, as a substrate, we have purified to homogeneity from unfertilized Xenopus eggs a 41-kDa serine/threonine kinase termed rsk kinase. Both microtubule-associated protein-2 and myelin basic protein are good substrates for rsk kinase, whereas alpha-casein, histone H1, protamine, and phosvitin are not. rsk kinase is inhibited by low concentrations of heparin as well as by beta-glycerophosphate and calcium. Activation of rsk kinase during Xenopus oocyte maturation is correlated with phosphorylation on threonine and tyrosine residues. However, in vitro, rsk kinase undergoes autophosphorylation on serine, threonine, and tyrosine residues, identifying it as a "dual specificity" enzyme. Purified rsk kinase can be inactivated in vitro by either a 37-kDa T-cell protein-tyrosine phosphatase or the serine/threonine protein phosphatase 2A. Phosphatase-treated S6KII can be reactivated by rsk kinase, and S6 kinase activity in resting oocyte extracts increases significantly when purified rsk kinase is added. The availability of purified rsk kinase will enhance study of the signal transduction pathway(s) regulating phosphorylation of ribosomal protein S6 in Xenopus oocytes.

Animals↗

PubMed1997

Evidence that a kinase distinct from protein kinase C and phosphatidylinositol 3-kinase mediates ligation-dependent serine/threonine phosphorylation of the T-lymphocyte co-stimulatory molecule CD28.

The CD28 cytoplasmic tail contains several potential phosphorylation sites for the serine/threonine kinase protein kinase C (PKC) and/or proline-directed serine/threonine kinases, such as extracellular signal-regulated kinases. We demonstrate that ligation of CD28 by B7.1 results in strong serine/threonine phosphorylation of CD28. It is unlikely that ligation-stimulated phosphorylation of CD28 is mediated via activation of PKC, since it was not prevented by pre-treatment of Jurkat cells with inhibitors of PKC, and it was not mimicked by treatment with PKC activators such as PMA. Nevertheless, despite for lack of detectable effects of PMA treatment on CD28 phosphorylation, PMA did partially inhibit the association of CD28 with the putative signalling molecule phosphatidylinositol 3-kinase (PI 3-kinase) and the subsequent accumulation of PtdIns(3,4,5)P3. PI 3-kinase exhibits dual specificity as both a lipid kinase and a protein serine kinase, and site-specific mutagenesis of the Tyr173 residue in the CD28 cytoplasmic tail, which abolishes CD28 coupling to PI 3-kinase [Pages, Ragueneau, Rottapel, Truneh, Nunes, Imbert and Olive (1994) Nature (London) 369, 327-329], also prevents ligation-stimulated phosphorylation of CD28. However, the two PI 3-kinase inhibitors wortmannin and LY294002 had no effect on phosphorylation of CD28 after ligation by B7.1. This study therefore demonstrates that (1) a CD28-activated serine/threonine kinase distinct from both PKC and PI 3-kinase mediates ligation-stimulated CD28 phosphorylation, and (2) the PMA-stimulated down-regulation of the coupling of CD28 to PI 3-kinase is not due to PMA-stimulated phosphorylation of CD28.

B7-1 Antigen↗

PubMed1988

Phosphatidylinositol kinase, phosphatidylinositol-4-phosphate kinase and diacylglycerol kinase activities in rat brain subcellular fractions.

