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.
Division of Immunology, Department of Molecular Cell Biology and Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Jangan-gu, Chunchun-dong 300, Suwon 440-746, Gyeonggi-do, Republic of Korea.
Ji-Yeon
JY
Kim
Kyungmin
K
Huh
Rara
R
Jung
Tae Jin
TJ
Kim
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001402:B-Lymphocytes / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D050719:Crk-Associated Substrate Protein / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D057809:HEK293 Cells; D006412:Hematopoietic Stem Cells / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology; D013379:Substrate Specificity; D000072377:Syk Kinase
C493841:BCAR1 protein, human; D050719:Crk-Associated Substrate Protein; D047908:Intracellular Signaling Peptides and Proteins; D010455:Peptides; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase