The tyrosine phosphorylation of the Csk -associated protein p62dok in B lymphocytes
Abstract
Csk is a cytosolic protein tyrosine kinase that negatively regulates Src-family kinases in various cell types, including B cells. Unlike most tyrosine kinases, the activity of Csk is not regulated by tyrosine phosphorylation. Instead, Csk is believed to be regulated by its cellular localization and/or its associated proteins. Because Csk is primarily cytosolic and its Src-family substrates are predominantly localized at the plasma membrane, Csk is believed to re-localize in an activation-dependent manner to cell fractions containing Src-family kinases, such as Lyn. We investigated whether Csk translocates from the cytosol to the plasma membrane in response to B cell receptor-mediated stimulation or pervanadate-treatment in B cells. Our results indicated that Csk was constitutively present in the cytosol, membrane and cytosol fractions. Immunoprecipitation studies revealed that Csk was constitutively associated with a 62kDa protein in both membrane and cytosolic fractions. Using V8 protease mapping experiments, the 62kDa protein was identified as p62dok, the rasGap-associated protein. Csk/p62dok complexes were found in both cytosol and membrane fractions and the association of Csk with p62dok was found to be mediated by the SH2 domain of Csk. To investigate the role of the protein tyrosine kinases Lyn and Syk in the tyrosine phosphorylation of p62dok and the association of p62dok and Csk, experiments were performed using kinase-deficient DT40 cell lines. In the absence of both Lyn and Syk, the tyrosine phosphorylation of p62dok was undetectable in DT40 chicken B cells. Our results revealed that Lyn was primarily responsible for the tyrosine phosphorylation of p62dok. These results suggest that Lyn may play a role in the negative regulation of itself in BCR-mediated signaling, by facilitating the recruitment of its negative regulator, Csk.
Degree
Ph.D.
Advisors
Harrison, Purdue University.
Subject Area
Cellular biology|Biochemistry
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