Dimerize RACK1 upon transformation with oncogenic ras.
From our previous studies, we learned that syndecan-2/p120-GAP complex provided docking site for Src to prosecute tyrosine kinase activity upon transformation with oncogenic ras. And, RACK1 protein was reactive with syndecan-2 to keep Src inactivated, but not when Ras was overexpressed. In the present study, we characterized the reaction between RACK1 protein and Ras. RACK1 was isolated from BALB/3T3 cells transfected with plasmids pcDNA3.1-[S-ras(Q61K)] of shrimp Penaeus japonicus and RACK1 was revealed to react with GTP-K(B)-Ras(Q61K), not GDP-K(B)-Ras(Q61K). This selective interaction between RACK1 and GTP-K(B)-Ras(Q61K) was further confirmed with RACK1 of human placenta and mouse RACK1-encoded fusion protein. We found that RACK1 was dimerized upon reaction with GTP-K(B)-Ras(Q61K), as well as with 14-3-3beta and geranylgeranyl pyrophosphate, as revealed by phosphorylation with Src tyrosine kinase. We reported the complex of RACK1/GTP-K(B)-Ras(Q61K) reacted selectively with p120-GAP. This interaction was sufficient to dissemble RACK1 into monomers, a preferred form to compete for the binding of syndecan-2. These data indicate that the reaction of GTP-K(B)-Ras(Q61K) with RACK1 in dimers may operate a mechanism to deplete RACK1 from reaction with syndecan-2 upon transformation by oncogenic ras and the RACK1/GTP-Ras complex may provide a route to react with p120-GAP and recycle monomeric RACK1 to syndecan-2.
Chu
Ling-Yun
LY
Institute of Zoology, National Taiwan University, Taipei, Taiwan.
Chen
Yu-Hsun
YH
Chuang
Nin-Nin
NN
D000818:Animals; D015153:Blotting, Western; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D020690:GTPase-Activating Proteins / Q000378:metabolism; D011905:Genes, ras; D006160:Guanosine Triphosphate / Q000378:metabolism; D047468:Immunoprecipitation; D051379:Mice; D008807:Mice, Inbred BALB C; D009479:Neuropeptides / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D000075370:Receptors for Activated C Kinase
D020690:GTPase-Activating Proteins; D009479:Neuropeptides; C090294:RACK1 protein, mouse; D000075370:Receptors for Activated C Kinase; D006160:Guanosine Triphosphate