An enhanced association of RACK1 with Abl in cells transfected with oncogenic ras.
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 beta-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 beta-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.
Huang
Chin-Ching
CC
Institute of Zoology, National Taiwan University, Taipei, Taiwan.
Liu
Chia-Hao
CH
Chuang
Nin-Nin
NN
D000818:Animals; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism; D011905:Genes, ras; D019012:Integrin beta1 / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D009479:Neuropeptides / Q000302:isolation & purification / Q000378:metabolism*; D016374:Oncogene Proteins v-abl / Q000302:isolation & purification / Q000378:metabolism*; D000075370:Receptors for Activated C Kinase; D015398:Signal Transduction; D014162:Transfection; D018631:ras Proteins / Q000378:metabolism*
D019012:Integrin beta1; D009479:Neuropeptides; D016374:Oncogene Proteins v-abl; C090294:RACK1 protein, mouse; D000075370:Receptors for Activated C Kinase; D051417:Focal Adhesion Kinase 1; D018631:ras Proteins