Phosphorylation of grb10 regulates its interaction with 14-3-3.
Grb10 is a member of adapter proteins that are thought to play a role in receptor tyrosine kinase-mediated signal transduction. Grb10 expression levels can influence Akt activity, and Grb10 may act as an adapter involved in the relocalization of Akt to the cell membrane. Here we identified 14-3-3 as a binding partner of Grb10 by employing a yeast two-hybrid screen. The 14-3-3.Grb10 interaction requires phosphorylation of Grb10, and only the phosphorylated form of Grb10 co-immunoprecipitates with endogenous 14-3-3. We could identify a putative phosphorylation site in Grb10, which is located in a classical 14-3-3 binding motif, RSVSEN. Mutation of this site in Grb10 diminished binding to 14-3-3. Thus, Grb10 exists in two different states of phosphorylation and complexes with 14-3-3 when phosphorylated on serine 428. We provide evidence that Akt directly binds Grb10 and is able to phosphorylate Grb10 in an in vitro kinase assay. Based on these findings, we propose a regulatory circuitry involving a phosphorylation-regulated complex formation of Grb10 with 14-3-3 and Akt.
Urschel
Susanne
S
Department of Internal Medicine III, Laboratory of Leukemogenesis, Technical University of Munich, 81675 Munich, Germany.
Bassermann
Florian
F
Bai
Ren-Yuan
RY
Münch
Silvia
S
Peschel
Christian
C
Duyster
Justus
J
D048948:14-3-3 Proteins / Q000378:metabolism*; D020816:Amino Acid Motifs; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D019556:COS Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051383:GRB10 Adaptor Protein; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D008954:Models, Biological; D009154:Mutation; D019319:Okadaic Acid / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D014176:Protein Biosynthesis; D011506:Proteins / Q000378:metabolism*; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D013997:Time Factors; D020798:Two-Hybrid System Techniques; D000077191:Wortmannin
D048948:14-3-3 Proteins; D000730:Androstadienes; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D011506:Proteins; D051383:GRB10 Adaptor Protein; D019319:Okadaic Acid; D012694:Serine; D005982:Glutathione Transferase; D010744:Phosphoric Monoester Hydrolases; D000077191:Wortmannin