Protein kinase D (PKD) regulates the fission of vesicles from the trans-Golgi-network 1,2 . We show that phosphatidylinositol-4-kinase III beta (PI4KIIIβ), a key player in Golgi complex structure and function 3 , is a physiological substrate of PKD. Of the three PKD isoforms, only PKD1 and PKD2 phosphorylated PI4KIIIβ at a motif highly conserved from yeast to man. PKD mediated phosphorylation stimulated lipid kinase activity of PI4KIIIβ and enhanced VSV-G transport to plasma membrane. The identification of PI4KIIIβ as one of the PKD substrates should help to reveal the molecular events leading to transport carrier formation.

In order to investigate regulatory mechanisms and to identify potential substrates of a novel member of the protein kinase C (PKC) family, PKCp, specific antibodies have been raised against unique aminoand carboxy-terminal regions. PKCp kinase activity was studied upon immunoprecipitation from stably transfected cell lines as well as from the A549 carcinoma cell line expressing the endogeneous PKCp gene. Cell fractionation revealed that PKCp is predominantly found in the particulate fraction, suggesting an association with the membrane or membrane-bound structures. In vitro kinase assays with immunoprecipitated PKCp demonstrated a Ca2+ independent enhancement of constitutive autophosphorylation activity by phosphatidylserine. Despite a limited in vitro phorbol ester response, an apparent phorbol ester activation of PKCp was observed when cell cultures, instead of immunoprecipitated enzyme, were treated with either phorbol 12-myristate 13-acetate or 1,2 dioleoyl-sn-glycerol. Both in vitro autophosphorylation and substrate phosphorylation of myelin basic protein and histone I11 were enhanced under these conditions. However, long-term treatment with the phorbol ester did not result in downregulation of PKCp protein levels and kinase activity. Studies with several protein kinase inhibitors revealed a novel sensitivity profile of PKCp, with no inhibition by calphostin C, reduced sensitivity to staurosporine but, compared to other PKCs, an approximately 60-fold higher sensitivity to the selective PKA inhibitor H89. Together, the data presented here show that localization of PKCp and regulation of its kinase activity differ from that of other PKCs suggesting a novel function of PKCp in intracellular signal pathways.Keywords. Protein kinase Cp; activators, of protein kinase Cp; inhibitors of protein kinase Cp.Protein kinases C (PKC) are important regulatory enzymes involved in multiple cellular responses. They are typically activated by lipid second messengers such as diacylglycerol in response to various extracellular agonists like hormones, neurotransmitters, growth factors and cytokines [ l , 21. Molecular cloning of various PKC isoforms has established that PKC is a multigene family [3 -111. All isozymes share a characteristic conserved catalytic domain in the carboxy-terminal region and an amino-terminal regulatory site (Cl). To date, 11 members have been identified, which can be grouped into three major classes according to their different activation conditions. The first group, the conventional PKCs (cPKCa, p l , p2 and y ) require Ca2' to be activated in the presence of phosphatidylserine [3] whereas the second group, the novel PKCs (nPKCs) lack the C2 domain of cPKCs and are Ca2+ independent [4-6, 8, 91. Common features within the C l domain of cPKCs and nPKCs are a conserved pseudosubstrate site and two adjacent aminoterminal cysteine clusters that are involved in phorbol ester binding [2]. Members of the third subgroup have been termed atypical PKCs (aPKCC, and A) because they lack the C2 region and contain o...

Protein kinase D (PKD) has been identified as a negative regulator of epithelial cell migration; however, its molecular substrates and downstream signaling pathways that mediate this activity have remained elusive. In this study, we provide evidence that the cofilin phosphatase slingshot 1 like (SSH1L), an important regulator of the complex actin remodeling machinery, is a novel in vivo PKD substrate. PKD-mediated phosphorylation of serines 937 and 978 regulates SSH1L subcellular localization by binding of 14-3-3 proteins and thus impacts the control of local cofilin activation and actin remodeling during cell migration. In line with this, we show that the loss of PKD decreases cofilin phosphorylation, induces a more spread cell morphology, and stimulates chemotactic migration of breast cancer cells in an SSHL1-dependent fashion. Our data thus identify PKD as a central regulator of the cofilin signaling network via direct phosphorylation and regulation of SSH1L. [Cancer Res 2009;69(14):5634-8]

Protein kinase C (PKC), a family of lipid-activated serine kinases, is involved in multiple functions in the regulation of growth control. The PKC-related isoform PKC/PKD has been implicated in mitogenic signal cascades because of the activation of p42/p44 MAPK leading to Elk1-mediated gene transcription, and PKC/PKD has been shown to be activated via a PKC-dependent pathway. By using confocal analyses, we demonstrate here that PKC partially colocalizes with PKC in different cell types. Colocalization depends on the presence of the PKC pleckstrin homology domain. Coexpression of constitutively active PKC with PKC leads to a significant enhancement of the PKC substrate phosphorylation capacity as a result of an increased phosphorylation of the activation loop Ser 738/742 of PKC, whereas Ser 910 autophosphorylation remains unaffected. In vitro phosphorylation experiments show that PKC directly phosphorylates PKC on activation loop serines. Consequently, the p42 MAPK cascade is triggered leading to an increase in reporter gene activity driven by a serum-responsive element in HEK293 cells. At the same time, PKC-mediated JNK activation is reduced, providing evidence for a mutual regulation of PKC/PKC affecting different arms of the p38/ERK/ JNK pathways. Our data provide evidence for the sequential involvement of selective PKC isoforms in kinase cascades and identify the relevant domains in PKC for interaction with and activation by PKC as pleckstrin homology domain and activation loop.

Recent studies have documented direct interaction between 14-3-3 proteins and key molecules in signal transduction pathways like Ras, Cbl, and protein kinases. In T cells, the 14-3-3 isoform has been shown to associate with protein kinase C and to negatively regulate interleukin-2 secretion. Here we present data that 14-3-3 interacts with protein kinase C (PKC), a subtype that differs from other PKC members in structure and activation mechanisms. Specific interaction of PKC and 14-3-3 can be shown in the T cell line Jurkat by immunocoprecipitiation and by pulldown assays of either endogenous or overexpressed proteins using PKC-specific antibodies and GST-14-3-3 fusion proteins, respectively. Using PKC deletion mutants, the 14-3-3 binding region is mapped within the regulatory C1 domain. Binding of 14-3-3 to PKC is significantly enhanced upon phorbol ester stimulation of PKC kinase activity in Jurkat cells and occurs via a Cbl-like serine containing consensus motif. However, 14-3-3 is not a substrate of PKC. In contrast 14-3-3 strongly down-regulates PKC kinase activity in vitro. Moreover, overexpression of 14-3-3 significantly reduced phorbol ester induced activation of PKC kinase activity in intact cells. We therefore conclude that 14-3-3 is a negative regulator of PKC in T cells.