PROTEIN-KINASE C-THETA ISOENZYME-SELECTIVE STIMULATION OF THE TRANSCRIPTION FACTOR COMPLEX AP-1 IN T-LYMPHOCYTES

T-lymphocyte stimulation requires activation of several protein kinase s, including the major phorbol ester receptor protein kinase C (PKC), ultimately leading to induction of lymphokines, such as interleukin-2 (1L-2). The relevant PKC isoforms which are involved in the activation cascades of nuclear transcription factors involved in IL-2 production have not get been clearly defined, We have examined the potential rol e of two representative PKC isoforms in the induction of the IL-2 gene , i.e., PKC-alpha and PKC-theta, the latter being expressed predominan tly in hematopoietic cell lines, particularly T cells, Similar to that of PKC-alpha, PKC-theta overexpression in murine EW thymoma cells cau sed a significant increase in phorbol 12-myristate 13-acetate (PMA)-in duced transcriptional activation of full-length IL-2-chloramphenicol a cetyltransferase (CAT) and NF-AT-CAT but not of NF-IL2A-CAT or NF-kapp a B promoter-CAT reporter gene constructs, Importantly, the critical A P-1 enhancer element was differentially modulated by these two distinc t PKC isoenzymes, since only PKC-theta but not PKC-alpha overexpressio n resulted in an approximate to 2.8-fold increase in AP-1-collagenase promoter CAT expression in comparison with the vector control. Deletio n of the AP-1 enhancer site in the collagenase promoter rendered it un responsive to PKC-theta, Expression of a constitutively active mutant PKC-theta A148E (but not PKC-alpha A25E) was sufficient to induce acti vation of AP-1 transcription factor complex in the absence of PMA stim ulation. Conversely, a catalytically inactive PKC-theta K409R (but not PKC-alpha K368R) mutant abrogated endogenous PMA-mediated activation of AP-1 transcriptional complex, Dominant negative mutant Ha-RasS17N c ompletely inhibited the PKC-theta A148E-induced signal, identifying PK C-theta as a specific constituent upstream of or parallel to Ras in th e signaling cascade leading to AP-1 transcriptional activation.