Identifying Non-Receptor Tyrosine Kinases which Regulate the Pro-Apoptotic Function of PKCdelt

The novel Protein Kinase C isoform PKCdelta is a ubiquitously expressed lipid regulated serine/threonine kinase, which regulates a wide range of cellular functions including proliferation, differentiation and apoptosis. The ability of PKCdelta to regulate such a variety of cellular functions depends largely on its strict subcellular localization. In resting non-transformed cells PKCdelta is localized within the cytoplasm and perinuclear region. In response to DNA damage PKCdelta undergoes nuclear translocation where it phosphorylates nuclear substrates, resulting in breakdown of the nuclear membrane and amplification of apoptotic signaling. Recent studies from our lab have elucidated the regulatory process that controls the dynamic subcellular localization of PKCdelta. In resting cells, PKCdelta is maintained in a closed but catalytically competent conformation where its C-terminal nuclear localization sequence is occluded from binding to importin-&agr;. Intra-molecular interactions necessary for maintaining a closed conformation have been mapped to the C2 domain and the C-terminal V5 region. In response to DNA damage, PKCdelta becomes phosphorylated at Y64 and Y155 resulting in structural rearrangements that convert PKCdelta into an open conformation with an exposed NLS. Recognition of the NLS by importin-&agr; is necessary for nuclear translocation. Our current study shows that the kinetics of Y64 and Y155 phosphorylation match the kinetics of c-Src and c-Abl activation, suggesting a role for non-receptor tyrosine kinases in PKCdelta activation. Our studies also show that pharmacological inhibition or knockdown of c-Src and c-Abl directly blocks phosphorylation of Y64 and Y155 respectively. Curiously, we demonstrate that c-Abl inhibition results in a decrease of Y155 and Y64 phosphorylation, suggesting a hierarchical relationship between phosphorylation of Y64 and Y155. Blocking Y155 phosphorylation by mutating the site to a phenylalanine resulted in abrogation of Y64 phosphorylation. On the contrary, mutation of Y64 to a phenylalanine failed to reduce Y155 phosphorylation. The role of both c-Src and c-Abl in the phosphorylation of Y64 and Y155 was confirmed through the expression of tyrosine kinase inhibitor resistant gatekeeper mutations for both c-Src (T341I) and c-Abl (T315I), which resulted in the reconstitution of Y64 and Y155 phosphorylation respectively. As phosphorylation of PKCdelta at Y64 and Y155 is necessary for nuclear import, we observed that inhibition of c-Src and c-Abl activity resulted in decreased apoptosis both in vitro and in vivo in response to radiation. In summary, our studies expand our understanding of the mechanism that regulates the pro-apoptotic function of PKCdelta. Additionally, we demonstrate a novel use of tyrosine kinase inhibitors as a prophylactic against radiation induced apoptosis.