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.