Properties, regulation and in vivo functions of novel C. elegans protein kinases (PKC4 and PKC5)

Abstract

I characterized cDNAs that encode novel C. elegans Ser/Thr protein kinases named PKC4 and PKC5. Both phosphotransferases are members of the PKD/PKCmu family: PKC5 is composed of 1068 amino acids and is encoded by a novel 3.9 kb cDNA. PKC5 and PKC4 (previously identified in the Rubin lab) contain tandem Cys-rich and PH regulatory domains, as well as catalytic domains that are homologous with corresponding regions in mammalian PKD/PKCmu.;Two Cys-rich regions near the N termini of PKC4 and PKC5 fold into Zn-stabilized domains that bind diacylglycerol (DAG) and phorbol esters (TPA). Incubation cytoplasm to plasma membrane, thereby promoting kinase activation via interaction with phosphatidylserine (PS) and possibly, upstream activating kinases. Subsequently, PKCs 4 and 5 are internalized and degraded in a phosphorylation dependent manner.;Mutation of conserved residues within the PH module (K298 or W396 to A) or PH domain deletion abrogates 7-fold activation of phosphotransferase activity in PKC4 elicited by DAG/PS. The PKC4 PH domain may function as an "internal adapter" that either (a) transmits activating conformational changes from Cys-rich regulatory regions to the C terminal catalytic domain; (b) stabilizes the DAG/PS-induced active conformation of catalytic and substrate binding sites or (c) targets PKC4 to the vicinity of a upstream kinase allowing PKC4 activation.;I also discovered that two conserved residues Ser925 and Ser929, in the PKC5 activation loop are critical regulators of activity and stability of the enzyme. Application of phospho-specific antibodies, PKC inhibitors and assays revealed that phosphorylation of Ser925 by a PKC (note that classical, novel and atypical PKCs, such as PKCalpha, PKCepsilon of Ser925 by a PKC (note that classical, novel and atypical PKCs, such as PKCalpha, PKCepsilon and PKCxi, belong to a partly-related, but distinct family of signaling kinases) is essential for PKC5 activation by growth factors.;Cellular and temporal patterns of PKC5 gene transcription were determined to provide insight into physiological functions of the kinase. The single-copy PKC5 gene was mapped to chromosome V. A 1.4 kbp DNA fragment (promoter/enhancer) that flanks the 5' end of the PKC5 gene was inserted upstream from nucleus-directed, GFP reporter cDNA in a C. elegans expression plasmid (designated pkc5P