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 (Rho⅐PRK⅐PDK1) 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.The protein kinase C-related protein kinases (PRKs) 1 are a subfamily of serine/threonine-specific kinases independently identified by molecular cloning, protein purification, and polymerase chain reaction-based screens for novel PKC isoforms (1-3). Two members of this subfamily have been fully cloned and characterized: PRK1 (also termed protein kinase N) and PRK2. They are activated by fatty acids and phospholipids in vitro, although the in vivo significance of this potential is as yet uncharacterized (4, 5). Two-hybrid screens and affinity chromatography identified the PRKs as potential effectors of Rho family GTPases (6 -8). The novel amino-terminal HR1 domain (9) was subsequently identified as the Rho-interacting region (10, 11), an interaction that is presumed to disrupt the autoinhibitory effect produced by a pseudosubstrate-catalytic domain contact (12). Indeed, the GTPase interaction has been shown to increase modestly the activity of the intact kinase (6 -8). The observed proteolytic activation of these kinases would support the role of an allosteric amino-terminal GTPase interaction in the regulation of activity (13,14). In addition to any allosteric component, Rho GTPases can also be responsible for the location of these kinases, with RhoB causing localization to an endosomal compartment in fibroblasts (15), a translocation event that is associated with the accumulation of a hyperphosphorylated form of the kinase.In addition to GTPases, other PRK interactions have been identified. The adapter protein NCK has been shown to interact with a proline-rich region just N-terminal of the kinase domain of PRK2 (16). A similar region is absent in PRK1, suggesting a specificity in the upstream recruitment of these kinases. A potential role for PRKs in the regulation of the cytoskeleton has been proposed following the demonstrated disruption of fibroblast actin stress fibers by the expression of a catalytically inactive PRK2 (8) and the observed PRK1 interaction with the head domain of intermediate fil...

Treatment of Swiss 3T3 cells with bombesin rapidly induced tyrosine phosphorylation of the p130 Crk-associated substrate (p130 cas ). Vasopressin, endothelin, bradykinin, lysophosphatidic acid, sphingosylphosphorylcholine, and phorbol 12,13-dibutyrate also stimulated p130 cas tyrosine phosphorylation. Bombesin-induced p130 cas tyrosine phosphorylation could be dissociated from both protein kinase C activation and Ca 2؉ 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 p130 cas via a pathway that depended on the integrity of the actin cytoskeleton. The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 prevented tyrosine phosphorylation of p130 cas 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 p130 cas tyrosine phosphorylation also promoted the formation of a p130 cas ⅐Crk complex in intact Swiss 3T3 cells. Thus, our results identified distinct signal transduction pathways that lead to tyrosine phosphorylation of p130 cas in the same cells and suggest that p130 cas could play a role in mitogen-mediated signal transduction.Neuropeptides stimulate DNA synthesis and proliferation in cultured cells and are implicated as growth factors in a variety of biological processes, including development, tissue regeneration, and tumorigenesis (1-3). In particular the neuropeptides bombesin, vasopressin, and endothelin are potent mitogens for Swiss 3T3 cells (1-4), a useful model system for the elucidation of signal transduction pathways leading to cell proliferation (4). Tyrosine phosphorylation has recently been implicated in the action of neuropeptides that act as cellular growth factors through G protein-coupled receptors (5). Addition of bombesin, vasopressin, endothelin, and bradykinin to Swiss 3T3 cells stimulates tyrosine phosphorylation of multiple substrates including proteins migrating in the M r 110,000 -130,000 range (6 -8). The cytosolic tyrosine kinase p125 fak1 (9, 10) and the adaptor protein paxillin (11, 12), which are associated with focal adhesion plaques, have been identified as prominent tyrosine-phosphorylated proteins in neuropeptide-stimulated Swiss 3T3 cells (13-15). (21,25). Recently, the organization of the actin cytoskeleton and the functional PI3Ј-kinase have been implicated in mitogen-stimulated tyrosine phosphorylation of p125 fak and paxillin (17-19, 26 -28). Addition of bombesin (13), LPA (16), or PDGF (18) also induces tyrosine phosphorylation of the previously reported p130 substrate of pp60 v-src (25,29,30). Subsequently, this protein has been shown to be closely related or identical to the most prominent tyrosine-phosphorylated protein in v-Crk transformed cells (31-33). The molecular clon...

The mammalian protein kinase N (PKN) family of Serine/Threonine kinases comprises three isoforms, which are targets for Rho family GTPases. Small GTPases are major regulators of the cellular cytoskeleton, generating interest in the role(s) of specific PKN isoforms in processes such as cell migration and invasion. It has been reported that PKN3 is required for prostate tumour cell invasion but not PKN1 or 2. Here we employ a cell model, the 5637 bladder tumour cell line where PKN2 is relatively highly expressed, to assess the potential redundancy of these isoforms in migratory responses. It is established that PKN2 has a critical role in the migration and invasion of these cells. Furthermore, using a PKN wild-type and chimera rescue strategy, it is shown that PKN isoforms are not simply redundant in supporting migration, but appear to be linked through isoform specific regulatory domain properties to selective upstream signals. It is concluded that intervention in PKNs may need to be directed at multiple isoforms to be effective in different cell types.

SummaryIn animals, the protein kinase C (PKC) family has expanded into diversely regulated subgroups, including the Rho family-responsive PKN kinases. Here, we describe knockouts of all three mouse PKN isoforms and reveal that PKN2 loss results in lethality at embryonic day 10 (E10), with associated cardiovascular and morphogenetic defects. The cardiovascular phenotype was not recapitulated by conditional deletion of PKN2 in endothelial cells or the developing heart. In contrast, inducible systemic deletion of PKN2 after E7 provoked collapse of the embryonic mesoderm. Furthermore, mouse embryonic fibroblasts, which arise from the embryonic mesoderm, depend on PKN2 for proliferation and motility. These cellular defects are reflected in vivo as dependence on PKN2 for mesoderm proliferation and neural crest migration. We conclude that failure of the mesoderm to expand in the absence of PKN2 compromises cardiovascular integrity and development, resulting in lethality.