P120-GAP associated with syndecan-2 to function as an active switch signal for Src upon transformation with oncogenic ras.
BALB/3T3 cells transfected with plasmids pcDNA3.1-[S-ras(Q(61)K)] of shrimp Penaeus japonicus were applied to reveal a complex of p120-GAP/syndecan-2 being highly expressed upon transformation. Of interest, most of the p120-GAP/syndecan-2 complex was localized at caveolae, a membrane microdomain enriched with caveolin-1. To confirm the molecular interaction between syndecan-2 and p120-GAP, we further purified p120-GAP protein from mouse brains by using an affinity column of HiTrap-RACK1 and expressed mouse RACK1-encoded fusion protein and mouse syndecan-2-encoded fusion protein in bacteria. We report molecular affinities exist between p120-GAP and RACK1, syndecan-2 and RACK1 as well as p120-GAP and syndecan-2. The selective affinity between p120-GAP and syndecan-2 was found to be sufficient to detach RACK1. The p120-GAP/syndecan-2 complex was demonstrated to keep Src tyrosine kinase in an activated form. On the other hand, the syndecan-2/RACK1 complex was found to have Src in an inactivated form. These data indicate that the p120-GAP/syndecan-2 complex at caveolae could provide a docking site for Src to transmit tyrosine signaling, implying that syndecan-2/p120-GAP functions as a tumor promoter upon transformation with oncogenic ras of shrimp P. japonicus.
Huang
Jin-Wen
JW
Division of Biochemistry and Molecular Science, Institute of Zoology, Academia Sinica, Nankang, 11529 Taipei, Taiwan.
Chen
Chia-Lin
CL
Chuang
Nin-Nin
NN
D000818:Animals; D041702:BALB 3T3 Cells; D001665:Binding Sites; D021941:Caveolae / Q000378:metabolism*; D002478:Cells, Cultured; D019204:GTP-Binding Proteins; D005786:Gene Expression Regulation / Q000502:physiology; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D033561:Penaeidae / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011509:Proteoglycans / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D053669:Syndecan-2; D014170:Transformation, Genetic / Q000235:genetics; D020729:p120 GTPase Activating Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism*
D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; D011509:Proteoglycans; C419368:RACK1 protein, human; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; C508245:SDC2 protein, human; C508246:Sdc2 protein, mouse; D020729:p120 GTPase Activating Protein; D053669:Syndecan-2; D019061:src-Family Kinases; D019204:GTP-Binding Proteins; D018631:ras Proteins