No abstract

The importance of activation loop phosphorylation in the regulation of protein kinase D (PKD/protein kinase C (PKC) ) activity has become controversial. In order to clarify the mechanism(s) of PKD activation, we developed a novel phosphospecific antibody recognizing phosphorylated Ser 748 in PKD (pS748

Protein kinase D (PKD)/protein kinase C (PKC) is a serine/threonine protein kinase that can be activated by physiological stimuli like growth factors, antigen-receptor engagement and G protein-coupled receptor (GPCR) agonists via a phosphorylation-dependent mechanism that requires PKC activity. In order to investigate the dynamic mechanisms associated with GPCR signaling, the intracellular translocation of a green fluorescent protein-tagged PKD was analyzed by real-time visualization in fibroblasts and epithelial cells stimulated with bombesin, a GPCR agonist. We found that bombesin induced a rapidly reversible plasma membrane translocation of green fluorescent protein-tagged PKD, an event that can be divided into two distinct mechanistic steps. The first step, which is exclusively mediated by the cysteine-rich domain in the N terminus of PKD, involved its translocation from the cytosol to the plasma membrane. The second step, i.e. the rapid reverse translocation of PKD from the plasma membrane to the cytosol, required its catalytic domain and surprisingly PKC activity. These findings provide evidence for a novel mechanism by which PKC coordinates the translocation and activation of PKD in response to bombesininduced GPCR activation.

(8,9). In addition to its role as sensor of [Ca 2ϩ ] o , the CaR is also stimulated by aromatic amino acids (10) that, like [Ca 2ϩ ] o , induce striking and lasting CaR-mediated [Ca 2ϩ ] i oscillations (9, 11). However, the patterns of [Ca 2ϩ ] i oscillations induced by these agonists are different. Aromatic amino acid stimulation of the CaR induces repetitive, low frequency [Ca 2ϩ ] i spikes that return to the base-line level, a pattern known as transient oscillations. In contrast, [Ca 2ϩ ] o -elicited CaR activation produces high frequency sinusoidal oscillations upon a raised plateau level of [Ca 2ϩ ] i (9, 11). The amplitude, frequency, and duration of [Ca 2ϩ ] i oscillations are increasingly recognized as encoding important information for a variety of biological processes, and, consequently, there is intense interest in understanding the underlying mechanisms (12).Our previous results produced several lines of evidence indicating that PKCs negatively regulate the frequency of [Ca 2ϩ ] i oscillations induced by activation of the CaR by increases in [Ca 2ϩ ] o (11). We hypothesized that periodic phosphorylation of the CaR by PKCs provides the negative feedback needed to cause [Ca 2ϩ ] o -induced sinusoidal [Ca 2ϩ ] i oscillations. Intriguingly, the transient [Ca 2ϩ ] i oscillations produced by the CaR in response to amino acid stimulation appear to be mediated by a different pathway, but the mechanism(s) involved remained poorly understood.In the present study, we examined whether sinusoidal and transient [Ca 2ϩ ] i oscillations produced by the CaR in response to Ca 2ϩ or L-phenylalanine are mediated by different pathways. Using real time imaging of changes in phosphatidylinositol 4,5-biphosphate hydrolysis and generation of Ins(1,4,5)P 3 in single cells, we found that [Ca 2ϩ ] o -induced CaR activation

and Ser 748 is the primary mechanism involved in early phase PKD activation, whereas PKD autophosphorylation on Ser 748 is a major mechanism contributing to the late phase of PKD activation occurring in cells stimulated by GPCR agonists. The present studies identify a novel mechanism induced by GPCR activation that leads to late, PKC-independent PKD activation.

The protein kinase D (PKD) family consists of three serine/threonine kinases: PKC/PKD, PKD2, and PKC/PKD3. Whereas PKD has been the focus of most studies, virtually nothing is known about the effect of G protein-coupled receptor agonists (GPCR) on the regulatory properties and intracellular distribution of PKD3. Consequently, we examined the mechanism that mediates its activation and intracellular distribution. GPCR agonists induced a rapid activation of PKD3 by a protein kinase C (PKC)-dependent pathway that leads to the phosphorylation of the activation loop of PKD3. Comparison of the steady-state distribution of endogenous or tagged PKD3 versus PKD and PKD2 in unstimulated cells indicated that whereas PKD and PKD2 are predominantly cytoplasmic, PKD3 is present both in the nucleus and cytoplasm. This distribution of PKD3 results from its continuous shuttling between both compartments by a mechanism that requires a nuclear import receptor and a competent CRM1-nuclear export pathway. Cell stimulation with the GPCR agonist neurotensin induced a rapid and reversible plasma membrane translocation of PKD3 that is PKCdependent. Interestingly, the nuclear accumulation of PKD3 can be dramatically enhanced in response to its activation. Thus, this study demonstrates that the intracellular distribution of PKD isoenzymes are distinct, and suggests that their signaling properties are regulated by differential localization.

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...

Several studies have provided evidence that the cellular cytoskeleton may be involved in the assembly and budding of retroviruses. In fractionation studies of HIV-1-infected CEM cells, the majority of the unprocessed Gag polyprotein cofractionated with the cellular cytoskeleton. In vivo and in vitro analyses of this interaction indicated that the unprocessed Gag polyprotein is capable of association with polymerized actin (F-actin). Binding of Gag to F-actin may be involved in the assembly or budding of HIV-1.