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PubMed2000

Follicle-Stimulating hormone (FSH) stimulates phosphorylation and activation of protein kinase B (PKB/Akt) and serum and glucocorticoid-lnduced kinase (Sgk): evidence for A kinase-independent signaling by FSH in granulosa cells.

FSH stimulates in ovarian granulosa cells diverse, differentiation-dependent responses that implicate activation of specific cellular signaling cascades. In these studies three kinases were investigated to determine their relationship to FSH, cAMP, and A kinase signaling: protein kinase B (PKB/Akt), serum and glucocorticoid-induced kinase (Sgk), and p38 mitogen-activated protein kinase (p38MAPK). The phosphorylation (activation) of these kinases was analyzed by using selective agonists/inhibitors: forskolin/H89 for cAMP-dependent protein kinase (A kinase), insulin-like growth factor I (IGF-I)/LY294002 and wortmannin for phosphatidylinositol-dependent kinase (PI3-K), and phorbol myristate (PMA)/GF109203X for diacylglycerol and Ca++-dependent kinases (C kinases). An inhibitor (PD98059) of MEK1, which regulates extracellular regulated kinases (ERKs), and SB203580, which inhibits p38MAPK, were also used. In addition, we analyzed the expression of the recently described, cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFI and GEFII) that impact Ras-related GTPases and Raf kinases, known regulators of various protein kinase cascades. We provide evidence that FSH, forskolin, and 8-bromo-cAMP stimulate phosphorylation of PKB by mechanisms involving PI3-K (LY294002/wortmannin sensitive) not A kinase (H89 insensitive), a pattern of response mimicking that of IGF-I. In contrast, FSH induction and phosphorylation of Sgk protein requires A kinase (H89 sensitive) but also involves PI3-K (LY294002 sensitive) as well as p38MAPK (SB203580 sensitive) pathways. PMA (C kinase) abolished FSH-mediated (but not IGF-I-mediated) phosphorylation of PKB at a step(s) upstream of PI3-K and independent of A kinase. Lastly, FSH-mediated phosphorylation of p38MAPK is negatively affected by A kinase and PI3-K, suggesting that it may be downstream of specific members of the cAMP-GEF/Rap/Raf pathway. We propose that cAMP activation of A kinase is obligatory for transcription of Sgk in granulosa cells whereas cAMP (IGF-I-like)-mediated phosphorylation (activation) of PKB and Sgk (via PI3-K), as well as p38MAPK, involves other cellular events. These results provide new and exciting evidence that cAMP acts in granulosa cells by A kinase-dependent and -independent mechanisms, each of which controls specific kinase cascades.

Animals↗

PubMed1984

Phosphorylation of mammalian myosin light chain kinases by the catalytic subunit of cyclic AMP-dependent protein kinase and by cyclic GMP-dependent protein kinase.

The phosphorylation of the calmodulin-dependent enzyme myosin light chain kinase, purified from bovine tracheal smooth muscle and human blood platelets, by the catalytic subunit of cAMP-dependent protein kinase and by cGMP-dependent protein kinase was investigated. When myosin light chain kinase which has calmodulin bound is phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, 1 mol of phosphate is incorporated per mol of tracheal myosin light chain kinase or platelet myosin light chain kinase, with no effect on the catalytic activity. Phosphorylation when calmodulin is not bound results in the incorporation of 2 mol of phosphate and significantly decreases the activity. The decrease in myosin light chain kinase activity is due to a 5 to 7-fold increase in the amount of calmodulin required for half-maximal activation of both tracheal and platelet myosin light chain kinase. In contrast to the results with the catalytic subunit of cAMP-dependent protein kinase, cGMP-dependent protein kinase cannot phosphorylate tracheal myosin light chain kinase in the presence of bound calmodulin. When calmodulin is not bound to tracheal myosin light chain kinase, cGMP-dependent protein kinase phosphorylates only one site, and this phosphorylation has no effect on myosin light chain kinase activity. On the other hand, cGMP-dependent protein kinase incorporates phosphate into two sites in platelet myosin light chain kinase when calmodulin is not bound. The sites phosphorylated by the two cyclic nucleotide-dependent protein kinases were compared by two-dimensional peptide mapping following extensive tryptic digestion of the phosphorylated myosin light chain kinases. With respect to the tracheal myosin light chain kinase, the single site phosphorylated by cGMP-dependent protein kinase when calmodulin is not bound appears to be the same site phosphorylated in the tracheal enzyme by the catalytic subunit of cAMP-dependent protein kinase when calmodulin is bound. With respect to the platelet myosin light chain kinase, the additional site that was phosphorylated by cGMP-dependent protein kinase when calmodulin was not bound was different from that phosphorylated by the catalytic subunit of cAMP-dependent protein kinase.

