Discovery and characterization of two protein kinase C isoforms and a C-kinase anchor protein (C-KAP) from Caenorhabditis elegans

Abstract

Little is known about the expression, function, regulation and targeting of "atypical" protein kinase C (aPKC) and PKC{dollar}\mu{dollar}/PKD isoenzymes in vivo. To study these topics, I have characterized aPKC (PKC3) and PKC{dollar}\mu{dollar}/PKD (PKC4) isoforms from C. elegans.;C. elegans PKC3 (597 amino acids) is encoded by a 2.5 kbp cDNA. Structural/enzymic properties of PKC3 indicate that the C. elegans enzyme is a new member of the atypical subclass of PKC isoforms. In post-embryonic animals, a 647 bp promoter/enhancer directs pkc-3 gene transcription and PKC3 protein accumulation in {dollar}\sim{dollar}85 muscle, epithelial and hypodermal cells. These cells are incorporated into tissues involved in feeding, digestion, excretion and reproduction. PKC3 is also expressed in multiple cell types during embryogenesis. Ablation of PKC3 function by microinjection of anti-sense RNA elicits production of disorganized, developmentally-arrested and inviable embryos.;Biochemical analysis and immunofluorescence microscopy revealed that PKC3 accumulates near cortical actin cytoskeleton/plasma membrane at the apical surface of intestinal cells and in embryonic cells. An aPKC anchoring/targeting protein, PICK1{dollar}\rm\sb{lcub}CE{rcub}{dollar} has been identified and characterized. PICK1{dollar}\rm\sb{lcub}CE{rcub}{dollar} and PKC3 form complexes in cells and PICK1{dollar}\rm\sb{lcub}CE{rcub}{dollar} elicits translocation of PKC3 from cytosol to a particulate fraction in co-transfected mammalian cells.;A novel 2.3 kbp cDNA encodes C. elegans PKC4 (722 amino acids) which contains PH and tandem Cys-rich regulatory domains. PKC4 is a DAG-stimulated phosphotransferase that is most related to mammalian PKC{dollar}\mu{dollar}/PKD. Unlike PKC{dollar}\mu{dollar}/PKD, PKC4 lacks a signal sequence and transmembrane domain; moreover, it is highly susceptible to depletion via down-regulation. PKC4 is in cytosol at all stages of C. elegans development. PKC4 protein content is maximal in embryos and declines steadily thereafter. Expression of a PKC4 transgene in AV-12 cells yields a cytosolic enzyme. Phorbol ester (TPA) elicits translocation of PKC4 from cytoplasm to plasma membrane and the subsequent depletion of the kinase from all cell compartments. pkc-4 gene transcription is detected in {dollar}\sim{dollar}25 cells of C. elegans in situ. pkc-4 promoter activity is evident in hypodermal seam cells that are thought to generate the alae (treads essential for motility) in late embryos and young adults.