Regulated Nucleocytoplasmic Transport of Protein Kinase D in Response to G Protein-coupled Receptor Activation

Abstract

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...

Role of the Regulatory Domain of Protein Kinase D2 in Phorbol Ester Binding, Catalytic Activity, and Nucleocytoplasmic Shuttling

“…Furthermore, deletion of the C1a, the C1b, or the PH subdomain, respectively, leads to the constitutive activation of PKD1, suggesting that these subdomains have a negative/inhibitory impact on its catalytic activity . The PH domain also has been shown to mediate nuclear export, whereas the C1b domain regulates nuclear import of PKD1 (Rey et al, 2001). At present little is known about whether the various PKD isoforms are regulated and act in a similar manner or exhibit distinct regulatory and functional properties.…”

Protein kinase D: a new player among the signaling proteins that regulate functions in the nervous system

“…As no interactions have been found between the PH domain of PKD1 and phosphorylated inositol lipids, which are important ligands responsible for membrane localization, this domain is not required for plasma membrane translocation [31,32] or Golgi localization [33][34][35][36][37][38][39] like other PH-domain-containing AGC kinases, such as PKB and the GRKs. However, the PH domain is required for the nuclear export of PKD1 [22,40] .…”

“…For example, G-proteincoupled receptors or receptor tyrosine kinases that activate PLC and PKCε or PKCη cause the phosphorylation of PKD at Ser744 and Ser748 in the activation loop [16][17][18][19] . Second, Gβγ subunits directly activate PKD1 [20][21][22] . The precise mechanism of this activation in vivo needs to be defi ned.…”

“…As no interactions have been found between the PH domain of PKD1 and phosphorylated inositol lipids, which are important ligands responsible for membrane localization, this domain is not required for plasma membrane translocation [31,32] or Golgi localization [33][34][35][36][37][38][39] like other PH-domain-containing AGC kinases, such as PKB and the GRKs. However, the PH domain is required for the nuclear export of PKD1 [22,40] .The different lipid-binding specificity of the zinc-fingers C1A and C1B results in their different roles in targeting PKD1 to different destinations [29,33,[41][42][43] . Plasma membrane translocation of PKD1 is dependent on the C1B domain, while its Golgi localization requires the C1A domain and the phosphorylation of loop serines.…”

“…Plasma membrane translocation of PKD1 is dependent on the C1B domain, while its Golgi localization requires the C1A domain and the phosphorylation of loop serines. After activation by G-protein-coupled receptors, PKD1 can shuttle between nucleus and cytosol, requiring the C1B domain for import and the PH domain for export from the nucleus [22] . …”