Neuropeptides are implicated as growth factors in a variety of fundamental processes including development, inflammation, tissue regeneration, and neoplastic transformation (1-3). In particular, bombesin and its mammalian counterpart gastrin-releasing peptide bind to a GPCR 1 (4, 5) that promotes G␣ q -mediated activation of ␤ isoforms of phospholipase C (6 -8) to produce 2 s messengers: Ins(1,4,5)P 3 that mobilizes Ca 2ϩ from internal stores and diacylglycerol that activates conventional (␣, ␤ 1 , ␤ 2 , and ␥) and novel (␦, ⑀, , and ) PKCs (9, 10). The bombesin/gastrin-releasing peptide GPCR also interacts with members of the G 12 family leading to Rho-dependent actin remodeling and tyrosine phosphorylation of focal adhesion proteins, including FAK (10 -17). Subsequently, bombesin induces striking activation of serine phosphorylation cascades (11)(12)(13)(14) and promotes increased expression of immediate early response genes, stimulation of DNA synthesis, and cell proliferation (15-19). The mechanism(s) linking the early signaling pathways to the subsequent stimulation of cell proliferation remains incompletely understood.PKD (also initially known as PKC) is a serine/threonine protein kinase with structural, enzymology, and regulatory properties different from the PKC family members (20, 21). PKD most distinct characteristics are the presence of a catalytic domain distantly related to Ca 2ϩ -regulated kinases, a pleckstrin homology (PH) region that regulates enzyme activity and a highly hydrophobic stretch of amino acids in its Nterminal region (22)(23)(24). This N-terminal region also contains a tandem repeat of cysteine-rich, zinc finger-like motifs, which confers high affinity for phorbol esters and plays a negative role in the regulation of catalytic kinase activity (25-28). The identification of PKD-2 and PKD-3, similar in overall structure, primary amino acid sequence, and enzymology properties to PKD/PKC (29 -32), supports the notion that PKD isoenzymes constitute a separate family of serine protein kinases.PKD can be activated in intact cells through multiple G protein pathways, including G q , G i , and G 12 (33-39), as well as by biologically active phorbol esters, growth factors and antigen-receptor engagement (36, 37, 39 -45). In all these cases, rapid PKD activation is mediated by PKC-dependent phosphorylation of Ser-744 and Ser-748 within the activation loop of the catalytic domain of PKD (33, 46 -48). PKD activation is associated with its translocation to the plasma membrane and subsequent transient accumulation in the nucleus (25,28,50,51). These findings revealed that PKD is activated by multiple growth-promoting factors (22, 52) suggesting that it functions in mitogenic signaling. Indeed, we reported that PKD overexpression markedly potentiates DNA synthesis induced by the GPCR agonists bombesin and vasopressin in Swiss 3T3 cells (53), a cell line that has been used extensively as a model system to elucidate signal transduction pathways in the mitogenic action of GPCR agonists (1,54,55). Thes...

