Beta-tubulin binds Src homology 2 domains through a region different from the tyrosine-phosphorylated protein-recognizing site.
Src homology 2 (SH2) domains have been demonstrated to bind tyrosine-phosphorylated proteins that participate in signaling by growth factors and oncogenes by recognizing amino acid sequences containing phosphotyrosine residue. We found that SH2 domains such as Ash/Grb2, the 85-kDa subunit of phosphatidylinositol 3-kinase, and phospholipase Cgamma1 also bind beta-tubulin through a different region that recognizes phosphotyrosine in vitro and in vivo. Furthermore, binding occurs even when the SH2 domain is occupied by tyrosine-phosphorylated epidermal growth factor receptors. Using deleted constructs of Ash/Grb2 SH2, we found that carboxyl-terminal beta strands E and F, and alpha helix B (region "c") are required for binding. A synthetic peptide (FLWVVKFNSLNELVDYH) composed of region c inhibited the binding of beta-tubulin to the SH2 domains of Ash/Grb2, phosphatidylinositol 3-kinase, and phospholipase Cgamma1. The co-localization of SH2 proteins and microtubules is also confirmed by immunostaining. These data suggest that microtubules play important roles in the assembly of signaling molecules complexes containing SH2 proteins.
Department of Biochemistry, Institute of Medical Science, University of Tokyo, Tokyo 108, Japan.
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Itoh
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Miura
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Miki
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Takenawa
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism; D002417:Cattle; D066246:ErbB Receptors / Q000737:chemistry / Q000378:metabolism; D051380:GRB2 Adaptor Protein; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008870:Microtubules / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases; D051966:Phospholipase C gamma; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism*; D010738:Type C Phospholipases / Q000737:chemistry / Q000378:metabolism; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; D007527:Isoenzymes; D010446:Peptide Fragments; D011506:Proteins; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D019000:Phosphotyrosine; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D066246:ErbB Receptors; D010738:Type C Phospholipases; D051966:Phospholipase C gamma