Molecular cloning and expression of a cDNA encoding a novel isoenzyme of protein kinase C (nPKC). A new member of the nPKC family expressed in skeletal muscle, megakaryoblastic cells, and platelets.
At least seven bacteriophage lambda clones encoding structurally related but unique polypeptides with PKC activity have been isolated from mammalian brain, epidermis, and lung cDNA libraries. The possibility that additional isoenzymes are expressed in human blood platelets or megakaryoblastoid human erythroleukemia cells was examined by polymerase chain reaction amplification of reverse transcribed RNA employing oligonucleotide primers corresponding to conserved peptide sequences. cDNAs encoding a novel PKC-related sequence, designated PKC-theta, and four (alpha, beta, delta, and eta) previously identified isoenzymes were isolated from reverse transcribed total RNA of human erythroleukemia cells and platelets. PKC-theta lacks a conserved region (C2) that is present in the calcium-dependent isoenzymes and therefore belongs to the group of novel, or nPKC, isoenzymes. Significantly increased [3H] phorbol 12,13-dibutyrate binding and cytoskeleton-associated calcium-independent PKC activity were found in COS cells expressing the transfected cDNA. Northern transfer analysis of mRNA from various human tissues revealed high level expression of PKC-theta in skeletal muscle, lung, and brain, and minimal expression in cardiac muscle, placenta, and liver. These findings extend the PKC family and suggest a novel approach to the study of diversity within this pathway of intracellular signal transduction.
Chang
J D
JD
Harvard-Thorndike Laboratory, Department of Medicine (Cardiovascular Division), Beth Israel Hospital, Boston, Massachusetts 02215.
Xu
Y
Y
Raychowdhury
M K
MK
Ware
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001792:Blood Platelets / Q000201:enzymology*; D015152:Blotting, Northern; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA; D004730:Endothelium, Vascular / Q000201:enzymology; D015870:Gene Expression; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics*; D004915:Leukemia, Erythroblastic, Acute / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D005810:Multigene Family; D009132:Muscles / Q000201:enzymology*; D009838:Oligodeoxyribonucleotides; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D011061:Poly A / Q000302:isolation & purification / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D011493:Protein Kinase C / Q000096:biosynthesis* / Q000235:genetics*; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism; D012333:RNA, Messenger; D051381:Rats; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014407:Tumor Cells, Cultured; D014471:Umbilical Veins
D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D011061:Poly A; D015240:Phorbol 12,13-Dibutyrate; D012313:RNA; D004247:DNA; D011493:Protein Kinase C