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Protein kinase D (PKD)/protein kinase C is a serine/ threonine protein kinase activated by growth factors, antigen-receptor engagement, and G protein-coupled receptor (GPCR) agonists via a phosphorylationdependent mechanism that requires protein kinase C (PKC) activity. In order to investigate the dynamic mechanisms associated with GPCR signaling, the intracellular distribution of PKD was analyzed in live cells by imaging fluorescent protein-tagged PKD and in fixed cells by immunocytochemistry. We found that PKD shuttled between the cytoplasm and the nucleus in both fibroblasts and epithelial cells. Cell stimulation with mitogenic GPCR agonists that activate PKD induced a transient nuclear accumulation of PKD that was prevented by inhibiting PKC activity. The nuclear import of PKD requires its cys2 domain in conjunction with a nuclear import receptor, while its nuclear export requires its pleckstrin homology domain and a competent Crm1-dependent nuclear export pathway. This study thus characterizes the regulated nuclear transport of a signaling molecule in response to mitogenic GPCR agonists and positions PKD as a serine kinase whose kinase activity and intracellular localization is coordinated by PKC. Protein kinase D (PKD)1 /protein kinase C is a serine/threonine protein kinase with structural, enzymological, and regulatory properties different from other protein kinase C (PKC) family members (1, 2). The salient features of PKD structure include the presence of a catalytic domain distantly related to Ca 2ϩ -regulated kinases, a pleckstrin homology (PH) domain that regulates PKD activity, and a highly hydrophobic stretch of amino acids in its N-terminal region (1-4). The N-terminal region of PKD contains, in addition to the PH domain, a cysteine-rich domain (CRD) that confers high affinity binding to phorbol esters (5-7). The recent identification of additional cDNA clones, similar in overall structure, primary amino acid sequence, and enzymological properties to PKD (8, 9), supports the notion that PKD isoenzymes constitute a separate family of serine protein kinases.PKD can be activated in intact cells by pharmacological agents including biologically active phorbol esters and cellpermeant diacylglycerol (DAG) as well as by physiological stimuli including G protein-coupled receptors (GPCR) agonists, growth factors, and antigen-receptor engagement (10 -18). In all cases, PKD activation has been shown to be mediated by a PKC-dependent signal transduction pathway that involves the phosphorylation of Ser 744 and Ser 748 within the activation loop of the catalytic domain of PKD (19,20). These findings revealed a link between PKC and PKD in a novel signal transduction pathway activated by multiple growth-promoting factors (5, 21).PKD has been localized in the cytosol and in several intracellular compartments including Golgi, plasma membrane, and mitochondria (14,(22)(23)(24)(25). In addition, we recently found that bombesin, a mitogenic GPCR agonist, induced a rapid and reversible plasma membrane translocation of PK...

Oxidative stress has been implicated in aging and in a wide range of diseases affecting the nervous, respiratory, cardiovascular, and gastrointestinal systems in humans (1, 2). Exposure of cells to reactive oxygen species (ROS) 1 at high concentrations or for extended periods damages cellular DNA, widely hypothesized to either induce cell death via apoptosis/necrosis (3) or facilitate progression toward malignant transformation (4). In contrast, cell exposure to ROS for brief times and/or at limited concentrations induces signals that trigger diverse biological responses including proliferation, migration, or survival (1, 2). Recent studies (5-9) have also identified several mechanisms whereby stimuli or stresses including exposure to growth factors or ultraviolet light can induce ROS production within a variety of cell types. Consequently, the elucidation of cellular signal transduction pathways whereby ROS act as second messengers to mediate proliferation, differentiation, or transformation has recently become an area of intense investigation.Target molecules that mediate the initial sensing and subsequent propagation of ROS-induced signals include cellular protein-tyrosine phosphatases (10) and PTEN (11), a bifunctional tyrosine phosphatase/phosphatidylinositol 3-phosphate phosphatase. Reversible inhibition of these signal-regulating enzymes by ROS induces acute or aberrantly persistent increases in signal transmission through protein tyrosine phosphorylation and 3-phosphorylated inositol lipids, respectively. By modifying redox-sensitive functional domains, intracellular ROS also directly alter the activities of a wide range of other signal transduction proteins, including proximal receivers and transmitters of external signals, i.e. receptor and nonreceptor tyrosine kinases (12, 13) and monomeric and heterotrimeric G proteins (14, 15), and more distal elements including transcription factors and their regulatory serine-threonine protein kinases including ASK1, BMK1, and PKC (16).PKC, an enzyme family of at least 10 distinct isoforms with unique tissue distributions and dynamic subcellular localization, induces both short term alterations in cellular activities and long term effects such as differentiation, proliferation, and apoptosis (17). Multiple signal transduction pathways can be mobilized to mediate PKC activation during oxidative stress, including stimulation of phospholipase C␥ via enhanced tyrosine phosphoryla-