Characterization of Serine 916 as an in Vivo Autophosphorylation Site for Protein Kinase D/Protein Kinase Cμ

Abstract

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...

Protein kinase D1/2 is involved in the maturation of multivesicular bodies and secretion of exosomes in T and B lymphocytes

“…29 RO318220 treatment inhibited PMA-induced, PKCdependent phosphorylation of endogenous PKD1/2 and of PKD1 fused to GFP (GFP-PKD1) at the activation loop (pS744/S748) 30 (Supplementary Figure S1A); the effect was similar for a PKD1 kinase-deficient mutant (D733A; GFP-PKD1KD). 19,31 Inhibitor treatment also impaired PKD autophosphorylation (pS916) 27,29 induced by carbachol (CCh) (Supplementary Figure S1B) or by anti-TCR (data not shown). We pretreated J-HM1-2.2 cells with RO318220, followed by anti-TCR or CCh stimulation to induce exosome secretion.…”

“…Protein kinase C (PKC) phosphorylation at the PKD activation loop further promotes PKD autophosphorylation and activation. 27 Based on our previous studies showing DGKα regulation of DAG in MVB formation and exosome secretion, 9,14,28 and the identification of PKD1/2 association to MVB, 14 we hypothesized that DGKα control of DAG mediates these events, at least in part, through PKD. Here we explored whether, in addition to its role in vesicle fission from TGN, 19 PKD regulates other steps in the DAG-controlled secretory traffic pathway.…”

DAP kinase regulates JNK signaling by binding and activating protein kinase D under oxidative stress

“…Of note, since the use of purified proteins in vitro is limiting and can reflect only direct conformational changes resulting from the phosphorylation itself, it may be possible that Autophosphorylation of PKD on serine 910 reflects the status of its catalytic activity and is often used to determine PKD activation. 19 To further examine a role for DAPk in PKD activation, the effect of DAPk on PKD autophosphorylation levels was evaluated. Autophosphorylation of PKD at serine 910 was enhanced upon overexpression of DAPk by an average of 2.3-fold (Figure 2di and ii).…”

Protein kinase D displays intrinsic Tyr autophosphorylation activity: insights into mechanism and regulation

“…Known in vitro autophosphorylation sites for PKD include the activation loop Ser‐738/742 residues and the C‐terminal Ser‐910 residue 29, 31, 39, 40. In cells, PKD is phosphorylated on Ser‐738 mainly by upstream PKCs, while Ser‐742 phosphorylation can occur as a PKC‐mediated event, but under certain circumstances also as an autocatalytic event 41.…”