IDENTIFICATION OF THE MAJOR SITES OF AUTOPHOSPHORYLATION OF THE MURINE PROTEIN-TYROSINE KINASE SYK

The protein tyrosine kinase p72(syk) (Syk) is expressed in a variety o f hematopoietic cell types, including B cells, thymocytes, mast cells and others. Both the activity and phosphotyrosine content of this enzy me increase in these cells in response to engagement of the appropriat e cell surface receptors. Herein, we describe the cloning of murine Sy k and its expression in Sf9 cells as a catalytically active protein. F ull-length Syk and a catalytically active 42.5 kDa carboxyl terminal f ragment were also expressed as glutathione S-transferase fusion protei ns. Comparative reverse phase HPLC and 40% alkaline gel analysis of tr yptic 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 autophospho rylation were identified using a combination of Edman sequencing and m ass spectrometric analysis. Ten sites were identified. One site is loc ated in the amino terminal half of the molecule between the two tandem Src homology 2 (SH2) domains. Five sites are located in the hinge reg ion located between the carboxyl terminal SH2 domain and the kinase do main. 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 doc king site for other SH2 domain-containing proteins. Consistent with th is prediction, autophosphorylated Syk efficiently binds the carboxyl t erminal SH2 domain of phospholipase C-gamma 1.