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

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The C1 Domain in Cancer Signaling Molecules: Regulation by Lipids and Protein-Protein Interactions University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations Spring 2010 The C1 Domain in Cancer Signaling Molecules: Regulation by Lipids and Protein-Protein Interactions Hongbin Wang University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Pharmacology, Toxicology and Environmental Health Commons Recommended Citation Wang, Hongbin, "The C1 Domain in Cancer Signaling Molecules: Regulation by Lipids and Protein-Protein Interactions" (2010). Publicly Accessible Penn Dissertations. 141. https://repository.upenn.edu/edissertations/141 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/141 For more information, please contact [email protected]. The C1 Domain in Cancer Signaling Molecules: Regulation by Lipids and Protein- Protein Interactions Abstract 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 videncee 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 adiosensitizr es PC3 androgen- independent prostate cancer cells. Depletion of PKCepsilon but not PKCalpha and PKCdelta by shRNA, significantly adiosensitizr es PC3 cells. Confocal images demonstrated that gamma-irradiation-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) First Advisor Marcelo G. Kazanietz Keywords Signal transduction, Cysteine-rich (C1) domains, Beta2-chimaerin, Novel protein kinase C isozymes, p23/ Tmp21, Ionizing radiation Subject Categories Pharmacology, Toxicology and Environmental Health This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/141 DEDICATION This dissertation is dedicated to my wife Xia, my son Kevin and my daughter Anita who have supported and inspired me all the way since the beginning of my Ph.D. studies. Thanks for your understanding, patience, and love. Also to my parents, my brothers, and my parents-in-laws who are in China for always letting me know how proud you are of me. Finally, this dissertation is dedicated to those who believe in the richness of learning. ii ACKNOWLEDGEMENTS I would like to take the opportunity to express sincere appreciation to my dissertation advisor Dr. Marcelo G. Kazanietz for his guidance, sage advice, insightful criticisms, and encouragement. I also like to thank Dr. Meinkoth, Dr. Kennedy, Dr. Koumenis and Dr. Yang for willing to be my dissertation committee members and their valuable suggestions and comments. I am also very grateful for the support by radiation oncology NIH training grant (T32-CA-09677) during my Ph.D. study. Lastly, but not the least, I am thankful to all colleagues and friends who made my stay at the University of Pennsylvania a memorable and valuable experience. iii ABSTRACT THE C1 DOMAIN IN CANCER SIGNALING MOLECULES: REGULATION BY LIPIDS AND PROTEIN-PROTEIN INTERACTIONS HONGBIN WANG Marcelo G. Kazanietz. Ph.D. Cysteine-rich (C1) domains, present in PKC isozymes, Chimaerins (a family of small RacGTPase activating proteins), RasGRPs (a family of guanine nucleotide exchange factor for Ras/Rap1), PKDs (the serine/threonine kinase protein kinase D), Munc13s (the mammalian unc13), DGKs (the DAG kinases β and γ), and MRCKs (the serine/threonine kinase myotonic dystrophy kinase-related Cdc42-binding kinase), 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 β2-chimaerin. Glu227 and Leu248 in the β2- 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. PKCε 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 β2-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 PKCδ. I demonstrated that depletion of iv p23/Tmp21 significantly potentiates phorbol 12-myristate 13-acetate (PMA)-induced apoptosis in LNCaP cells. Remarkably, the effect was mediated by PKCδ as revealed by the fact that PKCδ RNAi depletion or PKC inhibitor GF 109203X can rescue the potentiating effect of p23/Tmp21 depletion. Immunoprecipitation and confocal microscopy analysis demonstrated that PKCδ and p23/Tmp21 formed a complex in LNCaP cells. Disruption of PKCδ-p23/Tmp21 association by depletion of p23/Tmp21 accelerates PMA-induced PKCδ 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 PKCδ- 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 PKCε but not PKCα and PKCδ by shRNA, significantly radiosensitizes PC3 cells. Confocal images demonstrated that γ-irradiation-induced PKCε membrane translocation was impaired by the EGFR inhibitor AG1478, the PLCγ1 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 PKCε 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. v Table of Contents Title Page………………………………………………………………….....i Dedication……………………………………………………………....…....ii Acknowledgements……………………………………………………..…..iii Abstract……………………………………………………………………....iv Table of Contents…………………………………………………………...vi List of Tables……………………………………………………………..…vii List of Figures…………………………………………………………….…vii Chapter 1: General Introduction…………………………………….……..1 Chapter 2: Introduction…………………………………………………....34 Results………………………………………………………...37 Discussion…………………………………………………….65 Materials and Methods…………………………………..…..72 Chapter 3: Introduction……………………………………………..….....82 Results…………………………………………….…..……...84 Discussion……………………………………….…..……….97 Materials and Methods…………………………...…….....100 Chapter 4: Introduction………………………………………………....105 Results…………………………………………...………....107 Discussion…………………………………….……..……..120 Materials and Methods…………………….………..…….123 Chapter 5: Final Remarks……………………………………………...126 Bibliography…..…………………………………….……………….…..134 vi List of Tables Table 2.1-Primers used to generate constructs.……………………...……………79 Table 4.1-Sequences of MISSION® shRNA lentiviral transduction particles used
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