Blood Platelets↗

PubMed1998

Phosphatidylinositol 3'-kinase associates with an insulin receptor substrate-1 serine kinase distinct from its intrinsic serine kinase.

Serine phosphorylation of insulin receptor substrate-1 (IRS-1) has been proposed as a counter-regulatory mechanism in insulin and cytokine signalling. Here we report that IRS-1 is phosphorylated by a wortmannin insensitive phosphatidylinositol 3'-kinase (PI 3-kinase)-associated serine kinase (PAS kinase) distinct from PI 3-kinase serine kinase. We found that PI 3-kinase immune complexes contain 5-fold more wortmannin-insensitive serine kinase activity than SH2-containing protein tyrosine phosphatase-2 (SHP2) and IRS-1 immune complexes. Affinity chromatography of cell lysates with a glutathione S-transferase fusion protein for the p85 subunit of PI 3-kinase showed that PAS kinase associated with the p85 subunit of PI 3-kinase. This interaction required unoccupied SH2 domain(s) but did not require the PI 3-kinase p110 subunit binding domain. In terms of function, PAS kinase phosphorylated IRS-1 and, after insulin stimulation, PAS kinase phosphorylated IRS-1 in PI 3-kinase-IRS-1 complexes. Phosphopeptide mapping showed that insulin-dependent in vivo sites of IRS-1 serine phosphorylation were comparable to those of PAS kinase phosphorylated IRS-1. More importantly, PAS kinase-dependent phosphorylation of IRS-1 reduced by 4-fold the ability of IRS-1 to act as an insulin receptor substrate. Taken together, these findings indicate that: (a) PAS kinase is distinct from the intrinsic serine kinase activity of PI 3-kinase, (b) PAS kinase associates with the p85 subunit of PI 3-kinase through SH2 domain interactions, and (c) PAS kinase is an IRS-1 serine kinase that can reduce the ability of IRS-1 to serve as an insulin receptor substrate.

Androstadienes↗

PubMed1992

Sequential activation of MAP kinase activator, MAP kinases, and S6 peptide kinase in intact rat liver following insulin injection.

An insulin-stimulated phosphorylation cascade was examined in rat liver after insulin injection via a portal vein by the use of immune complex kinase assays specific to the mitogen-activated protein (MAP) kinase and S6 kinase II homologue (rsk) kinase. We have prepared an antibody against the peptide consisting of a carboxyl-terminal portion of the extracellular signal-regulated kinase 1 (alpha C92), one of the MAP kinases, and an antibody against the peptide consisting of the carboxyl terminus of the mouse S6 kinase II homologue (alpha rsk(m)C). In alpha C92 immune complex assay, maximal activation of rat liver MAP kinases (approximately 4.3-fold) were observed 4.5 min after insulin injection. We also observed an insulin-stimulated MAP kinase activity (approximately 3-fold) in liver extracts from insulin-treated rat in fractions eluted from phenyl-Sepharose with 30-50% ethylene glycol. Kinase assay in myelin basic protein (MBP)-containing gel after sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by denaturation with 6 M guanidine HCl, and renaturation revealed that insulin injection stimulated the kinase activity of the 42- and 44-kDa proteins, which corresponded to the two distinct MAP kinases. In alpha rsk(m)C immune complex assay, maximal stimulation (approximately 5-fold) of the S6 peptide (Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala) kinase activity was observed 7.5 min after insulin injection. In addition, MAP kinases purified from insulin-treated rat liver were able to activate S6 peptide kinase activity in vitro in alpha rsk(m)C immunoprecipitates from untreated rat liver, accompanied by the appearance of several phosphorylated bands including a major band at 88 kDa. We also examined whether insulin injection stimulates the MAP kinase activator (Ahn, N. G., Seger, R., Bratlien, R. L., Diltz, C. D., Tonks, N. K., and Krebs, E. G. (1991) J. Biol. Chem. 266, 4220-4227) in rat liver. Using recombinant Xenopus MAP kinase, fractions of Q-Sepharose eluted early in the NaCl gradient were found to have MAP kinase activator activity accompanied by the phosphorylation of 42-kDa recombinant Xenopus MAP kinase. From these data, we demonstrate three tiers of a cascade composed of the MAP kinase activator, MAP kinases, and an S6 peptide kinase activity in rat liver under physiological conditions in the intact animal.

