The aim of this study was to investigate the influence of protein kinase C (PKC) alpha and beta on the TCR-CD28-stimulated protein kinase cascades participating in regulation of IL-2 gene transcription and secretion. Inhibition of the synthesis of PKCalpha and beta by specific phosphorothioate-modified antisense oligonucleotides (ODN) resulted in suppression of phosphorylation and activation of Raf-1, mitogen-activated extracellular-regulated kinase kinases and extracellular-regulated kinases in stimulated Jurkat T cells. Furthermore, a marked reduction of IkappaB kinase-alpha-catalyzed IkappaBalpha phosphorylation was observed in both PKCalpha- and beta-specific antisense oligonucleotide-treated cells. In sharp contrast, TCR-CD28-stimulated phosphorylation and activation of the Jun-N-terminal kinase (JNK) cascade was specifically suppressed upon treatment with PKCbeta-specific antisense ODN, suggesting that PKCbeta was a specific upstream regulator of the JNK protein kinase cascade. Significant inhibition of high-affinity NF-AT binding and transactivation, IL-2 gene expression, reduction of IL-2 mRNA synthesis, and, most impressively, a complete suppression of IL-2 secretion were observed in PKCbeta antisense ODN-treated cells. The data indicate a highly specific function of PKCbeta for regulation of TCR-CD28 induced-signaling, IL-2 gene expression and secretion in Jurkat T cells.