Activation of the serine kinase protein kinase D (PKD)/PKC is controlled by the phosphorylation of two serine residues within its activation loop via a PKC-dependent signaling cascade. In this study we have identified the C-terminal serine 916 residue as an in vivo phosphorylation site within active PKD/PKC. An antibody that recognized PKD/PKC proteins specifically phosphorylated on the serine 916 residue was generated and used to show that phosphorylation of Ser-916 is induced by phorbol ester treatment of cells. Thus, the pS916 antibody is a useful tool to study the regulation of PKD/PKC activity in vivo. Antigen receptor ligation of T and B lymphocytes also induced phosphorylation of the serine 916 residue of PKD/PKC. Furthermore the regulatory Fc␥RIIB receptor, which mediates vital negative feedback signals to the B cell antigen receptor complex, inhibited the antigen receptor-induced activation and serine 916 phosphorylation of PKD/PKC. The degree of serine 916 phosphorylation during lymphocyte activation and inhibition exactly correlated with the activation status of PKD/PKC. Moreover, using different mutants of PKD/PKC, we show that serine 916 is not trans-phosphorylated by an upstream kinase but is rather an autophosphorylation event that occurs following activation of PKD/PKC.The protein kinase C (PKC) 1 family of serine/threonine kinases has been implicated in a wide range of biological responses in a number of different cellular systems, including roles in the control of cell morphology, differentiation, and proliferation (1-5). There are multiple related PKC isoforms (5-8), which can be classified into three distinct subgroups on the basis of structural and regulatory differences: the conventional PKCs (␣, ␤ I , ␤ II , and ␥), which are regulated by calcium, diacylglycerol (DAG), and phospholipids; the novel PKCs (␦, ⑀, , and ), which are regulated by DAG and phospholipids; and the atypical PKCs ( and ), whose regulation is less characterized but that have been proposed to be regulated by D-3 phosphoinositides (9). The DAG-regulated PKC isoforms all bind phorbol esters and are the major cellular targets for this class of tumor promoter (10). All PKCs share a highly conserved catalytic domain, although each isoform has a different optimal substrate specificity (11), supporting the idea that each isoform has specific functions in vivo.A recently described PKC-related serine/threonine protein kinase is protein kinase D (PKD), also named PKC (12, 13). PKD/PKC contains a cysteine-rich domain that binds DAG and phorbol esters but lacks the C2 calcium binding domain seen in the classical PKCs. In contrast to other PKCs, (including mammalian, Drosophila, and yeast isoforms), the N-terminal regulatory region of PKD/PKC contains a pleckstrin homology (PH) domain that regulates enzyme activity (14) and lacks a sequence with homology to a typical PKC autoinhibitory pseudosubstrate motif. Moreover, the PKD/PKC catalytic domain shows little similarity to the highly conserved regions of the kinase subdomains of the...

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Mammalian PKD (protein kinase D) isoforms have been implicated in the regulation of diverse biological processes in response to diacylglycerol and PKC (protein kinase C) signalling. To compare the functions of PKD1 and PKD2 in vivo, we generated mice deficient in either PKD1 or PKD2 enzymatic activity, via homozygous expression of PKD1S744A/S748A or PKD2S707A/S711A ‘knockin’ alleles. We also examined PKD2-deficient mice generated using ‘gene-trap’ technology. We demonstrate that, unlike PKD1, PKD2 catalytic activity is dispensable for normal embryogenesis. We also show that PKD2 is the major PKD isoform expressed in lymphoid tissues, but that PKD2 catalytic activity is not essential for the development of mature peripheral T- and B-lymphocytes. PKD2 catalytic activity is, however, required for efficient antigen receptor-induced cytokine production in T-lymphocytes and for optimal T-cell-dependent antibody responses in vivo. Our results reveal a key in vivo role for PKD2 in regulating the function of mature peripheral lymphocytes during adaptive immune responses. They also confirm the functional importance of PKC-mediated serine phosphorylation of the PKD catalytic domain for PKD activation and downstream signalling and reveal that different PKD family members have unique and non-redundant roles in vivo.