Adenosine Triphosphate↗

PubMed1994

The level of insulin receptor tyrosine kinase activity modulates the activities of phosphatidylinositol 3-kinase, microtubule-associated protein, and S6 kinases.

The role of insulin receptor tyrosine kinase activity in stimulation of intracellular enzymes linked to insulin action [phosphatidylinositol 3-kinase (PtdIns 3-kinase), microtubule-associated protein (MAP) kinase, and S6 kinases] was studied in Chinese hamster ovary cells which overexpress wild type human insulin receptors, receptors with reduced kinase activity due to substitution of Phe for Tyr1146 (single-Phe), Tyr1150,1151 (double-Phe), and Tyr1146,1150,1151 (triple-Phe), or kinase-inactive receptors with a substitution of Ala for Lys1018 in the ATP binding site (A1018). We have previously shown that receptor autophosphorylation and kinase activity of these mutants were reduced by approximately 50, 65, 85, and 100%, respectively. Glycogen and DNA synthesis parallel the level of receptor autophosphorylation and kinase activity; however, receptor serine and threonine phosphorylation was independent of receptor tyrosine kinase activity and receptor internalization was completely dependent on maximal receptor kinase activity. Overexpression of the wild type insulin receptor increased both maximal insulin receptor substrate-1-associated and total insulin-stimulated PtdIns 3-kinase activity, as well as S6 and MAP kinase activities 2.0- to 3.6-fold. In addition there was a leftward shift of the dose-response curves for PtdIns 3-kinase and S6 kinases by approximately 10-fold. Expression of the single- and double-Phe mutant receptors also enhanced maximal PtdIns 3-kinase activity, but had no effect on insulin sensitivity, whereas expression of either the triple-Phe or kinase-inactive receptors did not enhance insulin stimulation or increase insulin sensitivity as compared to the control cells. When comparing the mutant and wild type receptors, differences in insulin sensitivity were least for insulin-stimulated MAP kinase and greatest for S6 kinase; with the latter there was greater than a 1000-fold difference in insulin sensitivity when cells that overexpress wild type vs. kinase-inactive insulin receptors were compared. Thus, the level of insulin receptor tyrosine autophosphorylation and kinase activity regulate both maximal activation and insulin sensitivity of these intracellular kinases in the insulin action pathway which may lead to glycogen and/or DNA synthesis. The differential sensitivity of these enzymes to changes in receptor activation suggests that they may be differently coupled to the receptor kinase.

Amino Acid Sequence↗

PubMed1997

Differential activation of mitogen-activated protein kinase by insulin and epidermal growth factor in 3T3-L1 adipocytes: a possible involvement of PI3-kinase in the activation of the MAP kinase by insulin.

Mitogen-activated protein (MAP) kinase plays crucial roles in cell growth and differentiation. It has recently been shown that the MAP kinase cascade in growth factor signaling diverges and cross-talks with other signaling pathways. In the present study, we examined the effects of wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3-kinase), on the activation of Ras, Raf-1 kinase, and MAP kinase by insulin and epidermal growth factor (EGF). The effect of LY294002, a structurally distinct PI3-kinase inhibitor, on the activation of Raf-1 kinase by both ligands was also examined. In 3T3-L1 adipocytes, 25 nmol/l wortmannin inhibited the insulin-induced activation of Raf-1 kinase to the basal level, whereas the same dose of wortmannin had little effect on the EGF-induced activation of Raf-1 kinase. One hundred micromol/l LY294002 blocked insulin-induced activation of Raf-1 kinase without affecting EGF-induced activation of this kinase. Twenty-five nmol/l wortmannin inhibited the insulin-induced activation of MAP kinase to the basal level with no effect on the EGF-induced activation of this kinase. But the same dose of wortmannin did not affect the formation of guanosine 5'-triphosphate (GTP)-bound Ras stimulated by either ligand. In KB cells, results similar to those in 3T3-L1 adipocytes were obtained. In contrast, in Chinese hamster ovary cells overexpressing the human insulin receptor (CHO-HIR cells), neither wortmannin nor LY294002 inhibited the insulin-induced activation of Raf-1 kinase, and wortmannin had little effect on the activation of MAP kinase by insulin. These results indicate that 1) PI3-kinase or wortmannin-sensitive molecules are involved in the interaction between activated Ras and Raf-1 kinase in the insulin signaling in 3T3-L1 adipocytes, 2) the involvement of PI3-kinase or wortmannin-sensitive molecules in the insulin-induced activation of MAP kinase appears to be cell-type specific, and 3) differential mechanisms to activate Raf-1 kinase and MAP kinase by insulin and EGF exist.

