scholarly journals Regulation of endothelin-1- and lysophosphatidic acid-stimulated tyrosine phosphorylation of focal adhesion kinase (pp125fak) in Rat-1 fibroblasts

1994 ◽  
Vol 301 (2) ◽  
pp. 407-414 ◽  
Author(s):  
M K Saville ◽  
A Graham ◽  
K Malarkey ◽  
A Paterson ◽  
G W Gould ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Lysophosphatidic Acid ◽  
Calcium Ionophore ◽  
Mitogen Activated Protein Kinase ◽  
Ionophore A23187 ◽  
Endothelin 1 ◽  
Kinase C

The characteristics of protein tyrosine phosphorylation were examined in Rat-1 fibroblasts in response to endothelin-1 (ET-1) and 1-oleoyl-lysophosphatidic acid (LPA). Both agonists stimulated the biphasic tyrosine phosphorylation of at least three major proteins of approx. 120 kDa (pp116, pp120 and pp130) and two of 80 kDa (pp80 and pp70). Immunoprecipitation experiments indicated that the pp120 protein corresponded to the recently described focal adhesion protein kinase pp125fak. Phorbol 12-myristate 13-acetate, alone or in combination with the calcium ionophore A23187, also stimulated the phosphorylation of pp125fak but to a smaller extent than LPA or ET-1. Removal of both extracellular and intracellular Ca2+ did not significantly reduce LPA- and ET-1-stimulated tyrosine phosphorylation of pp125fak. In cells where protein kinase C activity was down-regulated or inhibited, ET-1-stimulated tyrosine phosphorylation of pp125fak was reduced to a greater extent than phosphorylation in response to LPA. In addition, ET-1-stimulated tyrosine phosphorylation of pp80 was decreased by 50-70% in response to protein kinase C inhibition at both 2 and 60 min whereas LPA-stimulated tyrosine phosphorylation of this protein was only reduced at 2 min. Pretreatment with pertussis toxin reduced the tyrosine phosphorylation of pp42 and pp44 forms of mitogen-activated protein kinase in response to both ET-1 and LPA but reduced the tyrosine phosphorylation of pp125fak only in response to LPA. These results indicate agonist-specific differences in the regulation of pathways mediating the tyrosine phosphorylation of pp125fak and other target proteins.

scholarly journals Dissociation of mitogen-activated protein kinase activation from p125 focal adhesion kinase tyrosine phosphorylation in Swiss 3T3 cells stimulated by bombesin, lysophosphatidic acid, and platelet-derived growth factor.

1996 ◽  
Vol 7 (12) ◽  
pp. 1865-1875 ◽  
Author(s):  
T Seufferlein ◽  
D J Withers ◽  
D Mann ◽  
E Rozengurt
Keyword(s):  
Protein Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Lysophosphatidic Acid ◽  
Mitogen Activated Protein Kinase ◽  
Platelet Derived Growth Factor ◽  
3T3 Cells ◽  
Kinase C ◽  
Mitogen Activated Protein ◽  
Swiss 3T3

The experiments presented here were designed to examine the contribution of p125 focal adhesion kinase (p125FAK) tyrosine phosphorylation to the activation of the mitogen-activated protein kinase cascade induced by bombesin, lysophosphatidic acid (LPA), and platelet-derived growth factor (PDGF) in Swiss 3T3 cells. We found that tyrosine phosphorylation of p125FAK in response to these growth factors is completely abolished in cells treated with cytochalasin D or in cells that were suspended in serum-free medium for 30 min. In marked contrast, the activation of p42mapk by these factors was independent of the integrity of the actin cytoskeleton and of the interaction of the cells with the extracellular matrix. The protein kinase C inhibitor GF 109203X and down-regulation of protein kinase C by prolonged pretreatment of cells with phorbol esters blocked bombesin-stimulated activation of p42mapk, p90rsk, and MAPK kinase-1 but did not prevent bombesin-induced tyrosine phosphorylation of p125FAK. Furthermore, LPA-induced p42mapk activation involved a pertussis toxin-sensitive guanylate nucleotide-binding protein, whereas tyrosine phosphorylation of p125FAK in response to LPA was not prevented by pretreatment with pertussis toxin. Finally, PDGF induced maximum p42mapk activation at concentrations (30 ng/ml) that failed to induce tyrosine phosphorylation of p125FAK. Thus, our results demonstrate that p42mapk activation in response to bombesin, LPA, and PDGF can be dissociated from p125FAK tyrosine phosphorylation in Swiss 3T3 cells.

