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Protein kinase N 1 (PKN1), which in part resembles yeast protein kinase C, has been shown to be under the control of Rho GTPases and 3-phosphoinositide-dependent kinase 1 (PDK1). We show here that green fluorescent protein-tagged PKN1 has the ability to translocate in a reversible manner to a vesicular compartment following hyperosmotic stress. PKN1 kinase activity is not necessary for this translocation, and in fact the PKN inhibitor HA1077 is also shown to induce PKN1 vesicle accumulation. PKN1 translocation is dependent on Rac1 activation, although the GTPase binding HR1abc domain is not sufficient for this recruitment. The PKN1 kinase domain, however, localizes constitutively to this compartment, and we demonstrate that this behavior is selective for PKNs. Associated with vesicle recruitment, PKN1 is shown to undergo activation loop phosphorylation and activation. It is established that this activation pathway involves PDK1, which is shown to be recruited to this PKN1-positive compartment upon hyperosmotic stress. Taken together, our findings present a pathway for the selective hyperosmotic-induced Rac1-dependent PKN1 translocation and PDK1-dependent activation.

The protein kinase C-related protein kinases (PRKs) have been shown to be under the control of the Rho GTPases and influenced by autophosphorylation. In analyzing the relationship between these inputs, it is shown that activation in vitro and in vivo involves the activation loop phosphorylation of PRK1/2 by 3-phosphoinositide-dependent protein kinase-1 (PDK1). Rho overexpression in cultured cells is shown to increase the activation loop phosphorylation of endogenous PRKs and is demonstrated to influence this process by controlling the ability of PRKs to bind to PDK1. The interaction of PRK1/2 with PDK1 is shown to be dependent upon Rho. Direct demonstration of ternary (Rho.PRK.PDK1) complex formation in situ is provided by the observation that PDK1 is recruited to RhoB-containing endosomes only if PRK is coexpressed. Furthermore, this in vivo complex is maintained after phosphoinositide 3-kinase inhibition. The control of PRKs by PDK1 thus evidences a novel strategy of substrate-directed control involving GTPases.

Addition of insulin growth factor-I (IGF-I) to quiescent Swiss 3T3 cells rapidly induced tyrosine phosphorylation of the p130Crk-associated substrate (p130Cas), a novel adaptor protein localized at focal adhesions. Half-maximal effect was obtained at 0.6 nm. IGF-I also promoted the formation of a complex between p130Cas and c-Crk and elicited a parallel increase in the tyrosine phosphorylation of p125Fak and paxillin. IGF-I-induced p130Cas, p125Fak, and paxillin tyrosine phosphorylation could be dissociated from mitogen-activated protein kinase kinase, p70S6K, and protein kinase C activation. In contrast, the structurally unrelated phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 markedly attenuated the increase in tyrosine phosphorylation of p130Cas, p125Fak, and paxillin induced by IGF-I. Cytochalasin D, which disrupts the network of actin microfilaments, completely prevented tyrosine phosphorylation of p130Cas, p125Fak, and paxillin and the formation of a p130Cas·Crk complex in response to IGF-I. Thus, our results identified a phosphatidylinositol 3-kinase-dependent pathway that requires the integrity of the actin cytoskeleton to induce tyrosine phosphorylation of p130Cas, p125Fak, and paxillin in response to IGF-I and suggest that tyrosine phosphorylation of these focal adhesion proteins, together with the recruitment of c-Crk into a complex with p130Cas, may play a novel role in IGF-I signal transduction.

Treatment of Swiss 3T3 cells with bombesin rapidly induced tyrosine phosphorylation of the p130 Crk-associated substrate (p130cas). Vasopressin, endothelin, bradykinin, lysophosphatidic acid, sphingosylphosphorylcholine, and phorbol 12,13-dibutyrate also stimulated p130cas tyrosine phosphorylation. Bombesin-induced p130cas tyrosine phosphorylation could be dissociated from both protein kinase C activation and Ca2+ mobilization from intracellular stores. In contrast, cytochalasin D, which disrupts the network of actin microfilaments, completely prevented tyrosine phosphorylation of p130cas by bombesin. Platelet-derived growth factor, at low concentrations (1-5 ng/ml), also induced tyrosine phosphorylation of p130cas via a pathway that depended on the integrity of the actin cytoskeleton. The phosphatidylinositol 3′-kinase inhibitors wortmannin and LY294002 prevented tyrosine phosphorylation of p130cas in response to platelet-derived growth factor but not in response to neuropeptides, lysophosphatidic acid, sphingosylphosphorylcholine, or phorbol 12,13-dibutyrate. All agonists that induced p130cas tyrosine phosphorylation also promoted the formation of a p130cas·Crk complex in intact Swiss 3T3 cells. Thus, our results identified distinct signal transduction pathways that lead to tyrosine phosphorylation of p130cas in the same cells and suggest that p130cas could play a role in mitogen-mediated signal transduction.