Abstract
Activation of protein kinase C (PKC) in quiescent Swiss 3T3 cells using either the tumor promoter phorbol 12,13-dibutyrate (PDB) or diacylglycerols increased the tyrosine phosphorylation of p125 focal adhesion kinase (p125FAK) by 3.8-fold. PDB stimulation of p125FAK tyrosine phosphorylation was detected within 1 min and reached a maximum within 5 min, considerably slower than PDB stimulation of 80K/MARCKS phosphorylation which was maximal within 1 min. In sharp contrast, bombesin-induced tyrosine phosphorylation of p125FAK reached a maximum (8-fold stimulation) within 1 min after addition of the peptide and occurred with a half-maximal effect of 0.08 nM, 6-fold lower than the half-maximal effect of bombesin on 80K/MARCKS phosphorylation. Down-regulation of PKC by prolonged treatment with PDB blocked the effect of PDB on p125FAK tyrosine phosphorylation but had no effect on the response to bombesin. A selective inhibitor of PKC, GF 109203X, markedly inhibited the stimulation of p125FAK tyrosine phosphorylation by PDB but had little effect on the response to bombesin, vasopressin, and endothelin. Bombesin stimulation of tyrosine phosphorylation could also be dissociated from mobilization of Ca2+ from intracellular stores. Depletion of the intracellular Ca2+ pool by treatment with the tumor promoter thapsigargin completely blocked the ability of bombesin to transiently increase the cytosolic Ca2+ concentration but had no effect on bombesin stimulation of p125FAK tyrosine phosphorylation. In contrast, cytochalasin D, an agent which selectively disrupts the network of actin microfilaments, completely inhibited bombesin- and PDB-induced p125FAK tyrosine phosphorylation. Within the same concentration range (0.3-2 microM), the drug had no effect on other early events stimulated by bombesin, including Ca2+ mobilization and activation of PKC. These findings demonstrate that neither the PKC nor Ca2+ pathways are responsible for the rapid stimulation of p125FAK tyrosine phosphorylation by neuropeptide growth factors. Furthermore, the integrity of the actin cytoskeleton is essential for the effects of both PDB and bombesin.
Highlights
Ca2+mobilization, and cytoskeletal organization play a role in mediating the stimulation of tyrosine phosphorylation by bombesinand other neuropeptides.Wereport that direct activation of protein kinase C (PKC) by phorbol esteras nd membrane-permeant taken from cultures of Swiss 3T3 cells grown in DMEM with 10% fetal bovine serum that have attained quiescence and is depleted of growth-promoting activity
Stimulates ~ 1 2 5 ~P'h'o~sphorylation-To determine whether direct activation of PKC increased the tyrosine phosphorylation of p125FAK, quiescent Swiss 3T3 cells were treated for 10 min with 200 nM of PDB and lysed
The results presented iFnig. 1suggested that PKCactivation could providea potential mechanismby which bombesin and other neuropeptides stimulate tyrosine phosphorylation of ~ 1 2 5 C~on~seq~ue.ntlythe following experiments were designed to examine in detail the role of PKC in bombesin
Summary
Stimulates ~ 1 2 5 ~P'h'o~sphorylation-To determine whether direct activation of PKC increased the tyrosine phosphorylation of p125FAK, quiescent Swiss 3T3 cells were treated for 10 min with 200 nM of PDB and lysed. Over the besin-Stimuluted ~ 1 2 Ty5ros~ine ~Pho~sphorylation-Down- same concentration range the inhibitor had little effect on regulation of PKC by prolonged exposure of cells to PDB has bombesin stimulation of ~ 1 2 ty5rosi~nep~hos~phorylation been extensively used to dissect the role of this pathway in (Fig. 4B). Anti-Tyr(P) of GF109203X was examined.In accordwith theresults immunoblotting of mAb 2A7 immunoprecipitates showed that obtained for bombesin,the inhibitor, aatconcentration which stimulation of p12SFAKtyrosine phosphorylationeven by max- blocked the response to PD(B3.5 PM), had littleffect on the imal concentrationsof phorbol esterwas markedly reduced in increase in~ 1 2tyr5osi~ne ~pho~sphorylationcaused byeither cells which had previously been chronically exposed to PDB vasopressin or endothelin, but markedly reduced the stimu-. The maximum increase in focal adhesions which form at the termini of actin stress fibers [51],we reasoned that the integritoyf the cytoskeleton, is that elicited by 10 nM bombesin in unpretreated cells
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