3T3 Cells↗

PubMed1999

Platelet-derived growth factor-BB (PDGF-BB) regulation of migration and focal adhesion kinase phosphorylation in rabbit aortic vascular smooth muscle cells: roles of phosphatidylinositol 3-kinase and mitogen-activated protein kinases.

OBJECTIVE: Phosphatidylinositol 3'-kinase (PI3-kinase) is implicated in cell migration and focal adhesion kinase (FAK) phosphorylation. In contrast, it has been proposed that mitogen-activated protein (MAP) kinases are essential for proliferation but may be dissociated from chemotactic signalling. We investigated the roles of PI3-kinase and p42/p44 MAP kinases in cell migration and FAK tyrosine phosphorylation induced by platelet-derived growth factor-BB (PDGF-BB) in rabbit aortic vascular smooth muscle cells (VSMCs). The roles of PI3-kinase and MAP kinase pathways in the chemotactic response to insulin-like growth factor-I (IGF-I) were also examined. METHODS: The roles of PI3-kinase and p42/p44 MAP kinases were assessed using the PI3-kinase inhibitors, wortmannin and LY294002, and an inhibitor of MAP kinase kinase, PD98059. PI3-kinase activity was measured by phosphatidylinositol phosphorylation in anti-phosphotyrosine immunoprecipitates and by thin layer chromatography of phosphorylated products. Phosphorylation was assessed by immunoprecipitation with anti-phosphotyrosine antibodies and Western blotting with FAK-specific antibody. Migration was evaluated in a chemotaxis chamber using polycarbonate filters with an 8-mm pore size. RESULTS: Neither wortmannin nor LY294002 significantly reduced PDGF-BB stimulation of FAK tyrosine phosphorylation, chemotaxis or immunofluorescent staining of focal adhesions in VSMCs. PD98059, a specific inhibitor of MAP kinase activation, did not inhibit FAK tyrosine phosphorylation but markedly inhibited the migratory response of VSMCs to PDGF-BB. IGF-I also stimulated migration of VSMCs, and, relative to the effect of PDGF-BB, induced smaller increases in PI3-kinase and MAP kinase activities. Both wortmannin and PD98059 partially inhibited the migratory response to IGF-I. CONCLUSIONS: PDGF-BB stimulation of both FAK tyrosine phosphorylation and migration in VSMCs are not dependent on activation of PI3-kinase. While PDGF-BB stimulation of FAK tyrosine phosphorylation is not dependent on p42/p44 MAP kinase activation, PDGF-BB and IGF-I both stimulate p42/p44 MAP kinase activity and the chemotactic response to these factors is partially dependent on MAP kinase activation.

Androstadienes↗

PubMed1993

Phosphorylation and activation of human tyrosine hydroxylase in vitro by mitogen-activated protein (MAP) kinase and MAP-kinase-activated kinases 1 and 2.

Mitogen-activated protein-kinase (MAP) kinase-activated protein kinases 1 and 2 (MAPKAP kinase-1, MAPKAP kinase-2), were found to phosphorylate bacterially expressed human tyrosine hydroxylase in vitro at comparable rates to other proteins thought to be physiological substrates of these protein kinases. The phosphorylation of all four alternatively spliced forms of human tyrosine hydroxylase by MAPKAP kinases-1 and -2 reached plateau values at 1 mol/mol subunit and 2 mol/mol subunit, respectively; the sites of phosphorylation were identified as Ser40 (MAPKAP kinase-1) and Ser19 and Ser40 (MAPKAP kinase-2). In contrast to calmodulin-dependent protein kinase-II, which phosphorylates Ser19 faster than Ser40, MAPKAP kinase-2 phosphorylated Ser40 about twice as fast as Ser19. The maximal activation of tyrosine hydroxylase by MAPKAP kinase-1 or-2 was about 3-fold, and activation by MAPKAP kinases-1 and -2 or calmodulin-dependent protein kinase-II correlated with the extent of phosphorylation of Ser40. The four alternatively spliced forms of human tyrosine hydroxylase were phosphorylated at Ser31 by MAP kinase, but at markedly different rates (3 = 4 > 1 >> 2). Forms 3 and 4 were phosphorylated rapidly and stoichiometrically by MAP kinase doubling the activity, while phosphorylation of form 1 by MAP kinase to 0.4 mol/mol subunit increased activity by 40%. The effect on activity of phosphorylating both Ser31 and Ser40 was not additive. The possible roles of MAPKAP kinase-1, MAPKAP kinase-2 and MAP kinase in the regulation of tyrosine hydroxylase in vivo are discussed.

