Identification and characterization of the sites of mitotic serine phosphorylation in Lck

Kamala Padma Kesavan, Purdue University

Abstract

Lck is a member of the Src family of non-receptor protein tyrosine kinases that plays a critical role in T cell signaling and development. The function of Lck is regulated both by an irreversible, cotranslational modification in the form of myristoylation and by reversible posttranslational modifications in the forms of palmitoylation and tyrosine and serine phosphorylation. Serine phosphorylation of Lck is both constitutive and inducible. A wide range of T cell stimuli can induce phosphorylation of serine residues which is often accompanied by a decrease in the electrophoretic mobility of Lck on SDS-polyacrylamide gels. In addition, differential serine phosphorylation of Lck has been demonstrated in mitotic T cells. The differential phosphorylation of Lck at mitosis suggested a specific mitotic function for Lck. It was therefore of interest to identify the in vivo sites of mitotic serine phosphorylation on Lck. Using a genetic approach and taking advantage of the diagnostic shift in the electrophoretic mobility of Lck, we have identified serine 59 as the site of mitotic serine phosphorylation in Lck. Similar studies revealed that neither the catalytic activity nor the SH2 and SH3 domains of Lck were essential for this mitotic serine phosphorylation. These results suggested that the mitotic serine phosphorylation of Lck was induced by a signaling pathway distinct from that involved in its serine phosphorylation in response to T cell stimulation. Additionally, the presence of the tyrosine kinase ZAP-70 was found not to be essential for the mitotic serine phosphorylation of Lck. Studies using specific inhibitors of the classical MAPK pathway and Cdc2 revealed that the mitotic seine phosphorylation of Lck was most likely mediated by Cdc2. As a possible function for the mitotic serine phosphorylation of Lck was the regulation of its mitosis-specific protein interactions, the effect of the phosphorylation site defective mutants on the association of Lck with one such protein, Sam68 was tested. Although the serine phosphorylation defective mutants associated with Sam68 normally, we found that the association was compromised in Lck mutants that are defective in either the SH2 or the SH3 domain. In addition, we were able to demonstrate that although the catalytic activity of Lck was dispensable for its association with Sam68, the mitotic tyrosine phosphorylation of Sam68 was dependent on the catalytic activity of Lck. In more recent studies, a unique one-dimensional phosphopeptide map was established for Lck using alkaline 40% acrylamide gels. Analysis of the phosphopeptide mapping pattern of Lck from Jurkat cells metabolically labeled with [32P]-orthophosphate confirmed that the differential phosphorylation of Lck was a truly mitotic event that resulted from the phosphorylation of serine 59. Additional analysis of the serine phosphorylation defective mutants using the phosphopeptide mapping strategy revealed the existence of novel sites of serine phosphorylation and suggested interesting regulatory aspects of the phosphorylation of Lck at serine 194.

Degree

Ph.D.

Advisors

Harrison, Purdue University.

Subject Area

Biochemistry|Molecular biology|Immunology

COinS