scholarly journals Growth hormone and phorbol esters require specific protein kinase C isoforms to activate mitogen-activated protein kinases in 3T3-F442A cells

1997 ◽  
Vol 324 (1) ◽  
pp. 159-165 ◽  
Author(s):  
Simon MacKENZIE ◽  
Iona FLEMING ◽  
Miles D. HOUSLAY ◽  
Neil G. ANDERSON ◽  
Elaine KILGOUR
Keyword(s):  
Growth Hormone ◽  
Protein Kinase C ◽  
Map Kinase ◽  
Map Kinases ◽  
Phorbol Esters ◽  
The Other ◽  
Kinase Activation ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Mitogen Activated Protein

Previous studies have shown that the activation of p44 and p42 mitogen-activated protein (MAP) kinases (ERK1 and ERK2) by growth hormone (GH) and phorbol esters, but not by epidermal growth factor, in 3T3-F442A preadipocytes is dependent on protein kinase C (PKC). In the present study two approaches have been taken to determine the PKC isoform dependence of MAP kinase activation in these cells. By immunoblotting with specific antibodies, the cells were found to express PKC-α, -γ, -δ, -ϵ and -ζ. Treatment of cells with 500 nM PMA for 3 h led to the complete depletion of PKC-δ and the partial depletion of PKC-α but did not significantly affect the expression of the other PKC isoforms. In parallel, such treatment severely attenuated the ability of GH to activate MAP kinase. The degree of this attenuation was not increased by more prolonged PMA pretreatment, indicating that PKC-δ and perhaps PKC-α are important for MAP kinase activation by GH. These experiments further revealed that additional PKC isoforms were required for the full activation of MAP kinases by acute treatment with PMA. A second approach involved the use of anti-sense oligodeoxynucleotides (ODNs) to deplete the individual PKC isoforms selectively. Each of the ODNs used effectively depleted the relevant isoform to undetectable levels and did not affect the expression of the other PKC isoforms. Pretreatment of cells with PKC-δ anti-sense ODN, but not with anti-sense ODN to the other phorbol ester-sensitive isoforms, severely attenuated the activation of MAP kinases by GH. PKC-δ anti-sense ODN also blocked (by approx. 50%) the activation of MAP kinases by PMA. Furthermore a combination of PKC-δ and -ϵ anti-sense ODNs completely blocked the effect of PMA on MAP kinases. Collectively, these results indicate that the novel PKC-δ and -ϵ isoforms can couple to the MAP kinase pathway in 3T3-F442A cells but that the activation of MAP kinases by GH specifically involves PKC-δ.

scholarly journals Selective activation of p42 mitogen-activated protein (MAP) kinase in murine B lymphoma cell lines by membrane immunoglobulin cross-linking. Evidence for protein kinase C-independent and -dependent mechanisms of activation

1992 ◽  
Vol 287 (1) ◽  
pp. 269-276 ◽  
Author(s):  
M R Gold ◽  
J S Sanghera ◽  
J Stewart ◽  
S L Pelech
Keyword(s):  
Map Kinase ◽  
Tyrosine Phosphorylation ◽  
Cell Lines ◽  
Map Kinases ◽  
Phorbol Esters ◽  
Cross Linking ◽  
Kinase Activation ◽  
Membrane Immunoglobulin ◽  
Protein Map ◽  
Mitogen Activated Protein

