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Molecular cloning of a cDNA clone encoding a phosphoprotein component related to the Ig receptor-mediated signal

S Inui1, K Kuwahara, J Mizutani

  • 1Department of Immunology, School of Life Science, Faculty of Medicine, Tottori University, Yonago, Japan.

Journal of Immunology (Baltimore, Md. : 1950)
|March 15, 1995
PubMed

Insights

Researchers identified a novel protein, alpha 4, involved in immunoglobulin receptor (IgR) signaling in B cells. This protein, a 52-kDa phosphoprotein, associates with a kinase and plays a role in IgR-mediated signal transduction.

Area of Science:

  • Immunology
  • Molecular Biology
  • Cell Signaling

Background:

  • A 52-kDa phosphoprotein (p52) associated with the Ig receptor (IgR)-associated MB-1 protein was previously shown to be phosphorylated upon stimulation.
  • This p52 protein was found to co-precipitate with MB-1 and was inducibly phosphorylated by 12-O-tetradecanoyl-phorbol-13-acetate.
  • A monoclonal antibody (mAb), 19-14, was generated against p52, capable of immunoprecipitating p52 and its associated kinase.

Purpose of the Study:

  • To isolate and characterize the gene encoding the 52-kDa phosphoprotein (p52) associated with Ig receptor (IgR)-mediated signal transduction.
  • To investigate the role of the alpha 4 gene-encoded molecule in B cell signaling pathways.
  • To confirm the association of the alpha 4 protein with kinase activity and its involvement in IgR signaling.

Main Methods:

  • Isolation of a cDNA clone (alpha 4) from a murine bone marrow library using the 19-14 mAb.
  • Production of a glutathione S-transferase (GST)-alpha 4 fusion protein for antibody generation and binding studies.
  • Immunoprecipitation assays using the 19-14 mAb and anti-alpha 4 antibodies, followed by kinase assays and protein characterization (2D gel electrophoresis, V8 protease mapping).

Main Results:

  • The alpha 4 cDNA encodes a novel 340-amino acid protein with multiple potential phosphorylation sites, and its mRNA is expressed in various cell types and organs.
  • The 19-14 mAb binds to the GST-alpha 4 fusion protein, and antibodies against alpha 4 immunoprecipitate a 52-kDa phosphoprotein with associated kinase activity.
  • The immunoprecipitated 52-kDa phosphoprotein is identical to the previously identified p52 protein and is inducibly phosphorylated in response to anti-IgM stimulation, indicating functional involvement in IgR signaling.

Conclusions:

  • The alpha 4 gene encodes a novel protein that is identical to the 52-kDa phosphoprotein (p52) previously implicated in Ig receptor signaling.
  • The alpha 4 gene-encoded molecule is functionally involved in immunoglobulin receptor-mediated signal transduction in B cells, associating with kinase activity.
  • These findings identify alpha 4 as a key component in the B cell signaling cascade initiated by immunoglobulin receptors.

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