Structure and properties of a ubiquitously expressed protein kinase C, nPKC delta.
cDNA clones coding for novel protein kinase C delta (nPKC delta) were isolated from a mouse brain cDNA library. Mouse nPKC delta consists of 674 amino acid residues and has sequence identity of 95% with rat nPKC delta. Antiserum raised against a C-terminal peptide of rat nPKC delta identified a 79-kDa protein in COS cells transfected with a mouse nPKC delta cDNA expression plasmid. nPKC delta expressed in COS1 cells had phorbol-ester-binding activity and protein kinase activity in a phorbol-ester- or diacylglycerol-dependent manner, like conventional protein kinase C (cPKC) isozymes and nPKC epsilon. However, nPKC delta, like nPKC epsilon, is not activated by Ca2+, a known activator of cPKCs, and requires lower concentrations of Mg2+ for full activation than cPKCs. Moreover, apparent kinetic constants for synthetic oligopeptides (MBP4-14, EGFR peptide and epsilon-peptide) were quite different between nPKC delta and cPKC in two different conditions. Among various phospholipids tested, phosphatidylinositol is the most potent activator of nPKC delta, in clear contrast to cPKCs and nPKC epsilon. Limited proteolysis of nPKC delta generated a C-terminal active fragment with a cofactor-independent kinase activity. Northern blot analysis indicated that nPKC delta, like cPKC alpha, is widely distributed in almost all the tissues and cells examined and, in some cases such as fibroblast cells, exists as a major PKC type. These results suggest that nPKC delta is involved in fundamental cellular functions regulated by diacylglycerols and mimicked by phorbol esters.
Mizuno
K
K
Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science, Japan.
Kubo
K
K
Saido
T C
TC
Akita
Y
Y
Osada
S
S
Kuroki
T
T
Ohno
S
S
Suzuki
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D015723:Gene Library; D007106:Immune Sera; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides / Q000138:chemical synthesis / Q000276:immunology; D010449:Peptide Mapping; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D011493:Protein Kinase C / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection
D007106:Immune Sera; D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides; D011994:Recombinant Proteins; D015240:Phorbol 12,13-Dibutyrate; D004247:DNA; D011493:Protein Kinase C