scholarly journals A role for protein kinase C in the membrane fusion necessary for platelet granule secretion

Blood ◽  
1989 ◽  
Vol 74 (7) ◽  
pp. 2405-2413 ◽  
Author(s):  
JM Gerrard ◽  
LL Beattie ◽  
J Park ◽  
SJ Israels ◽  
A McNicol ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Membrane Fusion ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ultrastructural Changes ◽  
Ionophore A23187 ◽  
Kinase C ◽  
Granule Secretion ◽  
Dimethyl Amiloride

Abstract The addition of 1-oleoyl-2-acetylglycerol (OAG), or phorbol-12- myristate-13-acetate (PMA) to platelets induced the phosphorylation of a 47,000 dalton protein (47 Kd), fusion of granule membranes with membranes of the surface connected canalicular system, the formation of large vesicles and the secretion of serotonin. 1-(5- isoquinolinesulfonyl)-2-methyl-piperazine (H7), and sphingosine, inhibitors of protein kinase C, significantly inhibited the ultrastructural changes and the phosphorylation of 47 Kd. N-(2- guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), structurally similar to H7, but a weaker inhibitor of protein kinase C, did not attenuate these responses to OAG or to PMA. H7, but not HA1004, also markedly inhibited secretion induced by the synergistic combination of OAG and the calcium ionophore A23187. Amiloride and 5-(N,N dimethyl)- amiloride, inhibitors of the Na+/H+ transporter, did not inhibit the ultrastructural response and the protein phosphorylation induced by OAG, or the secretion induced by the combination of A23187 and OAG. The results link the activation of protein kinase C by diglycerides to the labilization and fusion of granule membranes important for secretion.

Protein kinase C activation and calcium mobilization decrease prolactin release from human decidual cells in early pregnancy

1993 ◽  
Vol 137 (2) ◽  
pp. 335-340 ◽  
Author(s):  
T. Kubota ◽  
S. Kamada ◽  
M. Taguchi ◽  
S. Sakamoto ◽  
T. Aso
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Early Pregnancy ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Calcium Mobilization ◽  
Ionophore A23187 ◽  
Prolactin Release ◽  
Kinase C ◽  
Decidual Cells

ABSTRACT The present study was undertaken to investigate the effects of protein kinase C (PKC) activation and calcium mobilization on the release of prolactin from human decidual cells in early pregnancy. Decidua obtained from patients in early pregnancy was enzymatically dispersed and cultured with phorbol myristate acetate (PMA) and calcium ionophore A23187 in a cell culture system. Prolactin in the medium was measured by enzyme-immunoassay. PMA, a PKC activator, dose-dependently attenuated the release of prolactin from cultured decidual cells, while a PKC inhibitor, H7, significantly (P < 0·001) diminished the effect of PMA on prolactin release. PMA had no effect on cell numbers or DNA synthesis in the decidual cells during culture. It did not significantly increase the generation of inositol phosphate in decidual cells prelabelled with myo[3H]inositol and it had no effect on intracellular calcium concentration ([Ca2 + ]i). Calcium ionophore A23187, a Ca2 +-mobilizing agent, also significantly (P<0·001) attenuated the release of prolactin and potentiated the PMA-induced suppression of prolactin release from decidual cells. These findings suggest that activation of PKC and mobilization of Ca2+ may be involved in regulating prolactin release from human decidual cells. The PMA-induced suppression of prolactin release is not triggered by phosphoinositide hydrolysis nor by the increase in [Ca2 + ]i in decidual cells. Journal of Endocrinology (1993) 137, 335–340

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 Inhibition of mitogen-activated protein kinase kinase does not impair primary activation of human platelets

1996 ◽  
Vol 318 (1) ◽  
pp. 207-212 ◽  
Author(s):  
Angelika G. BÖRSCH-HAUBOLD ◽  
Ruth M. KRAMER ◽  
Steve P WATSON
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Substrate Inhibition ◽  
Human Platelets ◽  
Mitogen Activated Protein Kinase ◽  
Ionophore A23187 ◽  
Kinase C ◽  
Mitogen Activated Protein

