MOLECULAR-CLONING AND EXPRESSION OF A CDNA-ENCODING A NOVEL ISOENZYMEOF 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 relat ed but unique polypeptides with PKC activity have been isolated from m ammalian brain, epidermis, and lung cDNA libraries. The possibility th at additional isoenzymes are expressed in human blood platelets or meg akaryoblastoid human erythroleukemia cells was examined by polymerase chain reaction amplification of reverse transcribed RNA employing olig onucleotide primers corresponding to conserved peptide sequences. cDNA s encoding a novel PKC-related sequence, designated PKC-theta, and fou r (alpha, beta, delta, and eta) previously identified isoenzymes were isolated from reverse transcribed total RNA of human erythroleukemia c ells and platelets. PKC-theta lacks a conserved region (C2) that is pr esent in the calcium-dependent isoenzymes and therefore belongs to the group of novel, or nPKC, isoenzymes. Significantly increased [H-3] ph orbol 12,13-dibutyrate binding and cytoskeleton-associated calcium-ind ependent PKC activity were found in COS cells expressing the transfect ed 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 li ver. These findings extend the PKC family and suggest a novel approach to the study of diversity within this pathway of intracellular signal transduction.