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.
Itoh
T
T
Department of Biochemistry, Institute of Medical Science, University of Tokyo, Tokyo 108, Japan.
Miura
K
K
Miki
H
H
Takenawa
T
T
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