Alternative Splicing↗

PubMed1995

Characterization of a Ca2+/calmodulin-dependent protein kinase cascade. Molecular cloning and expression of calcium/calmodulin-dependent protein kinase kinase.

Recent studies have demonstrated that Ca2+/calmodulin-dependent protein kinase IV (CaM-kinase IV) can mediate Ca(2+)-dependent regulation of gene expression through the phosphorylation of transcriptional activating proteins. We have previously identified and purified a 68-kDa rat brain CaM-kinase kinase that phosphorylates and increases total and Ca(2+)-independent activities of CaM-kinase IV (Tokumitsu, H., Brickey, D. A., Gold, J., Hidaka, H., Sikela, J., and Soderling, T. R. (1994) J. Biol. Chem. 269, 28640-28647). Using a partial amino acid sequence of the purified brain kinase, a CaM-kinase kinase cDNA was cloned from a rat brain cDNA library. Northern blot analysis showed that CaM-kinase kinase mRNA (3.4 kilobases) was expressed in rat brain, thymus, and spleen. Sequence analyses revealed that the cDNA encoded a 505-amino acid protein, which contained consensus protein kinase motifs and was 30-40% homologous with members of the CaM-kinase family. Expression of the cDNA in COS-7 cells yielded an apparent 68-kDa CaM-binding protein, which catalyzed in vitro activation in the presence of Mg2+/ATP and Ca2+/ CaM of CaM-kinases I and IV but not of CaM-kinase II. Co-expression of CaM-kinase kinase with CaM-kinase IV gave a 14-fold enhancement of cAMP-response element-binding protein-dependent gene expression compared with CaM-kinase IV alone. These results are consistent with the hypothesis that CaM-kinases I and IV are regulated through a unique signal transduction cascade involving CaM-kinase kinase.

Amino Acid Sequence↗

PubMed1995

Full activation of brain calmodulin-dependent protein kinase IV requires phosphorylation of the amino-terminal serine-rich region by calmodulin-dependent protein kinase IV kinase.

Rat brain calmodulin-dependent protein kinase IV (CaM-kinase IV) was activated approximately 20 to 30-fold by incubation with CaM-kinase IV kinase purified from rat brain under the Ca2+/calmodulin-dependent phosphorylation conditions. When CaM-kinase IV was incubated without CaM-kinase IV kinase, no significant activation was observed, indicating that the marked activation of CaM-kinase IV occurred as a result of the action of CaM-kinase IV kinase. More than 3 mol of phosphate were incorporated into 1 mol of the enzyme after incubation with CaM-kinase IV kinase at 30 degrees C for 20 min, but the activation occurred upon the initial incorporation of 1 mol of phosphate. The rate of the initial phosphorylation increased when the amount of CaM-kinase IV kinase added into the reaction mixture increased, but the rate of phosphorylation following the initial phosphorylation did not increase, suggesting that the initial phosphorylation was catalyzed by CaM-kinase IV kinase, and that subsequent phosphorylation was catalyzed by CaM-kinase IV itself activated by CaM-kinase IV kinase. The initial phosphorylation occurred in the amino-terminal serine-rich region of CaM-kinase IV. Kinetic analysis revealed that the increase in the activity upon phosphorylation was due mainly to an increase in the Vmax values.

Amino Acid Sequence↗

PubMed1994

Activation mechanisms for Ca2+/calmodulin-dependent protein kinase IV. Identification of a brain CaM-kinase IV kinase.

This manuscript examines the mechanisms by which Ca2+/calmodulin-dependent protein kinase IV (CaM-kinase IV) is activated through the binding of Ca2+/CaM and by phosphorylation. Studies with the synthetic autoinhibitory domain peptides of CaM-kinase II indicate that CaM-kinase IV has a similarly located autoinhibitory domain, and this was confirmed since site-directed mutagenesis of this region (HMDT308 to DEDD and FN317 to DD) generated fully active Ca2+/CaM-independent kinases. Total activities of purified, baculovirus-expressed wild type and mutant kinases were increased 2-fold by intramolecular autophosphorylation, but this reaction was extremely slow (1-2 h) and probably not physiological. However, CaM-kinase IV can be activated by brain CaM-kinase IV kinase resulting in large increases in both total (5-7-fold) and Ca2+/CaM-independent (> 20-fold) CaM-kinase IV activities. This activation reaction required Mg2+/ATP and Ca2+/CaM, was intermolecularly catalyzed, and was reversed by protein phosphatase 2A. Activation of CaM-kinase IV resulted in a 10-fold decrease in Km for syntide-2 with little effect on Km for ATP or Vmax. CaM-kinase IV kinase was highly purified from rat brain extract and was shown to be a 68-kDa monomer. The results of this study demonstrate that CaM-kinase IV does have an autoinhibitory domain within residues His305-Lys321 that suppresses kinase activity in the absence of Ca2+/CaM. CaM-kinase IV is not significantly activated by autophosphorylation, but it can be activated 10-fold by a CaM-kinase IV kinase. This kinase cascade activation mechanism may be important for the physiological function of CaM-kinase IV such as transcriptional regulation through phosphorylation of cAMP responsive element binding protein (Enslen, H., Sun, P., Brickey, D., Soderling, S. H., Klamo, E., and Soderling, T.R. (1994) J. Biol. Chem. 269, 15520-15527).

Amino Acid Sequence↗

PubMed1993

Diacylglycerol kinase is phosphorylated in vivo upon stimulation of the epidermal growth factor receptor and serine/threonine kinases, including protein kinase C-epsilon.

In signal transduction, diacylglycerol (DG) kinase attenuates levels of the second messenger DG by converting it to phosphatidic acid. A previously cloned full-length human 86 kDa DG kinase cDNA was expressed as fusion protein in Escherichia coli, to aid in the generation of DG-kinase-specific monoclonal antibodies suitable for immunoprecipitation experiments. To investigate whether phosphorylation of DG kinase is a possible mechanism for its regulation, COS-7 cells were transiently transfected with the DG kinase cDNA and phosphorylation of the expressed DG kinase was induced by various stimuli. Activation of both cyclic AMP-dependent protein kinase and protein kinase C (PKC) resulted in phosphorylation of DG kinase on serine residues in vivo, and both kinases induced this phosphorylation within the same tryptic phosphopeptide, suggesting that they may exert similar control over DG kinase. No phosphorylation was observed upon ionomycin treatment, intended to activate Ca2+/calmodulin-dependent kinases. Co-transfections of DG kinase with either PKC-alpha or PKC-epsilon cDNA revealed that both protein kinases, when stimulated, are able to phosphorylate DG kinase. For PKC-epsilon, DG kinase is the first in vivo substrate identified. Stimulation with epidermal growth factor (EGF) of COS-7 cells transfected with both DG kinase and EGF-receptor cDNA results mainly in phosphorylation of DG kinase on tyrosine. Since the EGF receptor has an intrinsic tyrosine kinase activity, this finding implies that DG kinase may be a direct substrate for the activated EGF receptor.

1-Methyl-3-isobutylxanthine↗

PubMed1995

Angiotensin II and other hypertrophic stimuli mediated by G protein-coupled receptors activate tyrosine kinase, mitogen-activated protein kinase, and 90-kD S6 kinase in cardiac myocytes. The critical role of Ca(2+)-dependent signaling.

Many hypertrophic stimuli such as angiotensin II (Ang II) activate phospholipases through G protein-coupled receptors in cardiac myocytes. However, it is not known whether these stimuli also activate the tyrosine phosphorylation-dependent signaling pathway, which plays an essential role in growth factor-induced mitogenic responses in other cell types. Serine/threonine kinases such as mitogen-activated protein (MAP) kinases and 90-kD S6 kinase (RSK) are activated in response to many growth stimuli and are important downstream signaling pathways of tyrosine kinases. Therefore, we examined whether Ang II activates these protein kinases in primary cultures of cardiac myocytes and fibroblasts from neonatal rats. Ang II rapidly induced tyrosine phosphorylation of multiple proteins, including 42-, 44-, 75- to 80-, and 120- to 130-kD proteins, in both cardiac myocytes and fibroblasts. This was accompanied by an increase in tyrosine kinase activity. The 42- and 44-kD proteins were immunologically related to an extracellular signal-regulated kinase family (MAP kinases). Ang II rapidly increased kinase activity of MAP kinases and their downstream kinase, RSK. The Ang II-induced tyrosine phosphorylation and activation of MAP kinases and RSK were AT1 receptor-mediated. Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate or an increase in intracellular Ca2+ by the Ca2+ ionophore A23187 was sufficient to cause tyrosine phosphorylation of multiple proteins and activation of MAP kinase and RSK. Although downregulation of PKC did not suppress Ang II-induced activation of MAP kinase and RSK, chelating intracellular Ca2+ by BAPTA-AM completely abolished Ang II-induced activation of these kinases. Activation of MAP kinases and RSK was also observed in myocytes stimulated with other agonists for Gq protein-coupled receptors, such as phenylephrine, norepinephrine, and endothelin 1, but not with agonists to Gs protein-coupled receptors, such as isoproterenol. These results suggest that Ang II and other hypertrophic stimuli, known to act through Gq protein-coupled receptors, rapidly cause tyrosine phosphorylation of several intracellular substrates through activation of tyrosine kinase and activate MAP kinases and RSK in cardiac myocytes as well as in cardiac fibroblasts. Furthermore, intracellular Ca2+, rather than PKC, seems to be critical for Ang II-induced activation of these protein kinases in cardiac myocytes.

