Molecular mechanism of the Syk activation switch.
Many immune signaling pathways require activation of the Syk tyrosine kinase to link ligation of surface receptors to changes in gene expression. Despite the central role of Syk in these pathways, the Syk activation process remains poorly understood. In this work we quantitatively characterized the molecular mechanism of Syk activation in vitro using a real time fluorescence kinase assay, mutagenesis, and other biochemical techniques. We found that dephosphorylated full-length Syk demonstrates a low initial rate of substrate phosphorylation that increases during the kinase reaction due to autophosphorylation. The initial rate of Syk activity was strongly increased by either pre-autophosphorylation or binding of phosphorylated immune tyrosine activation motif peptides, and each of these factors independently fully activated Syk. Deletion mutagenesis was used to identify regions of Syk important for regulation, and residues 340-356 of the SH2 kinase linker region were identified to be important for suppression of activity before activation. Comparison of the activation processes of Syk and Zap-70 revealed that Syk is more readily activated by autophosphorylation than Zap-70, although both kinases are rapidly activated by Src family kinases. We also studied Syk activity in B cell lysates and found endogenous Syk is also activated by phosphorylation and immune tyrosine activation motif binding. Together these experiments show that Syk functions as an "OR-gate" type of molecular switch. This mechanism of switch-like activation helps explain how Syk is both rapidly activated after receptor binding but also sustains activity over time to facilitate longer term changes in gene expression.
Tsang
Emily
E
Department of Inflammation Discovery, Roche Palo Alto LLC, Palo Alto, California 94304, USA.
Giannetti
Anthony M
AM
Shaw
David
D
Dinh
Marie
M
Tse
Joyce K Y
JK
Gandhi
Shaan
S
Ho
Hoangdung
H
Wang
Sandra
S
Papp
Eva
E
Bradshaw
J Michael
JM
D020816:Amino Acid Motifs; D001402:B-Lymphocytes / Q000378:metabolism; D017353:Gene Deletion; D006801:Humans; D007107:Immune System; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D016296:Mutagenesis; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013379:Substrate Specificity; D000072377:Syk Kinase; D014443:Tyrosine / Q000737:chemistry; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000737:chemistry
D047908:Intracellular Signaling Peptides and Proteins; D010455:Peptides; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human