Identification of BCAR-1 as a new substrate of Syk tyrosine kinase through a determination of amino acid sequence preferences surrounding the substrate tyrosine residue

Syk, a non-receptor tyrosine kinase, is an essential signaling molecule in B cells and other hematopoietic cells. Recently, its unexpected diverse functions were recognized in the regulation of cellular adhesion, innate immune recognition, vascular development, and carcinogenesis. Despite its pleiotropic role, only a few substrate proteins have been identified. To find new substrate proteins for Syk, we performed a systemic in vitro kinase assay using GST fusion peptides to determine the substrate specificity surrounding the tyrosine residue to be phosphorylated. Substitution of amino acid residues surrounding tyrosine 178 of BLNK, a principal Syk substrate in B cell receptor-mediated signaling, revealed that acidic residues at sites −5 to −1 were necessary for phosphorylation by Syk. Valine at site +1 was also influential in phosphorylation and a substitution of Pro on site +3 to a basic amino acid residue, Lys, resulted in attenuated phosphorylation. On the basis of these results, a general consensus phosphorylation motif for Syk was determined and several new candidate target proteins were identified in protein database searches. Of the candidate proteins, BCAR-1 (breast cancer anti-estrogen resistance 1) was confirmed to be phosphorylated by Syk in an in vitro kinase assay using a full-length protein of BCAR-1. Furthermore, BCAR-1 was tyrosine phosphorylated upon the overexpression of Syk in HEK-293T cells. These results suggest that more Syk substrates can be found using an in vitro kinase approach and show for the first time that BCAR-1 is a physiological substrate of Syk.

Graphical abstractFigure optionsDownload full-size imageDownload as PowerPoint slideResearch highlights▶ Syk phosphorylates substrates with a consensus sequence of “[ED]-[ED]-[ED]-X-[ED]-Y-X-X-{KR}”. ▶ BCAR-1 (p130Cas) is one of the Syk substrates with the consensus sequence.▶ BCAR-1 was confirmed to be phosphorylated by Syk in a co-transfection study in HEK-293T cells.