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The SH3 domain of Src can downregulate its kinase activity in the absence of the SH2 domain-pY527 interaction

Jan Brábek1, Dominik Mojzita, Marian Novotný

  • 1Department of Physiology and Developmental Biology, Charles University, Vinicna 7, 128 00 Prague 2, Czech Republic.

Biochemical and Biophysical Research Communications
|August 15, 2002
PubMed

Insights

Specific mutations in the SH3 domain of v-Src (viral sarcoma) explain its low kinase activity. These findings reveal how the SH3 domain influences Src kinase activity through direct interactions.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Oncology

Background:

  • The Src kinase family plays a critical role in cellular signaling pathways.
  • The regulatory mechanisms of Src kinase activity involve interactions between its SH2, SH3, and kinase domains.
  • Understanding these interactions is crucial for deciphering oncogenic transformation.

Purpose of the Study:

  • To investigate the specific role of the SH3 domain in regulating the kinase activity of v-Src (viral sarcoma).
  • To identify mutations within the SH3 domain responsible for the differential kinase activity observed in v-Src variants.
  • To elucidate the mechanism by which the SH3 domain influences Src kinase activity in the context of SH2 domain activation.

Main Methods:

  • Utilized v-Src variants Prague C (PRC) and Schmidt-Ruppin A (SRA) with differing kinase activities.
  • Employed in vitro kinase assays measuring phosphorylation by immunoprecipitated kinases produced in Saccharomyces cerevisiae.
  • Performed domain swapping experiments and site-directed mutagenesis to assess the impact of specific mutations on kinase activity.

Main Results:

  • Identified specific mutations (N117D, I96T, V124L) in the n-src- and RT-loops of the PRC v-Src SH3 domain as responsible for its low kinase activity.
  • Demonstrated that introducing corresponding mutations (D117N, R95W, T96I, L124V) into activated c-Src (carboxyl-terminal Src) significantly increased its activity by 2.5-fold.
  • Showed that mutations known to activate c-Src had no effect on the activity of "SH2-activated" Src kinases, indicating a distinct regulatory mechanism.

Conclusions:

  • The SH3 domain plays a significant role in modulating v-Src kinase activity.
  • Specific residues within the n-src- and RT-loops of the SH3 domain are critical determinants of Src kinase potency.
  • In "SH2-activated" Src forms, the SH3 domain continues to exert regulatory influence through direct interactions with the kinase N-terminal lobe.

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