Generation of an analog-sensitive Syk tyrosine kinase for the study of signaling dynamics from the B cell antigen receptor.
The Syk protein-tyrosine kinase is an essential component of the signaling machinery that couples the B cell receptor for antigen to multiple downstream signal transduction pathways. Syk is phosphorylated and activated rapidly and transiently following receptor engagement, but many signaling events, such as the activation of transcription factors occur over the course of several minutes or hours. To investigate a role for the continued activation of Syk in these processes, we generated an analog-sensitive mutant with an engineered ATP-binding pocket to render the kinase uniquely sensitive to an orthogonal inhibitor. Mutation of the gatekeeper residue in Syk yielded an enzyme with very low activity. Second-site mutations, selected based on structural comparisons between Syk and Src, were introduced that restored catalytic activity to the mutant Syk. Syk-deficient DT40 B cells were prepared expressing the analog-sensitive Syk (Syk-AQL). Inhibition of the activity of Syk prior to, concomitant with or shortly following receptor engagement led to the rapid inhibition of receptor-mediated tyrosine phosphorylation and blocked the activation of extracellular signal-regulated kinase, NF-kappaB, and NFAT. The receptor-mediated activation of NF-kappaB required active Syk for a relatively short period of time, whereas the activation of NFAT required active kinase for a prolonged (>1 h) period. Receptor cross-linking led to the recruitment of Syk to the clustered receptor. Retention of these receptor-kinase complexes on the cell surface was dependent on the continued activity of Syk. Thus, despite the apparent transient nature of the activation of Syk, the catalytic activity of the Syk was required for sustained signaling from ligated receptors.
Oh
Hyunju
H
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana, 47907; Purdue Cancer Center, Purdue University, West Lafayette, Indiana 47907.
Ozkirimli
Elif
E
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana, 47907; Purdue Cancer Center, Purdue University, West Lafayette, Indiana 47907.
Shah
Kavita
K
Purdue Cancer Center, Purdue University, West Lafayette, Indiana 47907; Department of Chemistry, Purdue University, West Lafayette, Indiana, 47907.
Harrison
Marietta L
ML
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana, 47907; Purdue Cancer Center, Purdue University, West Lafayette, Indiana 47907.
Geahlen
Robert L
RL
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana, 47907; Purdue Cancer Center, Purdue University, West Lafayette, Indiana 47907. Electronic address: geahlen@purdue.edu.
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002645:Chickens; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D049452:Green Fluorescent Proteins / Q000737:chemistry; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008958:Models, Molecular; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism; D050778:NFATC Transcription Factors / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism*; D015398:Signal Transduction; D000072377:Syk Kinase
D003432:Cross-Linking Reagents; D047908:Intracellular Signaling Peptides and Proteins; D016328:NF-kappa B; D050778:NFATC Transcription Factors; D011947:Receptors, Antigen, B-Cell; D049452:Green Fluorescent Proteins; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase