TY - JOUR
T1 - Association of Ash/Grb-2 with dynamin through the Src homology 3 domain
AU - Miki, Hiroaki
AU - Miura, Kenji
AU - Matuoka, Koozi
AU - Nakata, Takao
AU - Hirokawa, Nobutaka
AU - Orita, Satoshi
AU - Kaibuchill, Kozo
AU - Takai, Yoshimi
AU - Takenawa, Tadaomi
PY - 1994/2/25
Y1 - 1994/2/25
N2 - 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 (GPPPQVPSRPNRC, 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.
AB - 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 (GPPPQVPSRPNRC, 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.
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M3 - Article
C2 - 8119878
AN - SCOPUS:0028108262
SN - 0021-9258
VL - 269
SP - 5489
EP - 5492
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 8
ER -