Subcellular fractions isolated and purified from rat brain cerebral cortices were assayed for phosphatidylinositol (PI-), phosphatidylinositol-4-phosphate (PIP-), and diacylglycerol (DG-) kinase activities in the presence of endogenous or exogenously added lipid substrates and [gamma-32P]ATP. Measurable amounts of all three kinase activities were observed in each subcellular fraction, including the cytosol. However, their subcellular profiles were uniquely distinct. In the absence of exogenous lipid substrates, PI-kinase specific activity was greatest in the microsomal and non-synaptic plasma membrane fractions (150-200 pmol/min per mg protein), whereas PIP-kinase was predominantly active in the synaptosomal fraction (136 pmol/min per mg protein). Based on percentage of total protein, total recovered PI-kinase activity was most abundant in the cytosolic, synaptosomal, microsomal and mitochondrial fractions (4-11 nmol/min). With the exception of the microsomal fraction, a similar profile was observed for PIP-kinase activity when assayed in the presence of exogenous PIP (4 nmol/20 mg protein in a final assay volume of 0.1 ml). Exogenous PIP (4 nmol/20 mg protein) inhibited PI-kinase activity in most fractions by 40-70%, while enhancing PIP-kinase activity. PI- and PIP-kinase activities were observed in the cytosolic fraction when assayed in the presence of exogenously added PI or PIP, respectively, but not in heat-inactivated membranes containing these substrates. When subcellular fractions were assayed for DG-kinase activity using heat-inactivated DG-enriched membranes as substrate, DG-kinase specific activity was predominantly present in in the cytosol. However, incubation of subcellular fractions in the presence of deoxycholate resulted in a striking enhancement of DG-kinase activities in all membrane fractions. These findings demonstrate a bimodal distribution between particulate and soluble fractions of all three lipid kinases, with each exhibiting its own unique subcellular topography. The preferential expression of PIP-kinase specific activity in the synaptic membranes is suggestive of the involvement of PIP2 in synaptic function, while the expression of PI-kinase specific activity in the microsomal fraction suggests additional, yet unknown, functions for PIP in these membranes.

1-Phosphatidylinositol 4-Kinase↗

PubMed2004

Death-associated protein kinase (DAP kinase) alteration in soft tissue leiomyosarcoma: Promoter methylation or homozygous deletion is associated with a loss of DAP kinase expression.

The death-associated protein kinase (DAP kinase) was initially identified as a positive mediator of programmed cell death induced by interferon-gamma. To investigate the potential role and the alteration of the DAP kinase gene in soft tissue leiomyosarcoma (LMS), we first searched for homozygous deletion and promoter hypermethylation in 45 LMSs for which genomic DNA was available, using differential PCR and methylation-specific PCR, respectively. Promoter methylation was recognized in 10 of 45 cases (22%), and homozygous deletion was detected in 3 of 45 cases (7%). p53 mutation was detected in 11 of 45 LMS cases (24%). Cases with DAP kinase alteration or p53 mutation showed a close correlation with high French Federation of Cancer Centers grade or with poor prognosis (P = 0.0244, P = 0.0491, respectively). Next, to determine that DAP kinase promoter methylation or homozygous deletion is involved in the down-regulation of DAP kinase expression, we examined the expression of DAP kinase protein by immunohistochemistry. Decreased expression of DAP kinase protein was recognized in 13 of 45 LMS cases (29%). Seven of 13 cases (54%) with decreased expression of DAP kinase protein revealed promoter methylation or homozygous deletion of DAP kinase, and the methylation status or homozygous deletion of its gene showed a close correlation with decreased DAP kinase expression (P = 0.0300). In conclusion, although DAP kinase alteration was relatively rare, DAP kinase alteration and/or p53 mutation may associate with tumor progression in soft-tissue LMSs. Furthermore, although further detailed analyses are necessary, promoter methylation or homozygous deletion status of DAP kinase may present a major alternative mechanism of a loss of or decrease in DAP kinase expression.

Adult↗

PubMed2003

Effect of protein kinase C activator on mitogen-activated protein kinase and p34(cdc2) kinase activity during parthenogenetic activation of porcine oocytes by calcium ionophore.

The objective of this study was to elucidate the role of a [Ca2+]i rise and protein kinase C (PKC) activation on decreases of p34(cdc2) kinase and mitogen-activated protein (MAP) kinase activity during parthenogenetic activation of porcine oocytes. In oocytes treated with 50 microM Ca2+ ionophore, degradations of both p34(cdc2) kinase and MAP kinase activity were observed and half of these oocytes formed pronuclei. However, a supplement of PKC inhibitor, calphostin C, after 50 microM Ca2+ ionophore treatment, was sufficient to inhibit the inactivation of MAP kinase and pronuclear formation in the oocytes. These results showed that PKC played an important role in Ca2+-induced oocyte activation. On the other hand, 10 microM Ca2+ ionophore treatment could not affect the MAP kinase activity but induced a transient decrease of p34(cdc2) kinase activity, which resulted in recovery of p34(cdc2) kinase activity and progression to meiotic metaphase III stage. To investigate the effects of PKC activator on oocytes treated with 10 microM Ca2+ ionophore, matured oocytes were cultured with phorbol 12-myriatate 13-acetate (PMA), after 10 microM Ca2+ ionophore treatment. The additional treatment suppressed the recovery of p34(cdc2) kinase activity and rapidly induced a decrease of MAP kinase activity, and these low activities were maintained until 12-h cultivation. As a result, a significantly higher percentage of these oocytes (67%) had pronuclei at 12-h cultivation. Moreover, PMA treatment without Ca2+ ionophore treatment effectively led to a decrease of MAP kinase activity in a dose-dependent manner but not p34(cdc2) kinase activity in matured porcine oocytes. In conclusion, the parthenogenetic activation of porcine oocytes was mediated by the inactivation of p34(cdc2) kinase via a calcium-dependent pathway and thereafter by the inactivation of MAP kinase via a PKC-dependent pathway.

Animals↗

PubMed2002

Levamisole receptor phosphorylation: effects of kinase antagonists on membrane potential responses in Ascaris suum suggest that CaM kinase and tyrosine kinase regulate sensitivity to levamisole.

A two-micropipette current-clamp technique was used to record electrophysiological responses from the somatic muscle of Ascaris suum. Levamisole and acetylcholine were applied to the bag region of the muscle using a microperfusion system. Depolarizations produced by 10 s applications of 10 micro mol l(-1) levamisole or 20 s applications of 10 micro mol l(-1) acetylcholine were recorded. The effect on the peak membrane potential change of the kinase antagonists H-7, staurosporine, KN-93 and genistein was observed. H-7 (30 micro mol l(-1)), a non-selective antagonist of protein kinases A, C and G but which has little effect on Ca(2+)/calmodulin-dependent kinase II (CaM kinase II), did not produce a significant effect on the peak response to levamisole or acetylcholine. Staurosporine (1 micro mol l(-1)), a non-selective kinase antagonist that has effects on protein kinases A, C and G, CaM kinase and tyrosine kinase, reduced the mean peak membrane potential response to levamisole from 6.8 mV to 3.9 mV (P<0.0001) and the mean response to acetylcholine from 5.5 mV to 2.8 mV (P=0.0016). The difference between the effects of H-7 and staurosporine suggested the involvement of CaM kinase II and/or tyrosine kinase. KN-93, a selective CaM kinase II antagonist, reduced the mean peak response to levamisole from 6.2 mV to 2.7 mV (P=0.035) and the mean peak response of acetylcholine from 4.7 mV to 2.0 mV (P=0.0004). The effects indicated the involvement of CaM kinase II in the phosphorylation of levamisole and acetylcholine receptors. The effect of extracellular Ca(2+) on the response to levamisole was assessed by comparing responses to levamisole in normal and in low-Ca(2+) bathing solutions. The response to levamisole was greater in the presence of Ca(2+), an effect that may be explained by stimulation of CaM kinase II. Genistein (90 micro mol l(-1)), a selective tyrosine kinase antagonist, reduced peak membrane potential responses to levamisole from a mean of 6.4 mV to 3.3 mV (P=0.001). This effect indicated the involvement of tyrosine kinase in maintaining the receptor.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1988

Phosphorylation of myocardial fructose-6-phosphate,2-kinase: fructose-2,6-bisphosphatase by cAMP-dependent protein kinase and protein kinase C. Activation by phosphorylation and amino acid sequences of the phosphorylation sites.

Phosphorylation of pure fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase from bovine heart by cAMP-dependent protein kinase and protein kinase C was investigated. The major enzyme form (subunit Mr of 58,000) was rapidly phosphorylated by both cAMP-dependent protein kinase and protein kinase C, incorporating 0.8 and 1.0 mol/mol of subunit, respectively. The rate of phosphorylation of the heart enzyme by cAMP-dependent protein kinase was 10 times faster than that of the rat liver enzyme. The minor enzyme (subunit Mr of 54,000), however, was phosphorylated only by protein kinase C and was phosphorylated much more slowly with a phosphate incorporation of less than 0.1 mol/mol of subunit. Phosphorylation by either cAMP-dependent protein kinase or protein kinase C activated the enzyme, but each phosphorylation affected different kinetic parameters. Phosphorylation by cAMP-dependent protein kinase lowered the Km value for fructose 6-phosphate from 87 to 42 microM without affecting the Vmax, whereas the phosphorylation by protein kinase C increased the Vmax value from 55 to 85 milliunits/mg without altering the Km value. The phosphorylated peptides were isolated, and their amino acid sequences were determined. The phosphorylation sites for both cAMP-dependent protein kinase and protein kinase C were located in a single peptide whose sequence was Arg-Arg-Asn-Ser-(P)-Phe-Thr-Pro-Leu-Ser-Ser-Ser-Asn-Thr(P)-Ile-Arg-Arg-Pro. The seryl residue nearest the N terminus was the residue specifically phosphorylated by cAMP-dependent protein kinase, whereas the threonine residue nearest the C terminus was phosphorylated by protein kinase C.

Amino Acid Sequence↗

PubMed2006

Phosphorylation of Rho-associated kinase (Rho-kinase/ROCK/ROK) substrates by protein kinases A and C.

Rho-associated kinase (Rho-kinase/ROCK/ROK) is a serine/threonine kinase and plays an important role in various cellular functions. The cAMP-dependent protein kinase (protein kinase A/PKA) and protein kinase C (PKC) are also serine/threonine kinases, and directly and/or indirectly take part in the signal transduction pathways of Rho-kinase. They have similar phosphorylation site motifs, RXXS/T and RXS/T. The purpose of this study was to identify whether sites phosphorylated by Rho-kinase could be targets for PKA and PKC and to find peptide substrates that are specific to Rho-kinase, i.e., with no phosphorylation by PKA and PKC. A total of 18 substrates for Rho-kinase were tested for phosphorylation by PKA and PKC. Twelve of these sites were easily phosphorylated. These results mean that Rho-kinase substrates can be good substrates for PKA and/or PKC. On the other hand, six Rho-kinase substrates showing no or very low phosphorylation efficiency (<20%) for PKA and PKC were identified. Kinetic parameters (K(m) and k(cat)) showed that two of these peptides could be useful as substrates specific to Rho-kinase phosphorylation.

Amino Acid Motifs↗

PubMed1997

Possible involvement of differential splicing in regulation of the activity of Arabidopsis ANP1 that is related to mitogen-activated protein kinase kinase kinases (MAPKKKs).

Three types of Arabidopsis cDNA (cANP1, cANP2 and cANP3) have been isolated that encode putative protein kinases, designated ANP1, ANP2 and ANP3. These kinases exhibit a high degree of homology to NPK1, a tobacco protein that is a member of the family of mitogen-activated protein kinase kinase kinases (MAPKKKs), which appears to function in the proliferation of tobacco cells. The predicted amino acid sequences of the kinase domains in the amino-terminal halves of the ANPs were more than 80% identical to that of NPK1, while the kinase-unrelated regions in the carboxy-terminal halves exhibited relatively low homology. Two species of cANP1 were identified, ANP1L cDNA (cANP1L) and ANP1S cDNA (cANP1S), which were derived from a single ANP1 gene: the former had an intron-like sequence in the coding region for the kinase-unrelated region, while the latter did not include such an intron-like sequence. cANP1L encoded a putative protein with both kinase and kinase-unrelated domains, resembling NPK1, whereas cANP1S encoded only the amino-terminal kinase domain because the intron-like sequence was absent, with resulting elimination of most of the kinase-unrelated region. Genetic analysis with mutant yeast cells showed that over-expression of cANP1L or of cANP1S activated the mating pheromone-responsive signal pathway which is mediated by a MAP kinase cascade. Moreover, the extent of such activation by cANP1S was greater than that by cANP1L. These results predict that differential splicing of the intron-like sequence in the ANP1 transcript might be at least one of the molecular mechanisms involved in the generation of active ANP1 protein kinase.

Alternative Splicing↗