Protein kinase D (PKD; also known as PKCmicro) is a serine/threonine kinase activated by diacylglycerol signalling pathways in a variety of cells. PKD has been described previously as Golgi-localized, but herein we show that it is present within the cytosol of quiescent B cells and mast cells and moves rapidly to the plasma membrane after antigen receptor triggering. The membrane redistribution of PKD requires the diacylglycerol-binding domain of the enzyme, but is independent of its catalytic activity and does not require the integrity of the pleckstrin homology domain. Antigen receptor signalling initiates in glycosphingolipid-enriched microdomains, but membrane-associated PKD does not co-localize with these specialized structures. Membrane targeting of PKD is transient, the enzyme returns to the cytosol within 10 min of antigen receptor engagement. Strikingly, the membrane-recycled PKD remains active in the cytosol for several hours. The present work thus characterizes a sustained antigen receptor-induced signal transduction pathway and establishes PKD as a serine kinase that temporally and spatially disseminates antigen receptor signals away from the plasma membrane into the cytosol.

We have taken a knockout approach to interrogate the function of protein kinase D (PKD) serine/threonine kinases in lymphocytes. DT40 B cells express two PKD family members, PKD1 and PKD3, which are both rapidly activated by the B-cell antigen receptor (BCR). DT40 cells with single or dual deletions of PKD1 and/or PKD3 were viable, allowing the role of individual PKD isoforms in BCR signal transduction to be assessed. One proposed downstream target for PKD1 in lymphocytes is the class II histone deacetylases (HDACs). Regulation of chromatin accessibility via class II histone deacetylases is an important mechanism controlling gene expression patterns, but the molecules that control this key process in B cells are not known. Herein, we show that phosphorylation and nuclear export of the class II histone deacetylases HDAC5 and HDAC7 are rapidly induced following ligation of the BCR or after treatment with phorbol esters (a diacylglycerol mimetic). Loss of either PKD1 or PKD3 had no impact on HDAC phosphorylation, but loss of both PKD1 and PKD3 abrogated antigen receptor-induced class II HDAC5/7 phosphorylation and nuclear export. These studies reveal an essential and redundant role for PKD enzymes in controlling class II HDACs in B lymphocytes and suggest that PKD serine kinases are a critical link between the BCR and epigenetic control of chromatin.The protein kinase D (PKD) family comprises three different but closely related serine kinases, PKD1, PKD2, and PKD3, all of which have a highly conserved N-terminal regulatory domain containing two cysteine-rich diacylglycerol (DAG) binding domains and an autoinhibitory pleckstrin homology (PH) domain. PKD enzymes are highly expressed in hematopoietic cells, and they are selectively activated by the engagement of antigen receptors, including the B-cell antigen receptor (BCR), the T-cell receptor, and the FcεR1 in B cells, T cells, and mast cells, respectively (22,24). PKD family members are activated by a signaling pathway involving gamma phospholipase C activation, production of diacylglycerol, and activation of classical/novel PKCs. PKC-mediated phosphorylation of two conserved serine residues in the catalytic domains of PKDs is essential for their activation (13,22,45,48,49). In addition, binding of DAG to the regulatory domain of PKD contributes both to PKD1 activation (50) and to PKD subcellular localization (21,23,(38)(39)(40).PKD enzymes are predicted to play important functions in controlling lymphocyte biology, but most of the evidence that supports this hypothesis is indirect. For example, studies in transgenic mice have shown that constitutively active PKD1 can substitute for the pre-T-cell antigen receptor complex to regulate early thymocyte differentiation and proliferation (20). In other cell types, PKD enzymes have also been implicated in the regulation of Golgi organization and protein trafficking to the cell surface (1,8,12,14,18,53), cell survival (44), 43,44), glucose transport (5), and integrin activation/recycling (29, 51).In addition, it has been ...

Protein kinase D (PKD)/protein kinase CW W (PKCW W), a serine/threonine protein kinase with distinct structural and enzymological properties, is rapidly activated in intact cells via PKC. The amino-terminal region of PKD contains a cysteinerich domain (CRD) that directly binds phorbol esters with a high affinity. Here, we show that treatment of transfected RBL 2H3 cells with phorbol 12,13-dibutyrate (PDB) induces a striking CRD-dependent translocation of PKD from the cytosol to the plasma membrane, as shown by real time visualization of a functional green fluorescent protein (GFP)-PKD fusion protein.A single amino acid substitution in the second cysteine-rich motif of PKD (P287G) prevented PDB-induced membrane translocation but did not affect PKD activation. Our results indicate that PKD translocation and activation are distinct processes that operate in parallel to regulate the activity and localization of this enzyme in intact cells.z 1999 Federation of European Biochemical Societies.

Bryostatin 1 and phorbol esters are both potent activators of protein kinase C (PKC), although their specific biological effects can differ in many systems. Here, we report that bryostatin 1 activates protein kinase D (PKD), a novel serine/threonine protein kinase, in intact Swiss 3T3 cells and secondary mouse embryo fibroblasts and in COS-7 cells transiently transfected with a PKD expression construct. The dose response of PKD activation induced by bryostatin 1 follows a striking biphasic pattern with maximal activation achieved at a concentration of 10 nM. Higher concentrations of bryostatin 1 (100 nM) reduced PKD activation induced by phorbol 12,13-dibutyrate to levels stimulated by bryostatin 1 alone. Bryostatin 1-induced PKD activation was markedly attenuated by treatment of cells with the PKC inhibitors bisindolylmaleimide I and Ro 31-8220. However, these agents did not inhibit PKD activity when added directly to in vitro kinase assays, suggesting that bryostatin 1 stimulates PKD activation through a PKC-dependent pathway in intact cells. Our results raise the possibility that activated PKD in intact cells could mediate some of the multiple biphasic biological responses induced by bryostatin 1.Bryostatin 1 is a natural macrocyclic lactone with potent antineoplastic properties in a variety of animal models (1-3) and has entered clinical trials as a potential therapeutic agent (4, 5). Bryostatin family members bind to and activate classic and novel isoforms of protein kinase C (PKC) 1 (6 -9), the major cellular targets of the tumor-promoting phorbol esters (10). Despite appearing to bind to the same cellular targets, the biological responses induced by bryostatin 1 frequently differ from those induced by phorbol esters. For example, many bryostatin 1-mediated effects have unusual characteristics such as biphasic dose-response relationships, delayed kinetics, and the ability to inhibit phorbol ester-induced responses (7,(11)(12)(13)(14)(15)(16)). The precise mechanism(s) by which bryostatin 1 induces these biological effects remain poorly understood.The newly identified protein kinase D (PKD) is a mouse serine/threonine protein kinase with distinct structural features and enzymological properties (17)(18)(19). In particular, the catalytic domain of PKD, which is distantly related to Ca 2ϩ -regulated kinases, shows little homology to the highly conserved regions of the kinase domain of the PKC family. As a consequence of this, PKD does not phosphorylate a variety of known PKC substrates, indicating that PKD has a distinct substrate specificity (18, 19).The amino-terminal region of PKD contains a putative transmembrane domain, two cysteine-rich zinc finger-like motifs, and a pleckstrin homology domain. Unlike all known PKC isoforms, PKD does not contain a pseudosubstrate motif upstream of the cysteine-rich region, and the sequence separating the cysteine-rich repeats of PKD (95 amino acids) is substantially longer than that of classical and novel PKCs (28 and 35 amino acids, respectively). Additionally, residue...

Protein kinase D (PKD) is a serine/threonine kinase that binds phorbol esters in a phospholipid-dependent manner via a tandemly repeated cysteine-rich, zinc finger-like motif (the cysteine-rich domain, CRD). Here, we examined whether the individual cysteine-rich motifs of the CRD of PKD (referred to as cys1 and cys2) are functionally equivalent in mediating phorbol ester binding both in vivo and in vitro. Our results demonstrate that the cys1 and cys2 motifs of the CRD of PKD are functionally dissimilar, with the cys2 motif responsible for the majority of [ 3 H]phorbol 12,13-dibutyrate (PDB) binding, both in vivo and in vitro.z 1998 Federation of European Biochemical Societies.