We hypothesize that the oncogenic potential of Pim-1 arises in part from its contribution to cell survival by phosphorylating p21cip1/WAF1 (referred to as p21), a cyclin dependent kinase (CDK) inhibitor in vivo and, by doing so, causing p21's translocation out of the nucleus. This would serve to decrease cell cycle arrest and make p21 available for interaction with two other proteins in the cytoplasm, procaspase-3 and apoptosis signal-regulating kinase (ASK1). These p21/procaspase-3 and p21/ASK1 complexes would be important because they can block the processing of procaspase-3 into active caspase-3, one of the central proteolytic components of apoptosis (programmed cell death) and block the activation SAPK pathway via the inhibition of activation of ASK1 and subsequently JNK. To test this hypothesis, we propose: 1. To determine whether the sites of phosphorylation of p21 by Pim-1 in vivo are the same as those found to occur in vitro. A triple quadrupole mass spectrometer equipped with an electrospray ion source as well as specific antibodies to phosphorylated p21 residues will be used to analyze phosphorylation state of transfected HAp21 in p21 null cells as a result of either wild type or kinase dead Pim-1 expression. 2. To determine the extent to which apoptosis is inhibited as a result of the phosphorylation of p21 by Pim-1 in vivo. A combination of wild type and mutated proteins with tags (HA-tagged p21 and FLAG-tagged Pim-1) will be expressed from inducible vectors and used to assess the level of apoptotic activity. 3. To correlate resistance to apoptosis with complex formation mediated by Pim-1, in vivo phosphorylation of p21 by Pim-1 will be assessed in terms of p21/procaspase-3 and p21/ASK1 complex formation. Western blotting, immunoprecipitations and enzymatic assays will be used to determine complex formation and activity of caspase-3 and JNK 4. To demonstrate by immunohistochemistry in human breast and prostate tumor tissue over-expressing Pim-1 that p21 is localized predominantly in the cytoplasm. 5. To demonstrate with Pim-1 transgenic mice that the frequency of prostate and breast cancer generation is correlated with increased phosphorylation of p21 and cytoplasmic localization of p21. It is predicted that the results from this study will provide an explanation on the molecular level for the role of Pim-1 in promoting cancer cell survival that until this time has not been identified.
Showing the most recent 10 out of 16 publications
