Trap RACK1 with Ras to mobilize Src signaling at syndecan-2/p120-GAP upon transformation with oncogenic ras.
HiTrap-syndecan-2/p120-GAP and HiTrap-syndecan-2/RACK1 affinity columns were applied to reveal that Src tyrosine kinase was highly expressed in BALB/3T3 cells transfected with plasmids pcDNA3.1-[S-ras(Q(61)K)] of shrimp Penaeus japonicus. Both columns were effective to isolate Src tyrosine kinase. The selective molecular affinity for Src was found to be stronger with HiTrap-syndecan-2/RACK1, as revealed with competitive RACK1 to dislodge Src from HiTrap-syndecan-2/p120-GAP. We thus challenged the syndecan-2/p120-GAP and syndecan-2/RACK1 with GTP-K(B)-Ras(Q(61)K). The reaction between RACK1 and syndecan-2 was sustained in the presence of mutant Ras proteins, but not the reaction between p120-GAP and syndecan-2. In the presence of syndecan-2, GTP-K(B)-Ras(Q(61)K) exhibited sufficient reactivity with p120-GAP to discontinue the reaction between p120-GAP and syndecan-2. But the interference of mutant Ras disappeared when Src tyrosine kinase was introduced to stabilize the syndecan-2/p120-GAP complex. On the other hand, in the absence of syndecan-2, GTP-K(B)-Ras(Q(61)K) was found to react with RACK1. The reaction between GTP-K(B)-Ras(Q(61)K) and RACK1 could provide a mechanism to deprive RACK1 for the organization of syndecan-2/RACK1 complex and to facilitate the formation of syndecan-2/p120-GAP complex, as well as to provide docking sites for Src signaling upon transformation with oncogenic ras.
Huang
Jin-Wen
JW
Institute of Zoology, National Taiwan University, Taipei, Taiwan.
Chen
Chia-Lin
CL
Chuang
Nin-Nin
NN
D000818:Animals; D041702:BALB 3T3 Cells; D002471:Cell Transformation, Neoplastic / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006160:Guanosine Triphosphate / Q000235:genetics; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D009154:Mutation; D011485:Protein Binding; D011509:Proteoglycans / Q000096:biosynthesis* / Q000235:genetics; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D015398:Signal Transduction; D053669:Syndecan-2; D014162:Transfection; D020729:p120 GTPase Activating Protein / Q000096:biosynthesis / Q000378:metabolism*; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*; D019061:src-Family Kinases / Q000502:physiology*
D008562:Membrane Glycoproteins; D011509:Proteoglycans; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C508246:Sdc2 protein, mouse; D020729:p120 GTPase Activating Protein; D053669:Syndecan-2; D006160:Guanosine Triphosphate; D019061:src-Family Kinases; D018631:ras Proteins