Ligand regulation of bovine protein kinase C alpha response via either cysteine-rich repeat of conserved region C1.
Based on the finding by others that the conserved region C1 of conventional protein kinase C isoforms carries two independent, cysteine-rich phorbol ester binding sites, we have mapped the structural elements of the C1 region for their role in the phorbol ester- and phospholipid regulation of PKC alpha responses. We have prepared two amino terminal truncation mutants of bovine PKC alpha, ND91 lacking the first Cys-repeat of C1, and ND153 lacking both Cys-repeats of C1, as well as two internal deletion mutants, D162-245 lacking most of C2, and D109-263 lacking most of C2 and the second Cys-repeat of C1. The mutants were expressed in the yeast Saccharomyces cerevisiae which allows the rapid biochemical and physiological characterization of mammalian PKC isoforms. We found that all mutants displayed an elevated basal level of enzymatic activity in vitro but retained the basic catalytic PKC characteristics: regulation by Ca2+ and (except for ND153) by phospholipid or phorbol ester. In vivo we observed proportional physiological responses, the stimulation of Ca2+ uptake, and an increase in the cell doubling time for all mutants upon phorbol ester stimulation (constitutive for ND153) similar to the response of normal PKC alpha. Our findings indicate that after partial PKC activation by deletion mutagenesis, the presence of either Cys-repeat in C1 still allows phospholipid- and phorbol ester regulation of protein kinase C alpha responses.
Zhu
J
J
Section on Molecular Biology, Joslin Diabetes Center, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02215.
Hansen
H
H
Su
L
L
Shieh
H L
HL
Riedel
H
H
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002872:Chromosome Deletion; D017124:Conserved Sequence; D003545:Cysteine / Q000032:analysis*; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics*; D008024:Ligands; D008969:Molecular Sequence Data; D009154:Mutation; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D011493:Protein Kinase C / Q000096:biosynthesis / Q000235:genetics*; D051571:Protein Kinase C-alpha; D011994:Recombinant Proteins / Q000096:biosynthesis; D012091:Repetitive Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae
D007527:Isoenzymes; D008024:Ligands; D011994:Recombinant Proteins; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D003545:Cysteine