Cross-linking of membrane immunoglobulin (mIg), the B lymphocyte antigen receptor, with anti-receptor antibodies stimulates tyrosine phosphorylation of a number of proteins, including one of 42 kDa. Proteins with a similar molecular mass are tyrosine-phosphorylated in response to receptor stimulation in other cell types and have been identified as serine/threonine kinases, termed mitogen-activated protein (MAP) kinases or extracellular signal-regulated kinases (ERKs). The MAP kinases constitute a family of related kinases, at least three of which have molecular masses of 40-45 kDa. In this paper we show that mIg cross-linking stimulated the myelin basic protein phosphotransferase activity characteristic of MAP kinase in both mature and immature murine B cell lines. This enzyme activity co-purified on three different columns with a 42 kDa protein that was tyrosine-phosphorylated (pp42) in response to mIg cross-linking and which reacted with a panel of anti-(MAP kinase) antibodies. Although immunoblotting with the anti-(MAP kinase) antibodies showed that these B cell lines expressed both 42 kDa and 44 kDa forms of MAP kinase, only the 42 kDa form was activated and tyrosine-phosphorylated to a significant extent. Activation of protein kinase C (PKC) with phorbol esters also resulted in selective tyrosine phosphorylation and activation of the 42 kDa MAP kinase. This suggested that mIg-induced MAP kinase activation could be due to stimulation of PKC by mIg. However, mIg-stimulated MAP kinase activation and pp42 tyrosine phosphorylation was only partially blocked by a PKC inhibitor, the staurosporine analogue Compound 3. In contrast, Compound 3 completely blocked the ability of phorbol esters to stimulate MAP kinase activity and induce tyrosine phosphorylation of pp42. Thus mIg may activate MAP kinase by both PKC-dependent and -independent mechanisms.

scholarly journals Growth hormone activates mitogen-activated protein kinase and S6 kinase and promotes intracellular tyrosine phosphorylation in 3T3-F442A preadipocytes

1992 ◽  
Vol 284 (3) ◽  
pp. 649-652 ◽  
Author(s):  
N G Anderson
Keyword(s):  
Growth Hormone ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Tyrosine Phosphorylation ◽  
Phorbol Esters ◽  
Mitogen Activated Protein Kinase ◽  
S6 Kinase ◽  
Kinase C ◽  
Transient Activation ◽  
Mitogen Activated Protein

Physiological concentrations of growth hormone induced a rapid and transient activation of mitogen-activated protein kinase (MAP kinase) and S6 kinase in 3T3-F442A preadipocytes. These effects were abrogated by staurosporine and in cells chronically pretreated with phorbol esters, suggesting that protein kinase C is involved in the mechanism of activation. In addition, three cytosolic proteins exhibited a growth-hormone-dependent increase in tyrosine phosphorylation.

scholarly journals Endothelin rapidly stimulates mitogen-activated protein kinase activity in rat mesangial cells

1992 ◽  
Vol 287 (2) ◽  
pp. 589-594 ◽  
Author(s):  
Y Wang ◽  
M S Simonson ◽  
J Pouysségur ◽  
M J Dunn
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Map Kinase ◽  
Phorbol Ester ◽  
Map Kinases ◽  
Kinase Activity ◽  
Mesangial Cells ◽  
Kinase C ◽  
Mitogen Activated Protein

Mitogen-activated protein (MAP) kinases are regarded as switch kinases in the phosphorylation cascade initiated by various agonists. We have investigated whether endothelins (ET), which are constrictor and mitogenic isopeptides, can increase MAP kinase activity in rat mesangial cells, using bovine myelin basic protein (MBP) as a substrate for an in vitro kinase assay. Treatment of quiescent mesangial cells with ET-1 rapidly stimulated a kinase activity which phosphorylated exogenous MBP. This stimulation was dose-dependent, with threshold responses at 1 nM-ET-1. Epidermal growth factor and thrombin also activated this kinase in mesangial cells. We also examined the ET signal transduction pathways leading to activation of MBP kinase. Pertussis toxin had no effect on ET-stimulated MBP kinase activity. Stimulation of protein kinase C by phorbol ester increased MBP kinase activity, and down-regulation of PKC partially inhibited ET-stimulated MBP kinase as well as phorbol ester-stimulated MBP kinase activity. Interestingly, genestein, an inhibitor of protein tyrosine kinases, partially inhibited MBP kinase stimulated by ET but not by phorbol esters. These results suggest that ET stimulates MBP kinase activity in rat mesangial cells via at least two pathways: one which is protein kinase C-dependent and a second one that involves a protein tyrosine kinase. Finally, by raising rabbit antibodies against the two forms of MAP kinase, p44mapk and p42mapk, we demonstrated that both isoforms are expressed in mesangial cells. Antibody alpha 1 Cp42 specifically immunoprecipitated p42mapk and allowed us to demonstrate that ET stimulates MBP kinase activity in the p42mapk immunocomplex. In conclusion, we have provided evidence that, in rat mesangial cells, MAP kinases are rapidly activated by ET-1, a regulatory process that involves at least protein kinase C activation and also a contribution of a tyrosine kinase not yet characterized.

