Generation of an Analog-sensitive Syk Tyrosine Kinase for the Study of Signaling Dynamics from the B Cell Antigen Receptor

Abstract

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 analogsensitive 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-B, and NFAT. The receptor-mediated activation of NF-B 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. Upon engagement of the B cell antigen receptor (BCR)3 by binding polyvalent antigens, immunoreceptor tyrosine-based activation motifs in the cytoplasmic domains of Ig-␣ and Ig-␤ are phosphorylated leading to the recruitment of Syk to the receptor complex and its phosphorylation on tyrosine and subsequent activation (1). Syk is then required for coupling the BCR to the phosphorylation and activation of the downstream regulatory and adaptor molecules BLNK/SLP65, phosphoinositide 3-kinase, Btk, and phospholipase C-␥2, leading to the activation of Akt and protein kinase C, the mobilization of Ca 2ϩ , activation of the Ras/Raf/ERK and p38 mitogen-activated protein kinase (MAPK) signaling cascades, and activation of the transcription factors NF-B and NFAT.The kinetics of these BCR-initiated signaling events are of considerable interest as their magnitude and duration are thought to be critical factors in determining B cell survival, development and proliferation. The phosphorylation on tyrosine of BCR components, Syk and Btk all occur rapidly following receptor engagement (2-4). Phosphorylation is important for the activity of Syk and Btk and for the retention of BCRsignaling complexes on the cell surface (5); but these also are transient events. After immediate activation of membrane proximal signal...

Molecular Mechanism of the Syk Activation Switch

“…3B) The OR-gate behavior of Syk may help to explain recent findings that Syk activity is maintained over time after transient activation of the B cell receptor. Many signaling events after BCR ligation are rapidly reversed, but it has recently been found that Syk activity is required for more than 1 h to induce activation of NFAT (nuclear factor of activated T cells) transcription (33). Furthermore, sustained Syk activity has also been linked to store-operated Ca 2ϩ entry, which is important for longer term signaling events (12).…”

Syk Is Recruited to Stress Granules and Promotes Their Clearance through Autophagy

“…The cDNA for Syk-EGFP(R428Q/M429L/M442A) (Syk-AQL-EGFP) was amplified by PCR from the corresponding EGFP-N2 (Clontech) construct (32) and cloned into the Tet-inducible lentiviral vector pLVX-Tight-Puro (Clontech). Lentiviral particles were packaged in HEK293T cells.…”

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 is rapidly phosphorylated and activated upon BCR engagement, but its continued activity is required for B cell development and differentiation [39,40]. Activated Syk can move from plasma membrane to other cellular compartments and phosphorylate critical substrates such as BLNK for B cell proliferation and differentiation.…”