EXPRESSION AND BIOCHEMICAL-CHARACTERIZATION OF HUMAN PROTEIN-KINASE C-THETA

In this study, the recently identified human protein kinase C-Theta (P KC-Theta) isoform has been biochemically characterized in detail. An a ntiserum raised against the unique V3 domain of PKC-Theta identified a n 80-kDa protein in all human T-cell lines tested, in erythroleukemia K562 cells and in histiocytic lymphoma U-937 cells, but not in a B-lym phoma line (Raji) or in several melanoma, carcinoma, schwanoma or astr ocytoma lines, confirming, at the protein level, its predominant expre ssion in hematopoietic cell lines, in particular T cells. Immunoreacti ve PKC-B was detected almost exclusively in the cytosolic compartment of unstimulated Jurkat T cells. Stimulation with phorbol ester, howeve r, caused rapid translocation to the membrane. In order to compare the properties of PKC-Theta with a representative member of the Ca2+-depe ndent PKC enzymes, full-length cDNAs encoding PKC-Theta or PKC-alpha w ere transiently expressed in COS-1 cells, and recombinant enzymes were partially purified via a six-histidine peptide tag. The catalytic act ivity of these PKC enzymes was assayed against distinct substrates in the absence and presence of known PKC cofactors. Significant differenc es were found with respect to activation requirements and substrate pr eferences between PKC-Theta and PKC-alpha. Both enzymes were stimulate d by phospholipid and phorbol ester, and were active towards alpha PKC -derived substrate peptide corresponding to the pseudosubstrate site o f PKC. In contrast to PKC-alpha, however, full activation of PKC-Theta did not require Ca2+, and its basal activity towards histone H1 was n ot stimulated by lipid cofactors. Additionally, a myelin-basic-protein -(MBP)-derived peptide, which was readily phosphorylated by PKC-alpha, was a poor substrate for PKC-Theta. Similar to PKC-alpha, transient P KC-Theta overexpression in murine EL4 thymoma cells caused an approxim ately 2.5-fold increase in the phorbol-12-myristate-13-acetate-induced transcriptional activation of an interleukin-2 promoter-reporter gene construct. The unique expression and functional properties of PKC-The ta suggest that it may play a specialized role in T-cell signaling pat hways.