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Role of the Non-Receptor Tyrosine Kinase ACK2 in EGF Receptor Degradation
Annual summary rept. 28 Mar 2003-27 Mar 2006
CORNELL UNIV ITHACA NY
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Establishing a role for ACK2 and SH3PX1 in ErbB-2 receptor degradation is appealing based on the predictive property between receptor overexpression and breast cancer. Currently, we are interested in determining the significance of ACK2- dependent phosphorylation of SH3PX1 in cells. To address these objectives, we have carried out deletion analysis studies to delineate the region of the phosphorylation on SH3PX1. Based on these studies, we conclude that SH3PX1 phosphorylation is lost in the truncation mutant C84. In additional binding studies, ACK2 is unable to bind to any of the deletion mutants. Site-directed mutagenesis studies indicate that all conserved point-mutants of SH3PX1 retain tyrosine phosphorylation. Given these findings, we employed mass spectrometry as a means to determine the multiple phosphorylation sites on SH3PX1, recently identified as tyrosine residues 177, 239, 269, and 561. Additionally, recombinant forms of kinase and substrate were generated for in vitro kinase screens carried out at the high-throughput facility at Merck Co., Inc. The pyrido-pyrimidine compound from Park Davis PD158780 was shown to effectively block ACK2 in vitro with an IC50 of 80 pM. Low kinase activity of ACK2 in vivo and in vitro has been one of the greatest challenges in carrying out the research objectives in our statement of work. As a result, the DOD-sponsored research has led us to new and exciting discoveries, namely identifying two unreported kinases, FAK and Src, for SH3PX1. We believe that the phosphorylation state of SH3PX1 is critical in mediating its binding to a growing number of proteins involved in endocytosis, including the GTPase, dynamin. Our work is now focused on determining the biological outcome of SH3PX1 phosphorylation.
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