An enhanced association of RACK1 with Abl in cells transfected with oncogenic ras

Abstract The cellular RACK1 was shown in association with Abl in BALB/3T3 cells transfected with S- ras (Q 61 K) by immunoprecipitation. An identical finding was demonstrated with cells transfected with the embryonic E- ras , but not in cells without transformation. The Abl-RACK1 of transformed cells as resolvable with Triton X-114 was found with little affinity for FAK, PY 397 -FAK and integrin. Of interests, PY 397 -FAK in the membrane skeleton of transformed cells was shown in significant quantities on the Western blot. However the PY 397 -FAK of transformed cells was not functionally able to react with RACK1 and recruit cytokeratin-1, a substrate of Src, indicating that PY 397 -FAK is not operative to transmit integrin signals. In other words, the Abl-RACK1 of transformed cells cannot replace the Src-RACK1 of cells without transformation to bridge PY 397 -FAK and cytokeratin-1 for integrin signals, and the formation of Abl-RACK1 in transformed cells may block the association of PY 397 -FAK-RACK1. We characterized Abl and RACK1 from transformed cells by chromatography on a HiTrap-PEP Taxol affinity column, constructed from a β-tubulin peptide specific for Taxol binding (PEP Taxol ). However, the Triton X-100 cannot achieve the same resolution of Abl-RACK1 from plasma membrane as is shown with Triton X-114. A significant fraction of Abl was deposited at the membrane skeleton and was therefore not accessible with Triton X-100. Half of Abl resolved with Triton X-100 was demonstrated to have catalytic activity as shown with positive phosphotyrosine staining on the Western blot and competitive elution with a specific phosphate, such as sodium β-glycerophosphate, from HiTrap-PEP Taxol , but this was not associated with RACK1. No significant difference of RACK1 was found in Triton X-100 resolvable membrane preparations from cells with and without transformations. Future studies are planned to differentiate the mechanism operative for RACK1 associated and RACK1 freed Abl in cells transformed with oncogenic ras .