Interaction of Ash/Grb-2 via its SH3 domains with neuron-specific p150 and p65.
We found that 180 kDa, 150 kDa (p150), 110 kDa, 100 kDa and 65 kDa (p65) proteins comprise the major Ash/Grb-2-binding proteins in bovine brain. Among these proteins, 180 kDa and 100 kDa proteins have already been identified as Sos and dynamin respectively. Here, p150 and p65 were affinity-purified with glutathione S-transferase-Ash fusion protein and their partial amino acid sequences were determined. Analysis showed p150 and p65 to be new proteins. These two proteins bind to both the N-terminal SH3 domain and the C-terminal SH3 domain of Ash. It was found that p150 and p65 are expressed predominantly in brain, although Ash is widely distributed in all tissues examined by Western blots. Immunohistochemical staining of rat brain showed p150 and p65 to be localized in a variety of neurons in the cerebellum and hippocampus, with p65 being especially concentrated in the nerve terminal. When the Ash-binding-motif peptide of the epidermal growth factor receptor was used to detect complexes formed with Ash in vivo, 180 kDa, 150 kDa, 110 kDa, 100 kDa and 65 kDa proteins were also bound; this shows that these proteins form complexes with Ash in brain. In addition, p150 and p65 co-immunoprecipitated with Ash. All these results suggest that Ash may function as a regulator of synaptic vesicle transport through dynamin, p150 and p65.
Miura
K
K
Department of Molecular Oncology, Institute of Medical Science, University of Tokyo, Japan.
Miki
H
H
Shimazaki
K
K
Kawai
N
N
Takenawa
T
T
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D001923:Brain Chemistry; D002417:Cattle; D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D034281:Dynamins; D051380:GRB2 Adaptor Protein; D020558:GTP Phosphohydrolases / Q000378:metabolism; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D000906:Antibodies; D004268:DNA-Binding Proteins; D051380:GRB2 Adaptor Protein; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D011506:Proteins; D011993:Recombinant Fusion Proteins; C100394:neuron-specific p150; C100393:neuron-specific p65; D020558:GTP Phosphohydrolases; D034281:Dynamins