Characterization of serine 916 as an in vivo autophosphorylation site for protein kinase D/Protein kinase Cmu.
Activation of the serine kinase protein kinase D (PKD)/PKCmicro 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/PKCmu. An antibody that recognized PKD/PKCmu 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/PKCmu activity in vivo. Antigen receptor ligation of T and B lymphocytes also induced phosphorylation of the serine 916 residue of PKD/PKCmu. Furthermore the regulatory FcgammaRIIB 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/PKCmu. The degree of serine 916 phosphorylation during lymphocyte activation and inhibition exactly correlated with the activation status of PKD/PKCmu. Moreover, using different mutants of PKD/PKCmu, 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/PKCmu.
Matthews
S A
SA
Lymphocyte Activation Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, United Kingdom.
Rozengurt
E
E
Cantrell
D
D
D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000201:enzymology; D001483:Base Sequence; D019556:COS Cells; D017931:DNA Primers; D051379:Mice; D008807:Mice, Inbred BALB C; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D010748:Phosphopeptides; D011994:Recombinant Proteins; D015240:Phorbol 12,13-Dibutyrate; D012694:Serine; C089212:protein kinase D; D011493:Protein Kinase C