The C1 Domain in Cancer Signaling Molecules: Regulation by Lipids and Protein-protein Interactions

Cysteine-rich (C1) domains, present in PKC isozymes, Chimaerins, RasGRPs, PKDs, Munc13s, DGKs, and MRCKs, can bind the diacylglycerol (DAG) second messenger. In the present thesis research, I demonstrated that p23/Tmp21 acts as a C1-domain docking protein that mediates perinuclear translocation of beta2-chimaerin. Glu227 and Leu248 in the beta2-chimaerin C1-domain are crucial for binding p23/Tmp21 and perinuclear targeting. Isolated C1-domains from individual PKC isozymes or RasGRP1 differentially interact with p23/Tmp21. PKCepsilon interacts with p23/Tmp21 specifically via its C1b domain, however this association is lost in response to phorbol esters. These results demonstrate that p23/Tmp21 acts as an anchor that distinctively modulates compartmentalization of C1-domain-containing proteins, and it plays an essential role in beta2-chimaerin re-localization to the perinuclear region in response to phorbol esters. It has been established that apoptosis induced by phorbol esters in LNCaP cells is primarily mediated by the novel PKCdelta. I demonstrated that depletion of p23/Tmp21 significantly potentiates phorbol 12-myristate 13-acetate (PMA)-induced apoptosis in LNCaP cells. Remarkably, the effect was mediated by PKCdelta as revealed by the fact that PKCdelta RNAi depletion or PKC inhibitor GF 109203X can rescue the potentiating effect of p23/Tmp21 depletion. Immunoprecipitation and confocal microscopy analysis demonstrated that PKCdelta and p23/Tmp21 formed a complex in LNCaP cells. Disruption of PKCdelta-p23/Tmp21 association by depletion of p23/Tmp21 accelerates PMA-induced PKCdelta plasma membrane translocation and the activation of its downstream effector ROCK and JNK. Moreover, I demonstrated that depletion of p23/Tmp21 potentiates doxorubicin-mediated apoptosis of LNCaP cells. This work provided the first evidence that p23/Tmp21 negatively regulates PKCdelta-mediated apoptosis in LNCaP cells in response to PMA and doxorubicin. In addition, I demonstrated that the PKC inhibitor GF 109203X significantly radiosensitizes PC3 androgen-independent prostate cancer cells. Depletion of PKCepsilon but not PKCalpha and PKCdelta by shRNA, significantly radiosensitizes PC3 cells. Confocal images demonstrated that gammairradiation-induced PKCepsilon membrane translocation was impaired by the EGFR inhibitor AG1478, the PLCgamma1 inhibitor U73122, or the ROS scavenger N-acetyl-cysteine (NAC). The results revealed a potential role for DAG and C1-domain containing protein in the control of ionizing irradiation induced cell death/survival and also suggested that inhibition of PKCepsilon may be a useful therapeutic approach to radiosensitize prostate cancer cells. Taken together, in this work I present several novel findings that highlight the relevance of C1-domain containing proteins in cancer. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Pharmacological Sciences This dissertation is available at ScholarlyCommons: http://repository.upenn.edu/edissertations/141 First Advisor Marcelo G. Kazanietz