Angiotensin II↗

PubMed1995

Increased expression and subcellular translocation of the mitogen activated protein kinase kinase and mitogen-activated protein kinase in Alzheimer's disease.

The sequential activation of the mitogen-activated protein kinase kinase and its substrate, the mitogen-activated protein kinase is involved in a cascade of protein kinases which link a number of cell surface signals to intracellular changes in enzyme activity and gene expression. In vitro, mitogen-activated protein kinase is able to phosphorylate the microtubule-associated protein tau at Ser-Pro and Thr-Pro sites, thereby generating abnormally hyperphosphorylated tau species that are similar to paired helical filament-tau found in Alzheimer's disease. In the present study, we analysed the levels of immunoreactive mitogen-activated protein kinase kinase and mitogen-activated protein kinase in the temporal cortex (area 22) of patients with Alzheimer's disease by means of enzyme-linked immuno-sorbent assays and compared these changes with the content of abnormally phosphorylated paired helical filament-tau. The levels of immunochemically detected mitogen-activated protein kinase kinase and mitogen-activated protein kinase were both increased in Alzheimer's disease by between 35 and 40% compared with age-matched controls. Elevation of mitogen-activated protein kinase kinase was most pronounced during early stages of Alzheimer's disease and was inversely related to the tissue content of abnormally phosphorylated paired helical filament-tau. Pronounced immunoreactivity of mitogen-activated protein kinase kinase and mitogen-activated protein kinase was present in both tangle bearing neurons and unaffected neurons of the temporal cortex. Immunoreactive neurons were most often localized in the direct vicinity of neuritic plaques. In Alzheimer's disease, the subcellular distribution of mitogen-activated protein kinase kinase and mitogen-activated protein kinase showed a striking translocation from the cytoplasmic to the nuclear compartment. It is suggested that the activation of the mitogen-activated protein kinase cascade which appears to be an early feature of Alzheimer's disease might be critically involved in self-stimulating processes of neurodegeneration and aberrant repair under these conditions.

Aged↗

PubMed1994

Interleukin-1 activates p54 mitogen-activated protein (MAP) kinase/stress-activated protein kinase by a pathway that is independent of p21ras, Raf-1, and MAP kinase kinase.

In KB epidermoid cells, we previously showed that interleukin-1 alpha (IL-1) and various mitogens activate the mitogen-activated protein (MAP) kinases ERK1 and ERK2, which phosphorylate both myelin basic protein (MBP) and a peptide containing Thr669 of the epidermal growth factor receptor. In cell-free extracts made from gingival fibroblasts treated with platelet-derived growth factor or HepG2 hepatoma cells stimulated with phorbol myristate acetate, MBP and Thr669 kinase were both elevated 4-fold, and ERK1 and ERK2 were tyrosine-phosphorylated. In these cells IL-1 activated a kinase(s) that phosphorylated Thr669 peptide but not MBP and failed to cause tyrosine phosphorylation of ERK1/ERK2. Ceramide has been proposed as an intracellular mediator of IL-1 action, but C2-ceramide or sphingosine stimulated predominantly MBP-specific kinase activity in fibroblasts and had no effect in HepG2 cells. p54 MAP kinase (also called stress-activated protein kinase) is a c-Jun kinase first isolated from livers of cycloheximide-treated rats. After IL-1 stimulation, immunoprecipitates of lysates made from all three cell types with specific anti-p54 MAP kinase serum contained Thr669 and c-Jun phosphorylating activity, whereas precipitates from unstimulated cells contained no detectable p54 kinase activity. The major peak of IL-1-stimulated HepG2 Thr669 kinase activity co-chromatographed on Mono Q and phenyl-Superose with immunodetectable p54 MAP kinase. IL-1 did not cause p21ras activation in any cell type. Induction of Thr 669 kinase activity was not abrogated by elevation of cAMP levels, which has been shown to interfere with the activation of Raf-1. We could not detect MAP kinase kinase phosphorylating activity in unfractionated lysates made from IL-1-stimulated fibroblasts or HepG2 cells. KB cells contained a small amount of this activity, but it was not precipitated with an anti-Raf-1 antibody. We conclude that most of the IL-1-activated Thr669 kinase activity in fibroblasts and HepG2 cells, and a portion in KB cells, is due to p54 MAP kinase and that its activation is Ras-, Raf-, and MAP kinase kinase-independent.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1994

Activation of rat liver AMP-activated protein kinase by kinase kinase in a purified, reconstituted system. Effects of AMP and AMP analogues.