Mitogen-activated protein kinases (MAPKs), a family of protein serine/threonine kinases regulating cell growth and differentiation, are activated by a dual-specificity kinase through phosphorylation at threonine and tyrosine. We used a recently described selective inhibitor of the p42/p44mapk-activating enzyme, PD 98059 [2-(2´-amino-3´-methoxyphenyl)-oxanaphthalen-4-one], to investigate the role of the p42/p44mapk pathway in human platelets. PD 98059 inhibited p42/p44mapk activation in thrombin-, collagen- and phorbol ester-stimulated platelets, as determined from in-gel renaturation kinase assays, with an IC50 of approx. 5 µM (thrombin stimulation). It also prevented activation of MAPK kinase, which was measured in whole-cell lysates with glutathione S-transferase/p42mapk fusion protein (GST–MAPK) as substrate. Inhibition of p42/p44mapk did not affect platelet responses to thrombin or collagen such as aggregation, 5-hydroxytryptamine release and protein kinase C activation. In addition, PD 98059 did not interfere with release of arachidonic acid, a response mediated by cytosolic phospholipase A2 (cPLA2), or with cPLA2 phosphorylation. This suggests that platelet cPLA2 is not regulated by p42/p44mapk after stimulation with physiological agonists. In contrast, phorbol ester-induced phosphorylation of cPLA2 and potentiation of arachidonic acid release stimulated by Ca2+ ionophore A23187 were inhibited by PD 98059, indicating that p42/p44mapk phosphorylates cPLA2 after activation of protein kinase C by the non-physiological tumour promoter.

scholarly journals Protein kinase C regulates proliferation of mast cells and the expression of the mRNAs of fos and jun proto-oncogenes during activation by IgE-Ag or calcium ionophore A23187

Blood ◽  
1991 ◽  
Vol 78 (9) ◽  
pp. 2354-2364
Author(s):  
D Baranes ◽  
E Razin
Keyword(s):  
Protein Kinase C ◽  
Mast Cells ◽  
Protein Kinase ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Prolonged Treatment ◽  
Ionophore A23187 ◽  
Short Term ◽  
Kinase C ◽  
Short Term Exposure

Short-term stimulation (up to 16 hours) of interleukin-3 (IL-3)- dependent mouse bone marrow-derived mast cells, Abelson transformed mouse liver-derived mast cells, or rat basophilic leukemia cells by either IgE-Ag or calcium ionophore A23187 resulted in inhibition of their proliferation as measured by 3H-thymidine incorporation and MTT (tetrazolium) assays, and in accumulation of the mRNAs of c-fos, c-jun, junB and slightly of junD proto-oncogenes. The involvement of protein kinase C (PKC) in these responses was investigated by using several approaches of enzyme activity regulation. Direct activation of the PKC was achieved by short-term exposure of the cells to the PKC-specific activator phorbol 12-myristate-13-acetate (PMA). Inhibition of PKC activity was obtained by either prolonged treatment of the cells with PMA or by exposure of the cells to the PKC inhibitors H-7 and staurosporine. The results showed the following: (1) Short-term exposure of mast cells to PMA was sufficient to induce inhibition of proliferation. (2) An increase in PKC activity was associated with a decrease in the proliferation of IgE-dinitrophenol (DNP) or calcium ionophore A23187-stimulated cells. (3) A direct correlation was found between the increase in PKC activity and the increase in the level of the mRNAs of the jun proto-oncogenes in cells activated by both stimuli mentioned. (4) While an increase in PKC activity was associated with the upregulation of the level of c-fos mRNA during calcium ionophore A23187 stimulation, it showed the opposite effect on the expression of the mRNA of this proto-oncogene when the cells were triggered by IgE- DNP. Therefore, we concluded that PKC plays various roles in the expression of the mRNA of c-fos in activated mast cells depending on the stimulus involved. In addition, the expression of the mRNA of c-jun and junB proto-onogenes is not coordinately regulated with that of c- fos during immunologic stimulation. This discordancy, which is associated with the increase in PKC activity in mast cells, may play a role in the regulation of the transcription of AP-1-responsive genes, and therefore could be associated with the regulation of proliferation of these cells.

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