ID OF EFFECTORS FOR RAS FAMILY GTPASES & ELUCIDATION OF MECHANISM OF ACTION

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The Ras signaling pathway plays a crucial role in regulating cell growth and tumorigenesis. Ras is a member of a sub-family of related GTPases that functions by regulating a variety of downstream targets of effectors. Based on sequence homologies in the Ras Association domain, we have identified serveral new Ras family GTPase effectors and confirmed that these new proteins can interact with Ras when in its active, GTP-bound state. Very little, if anything, is known about these new Ras effectors. As a way of getting some insight into their function and mechanism of action, we are trying to identify the proteins they interact with within the cell. We have used the Tandem Affinity Purification (TAP) methodology to express and purify from colon cancer cells TAP-tagged versions of these new Ras effectors. After separation of the protein complexes by gel electrophoresis and coomassie blue staining, we can visualize several co-purifying protein bands that we would now like to identify by mass spectrometry.