Identification of the major site of autophosphorylation of the murine protein-tyrosine kinase Syk

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Abstract

The protein tyrosine kinase p72(syk) (Syk) is expressed in a variety of hematopoietic cell types, including B cells, thymocytes, mast cells and others. Both the activity and phosphotyrosine content of this enzyme increase in these cells in response to engagement of the appropriate cell surface receptors. Herein, we describe the cloning of murine Syk and its expression in Sf9 cells as a catalytically active protein. Full-length Syk and a catalytically active 42.5 kDa carboxyl terminal fragment were also expressed as glutathione S-transferase fusion proteins. Comparative reverse phase HPLC and 40% alkaline gel analysis of tryptic digests of phosphorylated Syk demonstrated that all of the major sites of autophosphorylation were also present in GST-Syk and all but one were contained in the 42.5 kDa fragment. The sites of autophosphorylation were identified using a combination of Edman sequencing and mass spectrometric analysis. Ten sites were identified. One site is located in the amino terminal half of the molecule between the two tandem Src homology 2 (SH2) domains. Five sites are located in the hinge region located between the carboxyl terminal SH2 domain and the kinase domain. Two sites lie in the kinase domain within the catalytic loop and two near the extreme carboxyl terminus. Sequences of phosphorylation sites located within the hinge region predict that Syk serves as a docking site for other SH2 domain-containing proteins. Consistent with this prediction, autophosphorylated Syk efficiently binds the carboxyl terminal SH2 domain of phospholipase C-γl.

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Furlong, M. T., Mahrenholz, A. M., Kim, K. H., Ashendel, C. L., Harrison, M. L., & Geahlen, R. L. (1997). Identification of the major site of autophosphorylation of the murine protein-tyrosine kinase Syk. Biochimica et Biophysica Acta - Molecular Cell Research, 1355(2), 177–190. https://doi.org/10.1016/S0167-4889(96)00131-0

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