Association of Ash/Grb-2 with dynamin through the Src homology 3 domain.
Ash/Grb-2 is an adaptor protein composed only of Src homology (SH) 2 and SH3 domains that is considered to be essential for Ras activation. To clarify the downstream of Ash signaling, we investigated Ash-bound proteins. Ash-glutathione S-transferase (GST) fusion proteins were used to affinity-purify proteins bound to Ash. We found 180-, 150-, 100-, and 70-kDa proteins bound to GST-Ash, among which the 100 kDa protein was found to be dynamin by amino acid sequencing and Western blot with anti-dynamin antibody. Next, the in vitro and in vivo associations between Ash and dynamin were examined using PC12 cells. Dynamin in PC12 cell lysates bound to GST-Ash independent of NGF treatment. Also, Ash and dynamin co-precipitated when cell lysates of PC12 were immunoprecipitated with anti-Ash antibody or anti-dynamin antibody. Using various GST-Ash constructs, we studied the importance of the individual domains in binding and found that the SH3 domain is necessary for binding. This binding was inhibited by a synthetic peptide (GPPQVPSRPNRC, amino acids 827-838 in dynamin). These data show that Ash SH3 domains bind to the proline-rich region of dynamin. Considering the function of dynamin in membrane trafficking, Ash may regulate endocytosis in addition to Ras activation.
Miki
H
H
Department of Molecular Oncology, University of Tokyo, Japan.
Miura
K
K
Matuoka
K
K
Nakata
T
T
Hirokawa
N
N
Orita
S
S
Kaibuchi
K
K
Takai
Y
Y
Takenawa
T
T
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D034281:Dynamins; D051380:GRB2 Adaptor Protein; D020558:GTP Phosphohydrolases / Q000378:metabolism; D016391:Genes, src; D005982:Glutathione Transferase / Q000378:metabolism; D008969:Molecular Sequence Data; D016716:PC12 Cells; D011392:Proline / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D017386:Sequence Homology, Amino Acid
D048868:Adaptor Proteins, Signal Transducing; D017931:DNA Primers; D051380:GRB2 Adaptor Protein; C495950:Grb2 protein, rat; D011506:Proteins; D011392:Proline; D005982:Glutathione Transferase; D020558:GTP Phosphohydrolases; D034281:Dynamins