A yeast mitogen-activated protein kinase homolog (Mpk1p) mediates signalling by protein kinase C

1993 ◽  
Vol 13 (5) ◽  
pp. 3067-3075 ◽  
Author(s):  
K S Lee ◽  
K Irie ◽  
Y Gotoh ◽  
Y Watanabe ◽  
H Araki ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Protein Kinases ◽  
Map Kinases ◽  
Pheromone Response ◽  
Pheromone Response Pathway ◽  
Kinase C ◽  
Mitogen Activated Protein ◽  
Kinase Cascade ◽  
Protein Kinase Cascade

Mitogen-activated protein (MAP) kinases are activated in response to a variety of stimuli through a protein kinase cascade that results in their phosphorylation on tyrosine and threonine residues. The molecular nature of this cascade is just beginning to emerge. Here we report the isolation of a Saccharomyces cerevisiae gene encoding a functional analog of mammalian MAP kinases, designated MPK1 (for MAP kinase). The MPK1 gene was isolated as a dosage-dependent suppressor of the cell lysis defect associated with deletion of the BCK1 gene. The BCK1 gene is also predicted to encode a protein kinase which has been proposed to function downstream of the protein kinase C isozyme encoded by PKC1. The MPK1 gene possesses a 1.5-kb uninterrupted open reading frame predicted to encode a 53-kDa protein. The predicted Mpk1 protein (Mpk1p) shares 48 to 50% sequence identity with Xenopus MAP kinase and with the yeast mating pheromone response pathway components, Fus3p and Kss1p. Deletion of MPK1 resulted in a temperature-dependent cell lysis defect that was virtually indistinguishable from that resulting from deletion of BCK1, suggesting that the protein kinases encoded by these genes function in a common pathway. Expression of Xenopus MAP kinase suppressed the defect associated with loss of MPK1 but not the mating-related defects associated with loss of FUS3 or KSS1, indicating functional conservation between the former two protein kinases. Mutation of the presumptive phosphorylated tyrosine and threonine residues of Mpk1p individually to phenylalanine and alanine, respectively, severely impaired Mpk1p function. Additional epistasis experiments, and the overall architectural similarity between the PKC1-mediated pathway and the pheromone response pathway, suggest that Pkc1p regulates a protein kinase cascade in which Bck1p activates a pair of protein kinases, designated Mkk1p and Mkk2p (for MAP kinase-kinase), which in turn activate Mpk1p.

MKK1 and MKK2, which encode Saccharomyces cerevisiae mitogen-activated protein kinase-kinase homologs, function in the pathway mediated by protein kinase C

1993 ◽  
Vol 13 (5) ◽  
pp. 3076-3083
Author(s):  
K Irie ◽  
M Takase ◽  
K S Lee ◽  
D E Levin ◽  
H Araki ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Map Kinase ◽  
Protein Kinases ◽  
Cell Lysis ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Kinase C ◽  
Mitogen Activated Protein

