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-κB, and NFAT. The receptor-mediated activation of NF-κB 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.

Nucleocytoplasmic Trafficking of the Syk Protein Tyrosine Kinase

ABSTRACT The protein tyrosine kinase Syk couples the B-cell receptor (BCR) for antigen to multiple intracellular signaling pathways and also modulates cellular responses to inducers of oxidative stress in a receptor-independent fashion. In B cells, Syk is found in both the nuclear and cytoplasmic compartments but contains no recognizable nuclear localization or export signals. Through the analysis of a series of deletion mutants, we identified the presence of an unconventional shuttling sequence near the junction of the catalytic domain and the linker B region that accounts for Syk's subcellular localization. This localization is altered following prolonged engagement of the BCR, which causes Syk to be excluded from the nucleus. Nuclear exclusion requires the receptor-mediated activation of protein kinase C and new protein synthesis. Both of these processes also potentiate the activation of caspase 3 in cells in response to oxidative stress in a manner that is dependent on the localization of Syk outside of the nucleus. In contrast, restriction of Syk to the nucleus greatly diminishes the stress-induced activation of caspase 3.