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Related Experiment Videos

Regulation of RasGRP via a phorbol ester-responsive C1 domain

C E Tognon1, H E Kirk, L A Passmore

  • 1Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada V5Z 4E6.

Molecular and Cellular Biology
|November 20, 1998
PubMed
Summary

Murine RasGRP (mRasGRP) transforms fibroblasts by activating MAP kinases, a process dependent on its C1 domain for membrane recruitment. This guanine nucleotide exchange factor links cell surface receptors to Ras activation.

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Area of Science:

  • Molecular Biology
  • Cell Signaling
  • Oncology

Background:

  • Ras guanine nucleotide exchange factors (GEFs) play critical roles in cell signaling pathways.
  • RasGRP is a GEF involved in Ras activation, but its precise mechanisms in mammalian cells are not fully understood.
  • Fibroblast transformation and mitogen-activated protein kinase (MAPK) activation are key indicators of cellular signaling dysregulation.

Purpose of the Study:

  • To identify and characterize the murine homolog of RasGRP (mRasGRP) and its role in fibroblast transformation.
  • To elucidate the structural domains and regulatory mechanisms governing mRasGRP activity.
  • To investigate the link between mRasGRP, diacylglycerol (DAG) signaling, and Ras activation in mammalian cells.

Main Methods:

  • cDNA library screening to isolate mRasGRP.

Related Experiment Videos

  • Site-directed mutagenesis to assess the function of mRasGRP domains (EF hands, C1 domain).
  • Co-expression studies with Ras proteins and dominant-negative mutants to analyze MAPK activation.
  • Phorbol ester treatment and serum stimulation to investigate mRasGRP regulation and membrane translocation.
  • Analysis of mRasGRP expression patterns in various tissues and cell lines.

    • Main Results:

    • A cDNA encoding murine RasGRP (mRasGRP) was isolated, and a specific point mutation abolished its transforming and MAPK-activating abilities.
    • The C1 domain and an adjacent basic amino acid cluster, but not the EF hands, were essential for mRasGRP's transforming activity.
    • Replacing the C1 domain with a prenylation signal or a protein kinase C C1 domain restored transforming activity, highlighting the importance of membrane localization.
    • Phorbol ester and serum stimulation induced mRasGRP translocation to cell membranes, dependent on the C1 domain, suggesting DAG-mediated recruitment.
    • mRasGRP is expressed in lymphoid tissues, brain, and lymphoid cell lines, indicating its potential role in these systems.

    Conclusions:

    • The C1 domain of mRasGRP is crucial for its transforming activity, primarily by mediating recruitment to diacylglycerol-enriched cell membranes.
    • mRasGRP acts as a direct link between phospholipase C signaling pathways generating DAG and the activation of Ras.
    • These findings reveal a key mechanism for Ras activation in fibroblasts and suggest a role for RasGRP in lymphoid tissues and the brain.