The PKC1 gene of Saccharomyces cerevisiae encodes a homolog of mammalian protein kinase C that is required for normal growth and division of yeast cells. We report here the isolation of the yeast MKK1 and MKK2 (for mitogen-activated protein [MAP] kinase-kinase) genes which, when overexpressed, suppress the cell lysis defect of a temperature-sensitive pkc1 mutant. The MKK genes encode protein kinases most similar to the STE7 product of S. cerevisiae, the byr1 product of Schizosaccharomyces pombe, and vertebrate MAP kinase-kinases. Deletion of either MKK gene alone did not cause any apparent phenotypic defects, but deletion of both MKK1 and MKK2 resulted in a temperature-sensitive cell lysis defect that was suppressed by osmotic stabilizers. This phenotypic defect is similar to that associated with deletion of the BCK1 gene, which is thought to function in the pathway mediated by PCK1. The BCK1 gene also encodes a predicted protein kinase. Overexpression of MKK1 suppressed the growth defect caused by deletion of BCK1, whereas an activated allele of BCK1 (BCK1-20) did not suppress the defect of the mkk1 mkk2 double disruption. Furthermore, overexpression of MPK1, which encodes a protein kinase closely related to vertebrate MAP kinases, suppressed the defect of the mkk1 mkk2 double mutant. These results suggest that MKK1 and MKK2 function in a signal transduction pathway involving the protein kinases encoded by PKC1, BCK1, and MPK1. Genetic epistasis experiments indicated that the site of action for MKK1 and MKK2 is between BCK1 and MPK1.

scholarly journals Novel members of the mitogen-activated protein kinase activator family in Xenopus laevis

1993 ◽  
Vol 13 (9) ◽  
pp. 5738-5748
Author(s):  
B M Yashar ◽  
C Kelley ◽  
K Yee ◽  
B Errede ◽  
L I Zon
Keyword(s):  
Protein Kinase ◽  
Xenopus Laevis ◽  
Map Kinase ◽  
Protein Kinases ◽  
Map Kinases ◽  
Yeast Cells ◽  
Amino Acid Sequence Similarity ◽  
Kinase Activation ◽  
Mitogen Activated Protein ◽  
Activation Pathways

Mitogen-activated protein (MAP) kinases comprise an evolutionarily conserved family of proteins that includes at least three vertebrate protein kinases (p42, p44, and p55 MAPK) and five yeast protein kinases (SPK1, MPK1, HOG1, FUS3, and KSS1). Members of this family are activated by a variety of extracellular agents that influence cellular proliferation and differentiation. In Saccharomyces cerevisiae, there are multiple physiologically distinct MAP kinase activation pathways composed of structurally related kinases. The recently cloned vertebrate MAP kinase activators are structurally related to MAP kinase activators in these yeast pathways. These similarities suggest that homologous kinase cascades are utilized for signal transduction in many, if not all, eukaryotes. We have identified additional members of the MAP kinase activator family in Xenopus laevis by a polymerase chain reaction-based analysis of embryonic cDNAs. One of the clones identified (XMEK2) encodes a unique predicted protein kinase that is similar to the previously reported activator (MAPKK) in X. laevis. XMEK2, a highly expressed maternal mRNA, is developmentally regulated during embryogenesis and expressed in brain and muscle. Expression of XMEK2 in yeast cells suppressed the growth defect associated with loss of the yeast MAP kinase activator homologs, MKK1 and MKK2. Partial sequence of a second cDNA clone (XMEK3) identified yet another potential MAP kinase activator. The pattern of expression of XMEK3 is distinct from that of p42 MAPK and XMEK2. The high degree of amino acid sequence similarity of XMEK2, XMEK3, and MAPKK suggests that these three are related members of an amphibian family of protein kinases involved in the activation of MAP kinase. Discovery of this family suggests that multiple MAP kinase activation pathways similar to those in yeast cells exist in vertebrates.

scholarly journals Bombesin and epidermal growth factor stimulate the mitogen-activated protein kinase through different pathways in Swiss 3T3 cells

