Syk-kinase protects Lyn-kinase from ubiquitination

Siba Prasad Bhattacharyya1 and Dean D. Metcalfe2

1Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institute of Health, Bethesda, MD 20892, 2Center for Biologics and Evaluation Research CBER, Food and Drug Administration, Bldg. 29 NIH Campus, Bethesda, MD 20892

Lyn is a member of the Src family of non-receptor protein tyrosine kinases and is associated with a number of cell surface receptors, including the B-cell antigen receptor and immunoglobulin E receptor (FceRI). Lyn is necessary for FceRI-mediated mast cell activation. To investigate how the level of Lyn is maintained in mast cell activation, we examined whether Lyn binds to ubiquitin and is ubiquitinated for proteasomal degradation. In the yeast two-hybrid system, Lyn specifically interacted with ubiquitin in vivo. Furthermore, Lyn bound to ubiquitin-conjugated Sepharose beads in vitro and was efficiently competed by soluble ubiquitin. Pulsechase experiments indicated intracellular degradation of Lyn was associated with the generation of a high molecular weight complex in the presence of proteasome-specific inhibitor, lactacystin. This high molecular weight complex cross-reacted with anti-Lyn and anti-ubiquitin, demonstrating the ubiquitination of Lyn. Overexpression of Lyn and ubiquitin in COS 7.2 cells also resulted in the ubiquitination of Lyn in the presence of lactacystin, supporting the ubiquitination of Lyn by proteasome specific pathway. The Syk-kinase specific inhibitor, piceatannol enhanced ubiqutination of Lyn-kinase. Syk-kinase also inhibited the interaction of Lyn with ubiquitin in the yeast two-hybrid system. These data suggest that Syk-kinase protects Lyn-kinase from ubiquitination.

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Molecular Pathology.