Regulation of Her2/neu by activation loop phosphorylation and ALG-2

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DESCRIPTION (provided by applicant): The candidate, Ron Bose, M.D., Ph.D., is a graduate of the Medical Scientist Training Program held jointly at Cornell University- Rockefeller University- Memorial Sloan-Kettering Cancer Center. His Ph.D. research, conducted at Memorial Sloan Kettering, studied a lipid second messenger pathway which induced apoptosis. Dr. Bose completed residency in Internal Medicine in 2002 and entered Medical Oncology fellowship at Johns Hopkins University. His goals are to pursue basic and translational research by applying proteomics to study signal transduction pathways in breast cancer. He has published a proteomic study of Her2/neu tyrosine kinase signaling (Appendix A) and systematically compared these results to proteomic studies of Epidermal growth factor receptor (EGFR) signaling already in the literature. In this proposal, he plans to pursue two related proteomic findings, Her2/neu activation loop phosphorylation on tyrosine 877 and Her2/neu induced phosphorylation of Apoptosis-linked gene 2 (ALG2), with focused mechanistic experiments. Both activation loop phosphorylation and ALG-2 phosphorylation may be regulatory mechanisms for Her2/neu function and may help control the receptor tyrosine kinase after dimerization has occurred. The activation loop is a major structural feature of kinase domains and has been shown to regulate the activity of insulin receptor kinase and Src. ALG-2 interacts with proteins involved in receptor internalization. Internalization and degradation of Her2/neu is a major mechanism of action of the anti-Her2/neu antibody, trastuzumab (Herceptin). In this proposal, the specific aims will test: 1) if recombinant Her2/neu kinase domain can in vitro autophosphorylate itself on the activation loop and the interaction of this process with a recent model of kinase domain dimerization, 2) if mutation of the activation loop site (Y877F) affects downstream signaling and trastuzumab-induced Her2/neu internalization, 3) if ALG-2 and its interacting protein Alix/AIP1 affect trastuzumab induced Her2/neu internalization and degradation. This research will be performed in the Department of Oncology, Johns Hopkins University. Relevance: Her2/neu and EGFR are protein kinases involved in multiple human cancers. Careful, mechanistic study of their regulation will result in better application of current targeted therapies and possible design of a new class of kinase inhibitors for the treatment of cancer.