Characterization of the protein-tyrosine kinase PTK72 and its association with immune recognition receptors on lymphocytes

Jill Elizabeth Hutchcroft, Purdue University

Abstract

Lymphocytes have receptors on their cell surfaces that are responsible for the detection of antigen. These signaling complexes include surface IgM (sIgM) and surface IgD (sIgD) on B lymphocytes and Fc$\varepsilon$RI on mast cells. Receptor engagement triggers a cascade of biochemical events beginning with the rapid phosphorylation of a handful of proteins on tyrosine. Prominent among these phosphoproteins is one of 72 kDa. Studies were performed to determine whether this protein corresponds to the protein-tyrosine kinase PTK72. In B lymphocytes, the 72 kDa phosphoprotein comigrated, in one- and two-dimensional polyacrylamide gel electrophoresis systems with PTK72. Both proteins showed identical binding and elution profiles on heparin agarose resin. Anti-phosphotyrosine and anti-PTK72 antibodies immunoprecipitated the same protein-tyrosine kinase from extracts of anti-IgM-activated cells as determined by immune complex kinase assays and one-dimensional phosphopeptide mapping. The 72 kDa phosphoprotein was also shown to contain an ATP-binding site by its ability to specifically bind the ATP analog FSBA. Additional studies established that in mast cells PTK72 was similarly phosphorylated on tyrosine in response to activation through Fc$\varepsilon$RI. These results indicate that tyrosine phosphorylation of PTK72 is an early event in the activation of B lymphocytes and mast cells via immune recognition receptors. The rapid phosphorylation of PTK72 following engagement of sIg or Fc$\varepsilon$RI was consistent with the idea that the kinase might be closely associated with these receptor complexes. In experiments performed to test this hypothesis, antibodies to sIgM or sIgD co-immunoprecipitated PTK72, as detected by its autophosphorylation in immune complexes prepared from B lymphocytes. In addition, anti-PTK72 immune complexes contained components of the antigen receptor complex including the sIgM-associated protein MB-1, which is a substrate for PTK72. Likewise, antibodies to the $\beta$ subunit of Fc$\varepsilon$RI co-immunoprecipitated PTK72 from antigen-stimulated mast cells. In both B cells and mast cells, engagement of the antigen receptors led to an increase in PTK72 activity as detected in anti-PTK72 immune complexes. These results provide additional support for the existence of common signaling pathways initiated by multichain immune recognition receptors.

Degree

Ph.D.

Advisors

Geahlen, Purdue University.

Subject Area

Biology

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