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Src-dependent phosphorylation of ASAP1 regulates podosomes

Sanita Bharti1, Hiroki Inoue, Kapil Bharti

  • 1Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA.

Molecular and Cellular Biology
|September 26, 2007
PubMed

Insights

ASAP1 protein is crucial for forming invadopodia and podosomes, cellular structures involved in tumor invasion. Its SH3 and BAR domains are essential for this process, highlighting ASAP1

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Cancer Research

Background:

  • Invadopodia are key cellular structures mediating tumor invasion.
  • ASAP1 (Arf GTPase-activating protein) is a Src substrate regulating invadopodia formation.
  • ASAP1 contains Src homology 3 (SH3) and Bin, amphiphysin, and RVS161/167 (BAR) domains.

Purpose of the Study:

  • To investigate the structural requirements of ASAP1 for invadopodia and podosome formation.
  • To determine the role of ASAP1 splice variants (ASAP1a and ASAP1b) in these cellular structures.
  • To elucidate ASAP1's function as a target of tyrosine kinase signaling.

Main Methods:

  • Utilized NIH 3T3 fibroblasts to study podosome formation.
  • Employed small interfering RNA (siRNA) to reduce ASAP1 levels.
  • Generated and tested recombinant ASAP1 variants (wild-type and mutants) for podosome formation.
  • Observed podosome dynamics and ASAP1 turnover using live-cell imaging.

Main Results:

  • Both ASAP1a and ASAP1b splice variants associate with invadopodia and podosomes.
  • Podosomes exhibit dynamic behavior with rapid turnover of ASAP1 and actin.
  • Reduction of ASAP1 levels via siRNA inhibits invadopodia and podosome formation.
  • Recombinant ASAP1a or ASAP1b can rescue podosome formation in ASAP1-depleted cells.
  • ASAP1 mutants lacking the Src binding site or GAP activity still support podosome formation.
  • ASAP1 lacking the BAR, SH3 domain, or Src phosphorylation site fails to support podosome formation.

Conclusions:

  • ASAP1 is a critical regulator of podosome and invadopodia formation.
  • The SH3 and BAR domains, along with the Src phosphorylation site, are essential for ASAP1 function in podosome formation.
  • ASAP1 acts as a coincidence detector, integrating signals through its SH3 domain and Src phosphorylation.

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