The shc adaptor protein forms interdependent phosphotyrosine-mediated protein complexes in mast cells stimulated with interleukin 3.
The Shc adaptor protein possesses 2 distinct phosphotyrosine (pTyr) recognition modules-the pTyr binding (PTB) domain and the Src homology 2 (SH2) domain-and multiple potential sites for tyrosine (Tyr) phosphorylation (Tyr residues 239, 240, and 317). On stimulation of hematopoietic cells with interleukin 3 (IL-3), Shc becomes phosphorylated and may therefore contribute to IL-3 signaling. We investigated the interactions mediated by the Shc modular domains and pTyr sites in IL-3-dependent IC2 premast cells. The Shc PTB domain, rather than the SH2 domain, associated both in vitro and in vivo with the Tyr-phosphorylated beta subunit of the IL-3 receptor and with the SH2-containing 5' inositol phosphatase (SHIP), and it recognized specific NXXpY phosphopeptides from these binding partners. In IL-3-stimulated mast cells, Shc phosphorylation occurred primarily on Tyr239 and 317 and was dependent on a functional PTB domain. Phosphorylated Tyr317, and to a lesser extent, Tyr239, bound the Grb2 adaptor and SHIP. Furthermore, a pTyr317 Shc phosphopeptide selectively recognized Grb2, Sos1, SHIP, and the p85 subunit of phosphatidylinositol 3' kinase from mast cells, as characterized by mass spectrometry. These results indicate that Shc undergoes an interdependent series of pTyr-mediated interactions in IL-3-stimulated mast cells, resulting in the recruitment of proteins that regulate the Ras pathway and phospholipid metabolism.
Velazquez
L
L
Programme in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
Gish
G D
GD
van Der Geer
P
P
Taylor
L
L
Shulman
J
J
Pawson
T
T
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D051380:GRB2 Adaptor Protein; D007377:Interleukin-3 / Q000494:pharmacology*; D007700:Kinetics; D008407:Mast Cells / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D010748:Phosphopeptides / Q000737:chemistry / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D019000:Phosphotyrosine / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495949:Grb2 protein, mouse; D007377:Interleukin-3; D010748:Phosphopeptides; D011506:Proteins; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D010744:Phosphoric Monoester Hydrolases; C098706:INPPL1 protein, human; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases