Protein kinase B (PKB), a ubiquitously expressed serine/threonine kinase, has central impact on several cellular processes including survival, proliferation and differentiation. In T cells, PKB is activated by growth factors, cytokines as well as TCR and CD28 stimulation. Previous studies performed with PKB transgenic (tg) mice, expressing a constitutively active version of PKBalpha (myrPKB) in the T cell lineage, revealed active PKB to influence TCR proximal signaling events. The aim of this study was to characterize the cross-talk of PKB with the transcription factor NFAT and the Src kinase Fyn at the biochemical and molecular level. Expression of a hyperactive form of the phosphatase calcineurin (deltaCam) in thymocytes increased nuclear NFAT levels and caused a block in early thymocyte differentiation. Co-expression of PKB in deltaCam tg thymocytes reduced NFAT activity, induced Rag and TCR beta-chain expression and abrogated the block in thymocyte differentiation. Rag2 promoter activity assays showed that NFATc1 as well as NFATc2 regulates the Rag2 promoter, NFAT factors thus being among the few transcription factors so far known to be involved in the regulation of rag expression in T cells. IL-2 promoter activity induced by NFAT was also down-modulated by active PKB. Furthermore, myrPKB enhanced the inhibition of NFAT activation in concert with the NFAT kinases PKA and GSK3 or the transcription factor Foxp3. Since recombinant PKB phosphorylated NFATc1 at (at least) two sites within the NFAT regulatory domain, NFAT could be a direct substrate of PKB. Compared with wild type cells, peripheral myrPKB tg CD4+ T cells showed enhanced proliferation after CD3 stimulation and in the presence of pharmacological Src kinase and MEK inhibitors. In addition, western blot analysis revealed enhanced Erk activity in myrPKB tg CD4+ T cells and in vitro kinase assays (IVKs) showed increased Fyn activity in myrPKB tg CD4+ T cells and thymocytes. By generating several GST-Fyn fusion proteins and mutagenesis of prospective PKB phosphorylation sites, a PKB phosphorylation site was identified in N-terminal Fyn, Fyn thus being a novel substrate of PKB in vitro. Furthermore, in transfected HEK 293T cells PKB and Fyn co-immunoprecipitated, supporting a direct interaction of PKB and Fyn in vivo. Interestingly, Fyn hyperactivity in PKB tg cells was not correlated with increased phosphorylation of the adapter molecule PAG at Y314, a known Fyn phosphorylation site and anchor for the recruitment of the kinase Csk, which inhibits Src kinase activity and, thereby, leads to the shut-down of T cell receptor signaling. It is known that increased Fyn activity causes/coincides with T cell anergy. Interestingly, in the model of ionomycin induced anergy, myrPKB tg CD4+ T cells were less susceptible to anergy induction. Whereas anergy, i.e. lack of proliferation after CD3 Ab restimulation, in wild type CD4+ T cells correlated with enhanced Fyn activity in comparison to untreated wild type cells, ionomycin treatment of myrPKB tg CD4+ T cells did not enhance Fyn activity. Altogether the data reveal a novel interaction and impact of PKB on Fyn activity. PKB mediated changes in Fyn activity, possibly also resulting in altered interaction of Fyn with certain substrates, may be important in regulatory processes like anergy, as our initial results indicate. Finally, the cross-talk of PKB with Fyn, both known proto-oncogenes, could also be important for transformation and tumorigenesis.