AMP-activated protein kinase, purified from rat liver as far as the diethylaminoethyl-Sepharose step, is inactivated by treatment with protein phosphatase 2C, and reactivated by an endogenous 'kinase kinase'. Further purification of AMP-activated protein kinase on Blue Sepharose removes the kinase kinase, but the system can be reconstituted by adding back the flow-through from the Blue-Sepharose column. The kinase kinase can be further purified by subjecting the flow-through from the Blue-Sepharose column to chromatography on a Mono-Q column. A single peak of kinase kinase activity is obtained. Using this fraction, and the most highly purified preparation of AMP-activated protein kinase, phosphorylation of the 63-kDa polypeptide, previously identified as the catalytic subunit of AMP-activated protein kinase, can be demonstrated. As previously shown in the partially purified system, phosphorylation of the 63-kDa polypeptide is markedly stimulated by AMP. The kinase and kinase kinase reactions exhibit similar dependence on AMP concentration. The structurally related AMP analogue, 8-aza-9-deazaadenosine-5'-monophosphate, mimics the effect of AMP on both allosteric activation and phosphorylation of the kinase, while adenosine (5')tetraphospho(5')adenosine antagonizes both effects. These results suggest that both the allosteric effect of AMP, and the promotion of phosphorylation and activation by the kinase kinase, are due to binding of AMP to a single site on the kinase.

AMP-Activated Protein Kinase Kinases↗

PubMed1995

Phosphatidylinositol 3-kinase: inhibition of intrinsic protein-serine kinase activity by phosphoinositides, and of lipid kinase activity by Mn2+.

Phosphatidylinositol (PI) 3-kinase is composed of 110 kDa catalytic and 85 kDa regulatory subunits. The 110 kDa subunit has two intrinsic kinase activities, i.e., Mn(2+)-dependent protein-serine kinase and Mg(2+)-dependent lipid kinase activities. These intrinsic kinases have been reported to be interdependent: protein-serine kinase phosphorylates the 85 kDa subunit of PI 3-kinase, which upon phosphorylation inhibits the lipid kinase activity of PI 3-kinase. We report here that phosphoinositides can selectively inhibit the protein-serine kinase activity of PI 3-kinase without affecting lipid kinase activity. This inhibition depends on the phosphorylation status of the phosphoinositides, i.e., PI 4,5-bisphosphate > PI 4-phosphate >> PI. Mn2+ (2 mM) protected protein kinase activity from phosphoinositides-mediated inhibition if added prior to interaction of PI 3-kinase with phosphoinositides. On the other hand, Mn2+ (2 mM) inhibited lipid kinase activity independent of its effect on the protein kinase activity of PI 3-kinase. The present study suggests that the protein-serine kinase and the lipid kinase activities of PI 3-kinase can be selectively inhibited by phosphoinositides and Mn2+ respectively.

Animals↗

PubMed1993

Complementation of byr1 in fission yeast by mammalian MAP kinase kinase requires coexpression of Raf kinase.

Intracellular signalling from receptor tyrosine kinases in mammalian cells involves the activation of a signal cascade which includes p21ras and the protein kinases p74raf-1, MAP kinase kinase and MAP kinases. In the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae the response to mating pheromones requires the Spk1 and KSS1/FUS3 kinases, which have sequence homology to vertebrate MAP kinases. The recent cloning of complementary DNAs for mammalian and frog MAP kinase kinases has shown that they are homologous to the S. pombe Byr1 (ref. 17) and S. cerevisiae STE7 (ref. 18) kinases, which have been proposed to function upstream of Spk1 and KSS1/FUS3, respectively. We have investigated whether these apparently similar kinase pathways are functionally conserved between vertebrates and S. pombe. We report here that expression of mammalian MAP kinase kinase alone fails to complement a byr1 mutant of S. pombe. When coexpressed with Raf kinase, however, MAP kinase kinase is activated by phosphorylation and the mating defect of the byr1 mutant is rescued. This suggests that the pathways are functionally homologous and that Raf kinase may directly phosphorylate and activate MAP kinase kinase.

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