1993 ◽  
Vol 289 (1) ◽  
pp. 283-287 ◽  
Author(s):  
L Pang ◽  
S J Decker ◽  
A R Saltiel
Keyword(s):  
Enzyme Activity ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Map Kinase ◽  
3T3 Cells ◽  
Kinase C ◽  
Mitogen Activated Protein ◽  
Epidermal Growth ◽  
Swiss 3T3 ◽  
Stimulation Of

Both bombesin and epidermal growth factor (EGF) are potent mitogens in Swiss 3T3 cells that nonetheless have dissimilar receptor structures. To explore possible common intracellular events involved in the stimulation of cellular growth by these two peptides, we have evaluated the regulation of the mitogen-activated protein (MAP) kinase. Exposure of Swiss 3T3 cells to bombesin, EGF or the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) causes the rapid and transient stimulation of the enzyme activity. Pretreatment of cells with the protein kinase inhibitor H-7, or down-regulation of cellular protein kinase C by prolonged exposure to PMA, causes a decrease of over 90% in the activation of MAP kinase by bombesin. In contrast, these treatments have no effect on the stimulation of MAP kinase by EGF. The stimulation of MAP kinase activity by bombesin is dose-dependent, occurring over a narrow concentration range of the peptide. Both EGF and bombesin stimulate the phosphorylation of an immunoprecipitable MAP kinase protein migrating at 42 kDa on SDS/PAGE. Phosphoamino acid analysis of this phosphorylated protein reveals that EGF and bombesin stimulate phosphorylation on tyrosine, threonine and serine residues. Tyrosine phosphorylation of the enzyme, as evaluated by antiphosphotyrosine blotting of the immunoprecipitated protein, reveals that the time course of phosphorylation by both mitogens correlates with stimulation of enzyme activity. These results provide further evidence for the convergence of discrete pathways emanating from tyrosine kinase and G-protein-linked receptors in the regulation of MAP kinase.

Cholecystokinin rapidly activates mitogen-activated protein kinase in rat pancreatic acini

1994 ◽  
Vol 267 (3) ◽  
pp. G401-G408 ◽  
Author(s):  
R. D. Duan ◽  
J. A. Williams
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Map Kinase ◽  
Threshold Concentration ◽  
Mitogen Activated Protein Kinase ◽  
Maximal Effect ◽  
Pancreatic Acini ◽  
Kinase C ◽  
Mitogen Activated Protein

The existence and activation of mitogen-activated protein (MAP) kinase in isolated pancreatic acini have been demonstrated. Immunoblotting and immunoprecipitation revealed two forms of MAP kinase in pancreatic acini, with relative molecular masses of approximately 42 and 44 kDa. Both forms of MAP kinase were activated by cholecystokinin (CCK). The threshold concentration of CCK was approximately 3 pM, and the maximal effect occurred at 1 nM, which enhanced MAP kinase activity by 2.5-fold, as determined in polyacrylamide gel copolymerized with substrate myelin basic protein. Activation of MAP kinase by CCK was rapid, reaching a maximum within 5-10 min that subsequently declined. Bombesin and carbachol but not secretin or vasoactive intestinal peptide also activated MAP kinase. CCK-induced activation of MAP kinase may be mediated by protein kinase C, since 12-O-tetradecanoylphorbol 13-acetate (TPA) mimicked the effect of CCK and staurosporine concentration dependently inhibited the action of CCK. Treatment of acini with thapsigargin, ionomycin, or ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid did not influence MAP kinase, indicating that mobilization of intracellular calcium by CCK is not important in activation of acinar MAP kinase. CCK and TPA increased tyrosine phosphorylation of both 42- and 44-kDa forms. Genistein and tyrphostin 23, the inhibitors of tyrosine kinase, suppressed the activation of MAP kinase by CCK. In conclusion, MAP kinase in pancreatic acini is activated by agonists related to hydrolysis of phosphoinositide, via a mechanism involving protein kinase C and tyrosine kinase.

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