Association of Ash/Grb-2 with dynamin through the Src homology 3 domain

Hiroaki Miki, Kenji Miura, Koozi Matuoka, Takao Nakata, Nobutaka Hirokawa, Satoshi Orita, Kozo Kaibuchill, Yoshimi Takai, Tadaomi Takenawa

Research output: Contribution to journalArticle

108 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)5489-5492
Number of pages4
JournalJournal of Biological Chemistry
Volume269
Issue number8
Publication statusPublished - 25-02-1994
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Association of Ash/Grb-2 with dynamin through the Src homology 3 domain'. Together they form a unique fingerprint.

  • Cite this

    Miki, H., Miura, K., Matuoka, K., Nakata, T., Hirokawa, N., Orita, S., Kaibuchill, K., Takai, Y., & Takenawa, T. (1994). Association of Ash/Grb-2 with dynamin through the Src homology 3 domain. Journal of Biological Chemistry, 269(8), 5489-5492.