Regulation of Syk by Phosphorylation on Serine in the Linker Insert

Abstract

The Syk protein-tyrosine kinase is phosphorylated on multiple tyrosines after the aggregation of the B cell antigen receptor. However, metabolic labeling experiments indicate that Syk is inducibly phosphorylated to an even greater extent on serine after receptor ligation. A combination of phosphopeptide mapping and mass spectrometric analyses indicates that serine 291 is a major site of phosphorylation. Serine 291 lies within a 23-amino acid insert located within the linker B region that distinguishes Syk from SykB and Zap-70. The phosphorylation of serine-291 by protein kinase C enhances the ability of Syk to couple the antigen receptor to the activation of the transcription factors NFAT and Elk-1. Protein interaction studies indicate a role for the phosphorylated linker insert in promoting an interaction between Syk and the chaperone protein, prohibitin.The Syk protein-tyrosine kinase is an essential component of the protein machinery that couples receptors bearing immunoreceptor tyrosine-based activation motifs to intracellular signal transduction pathways in hematopoietic cells (for a recent review, see Ref. 1). Syk is recruited via its N-terminal pair of Src homology 2 domains to a pair of tyrosines located in the cytoplasmic immunoreceptor tyrosine-based activation motifs of immune recognition receptors that become phosphorylated after ligand-induced aggregation. For example, clustering of the B cell receptor for antigen (BCR) 4 leads to the phosphorylation of the accessory molecules CD79a and CD79b, resulting in the recruitment of Syk to the clustered BCR complex. Receptor binding leads to the activation of Syk and to its phosphorylation on tyrosines that regulate its activity and interactions with downstream effectors that bear Src homology 2 or other phosphotyrosine binding domains. This in turn couples the BCR to the activation of, among others, the phospholipase ␥/PKC, PI3K/Akt, and Ras/Raf/Erk signaling pathways. In the absence of Syk, few if any signals are sent after BCR clustering (2-4).Syk contains a stretch of 23 amino acids known as the "linker insert" that lies within the linker B region that separates the tandem pair of Src homology 2 domains from the C-terminal catalytic domain. The presence of this insert distinguishes Syk from SykB, a shorter alternatively spliced isoform, and from ZAP-70, the Syk paralog found in T cells and natural killer cells (5, 6). The role of the linker insert in modulating the properties of Syk is poorly understood, but two functions have been proposed. In one, the linker insert harbors a signal sequence that allows the kinase to transit into the nucleus (7). In another, the insert enhances the ability of the kinase to interact with immunoreceptor tyrosine-based activation motif-bearing receptors (8). Within the linker insert (TWSPGGIISRIKSYSFPKPGHKK) are several potential sites of phosphorylation. The tyrosine (Tyr-290 in the murine Syk sequence) is a site of autophosphorylation in vitro (9) but to our knowledge is not known to be phosphorylated in response to r...

Complex phosphorylation dynamics control the composition of the Syk interactome in B cells

“…The compromised signaling response correlated with the inability of the S291A variant to associate with the chaperone prohibitin. No direct interaction between phosphorylated serine 291 and 14-3-3 proteins was observed in this study [47] despite the evolutionary conservation of the canonical mode 1 motif for 14-3-3 binding in murine and human Syk orthologes. The marked discrepancies to our data cannot be attributed to the use of different experimental systems.…”

“…While this manuscript was in preparation, Paris et al reported that protein kinase C phosphorylates murine Syk at serine 291, which corresponds to S297 in human Syk [47]. Using luciferasebased reporter gene assays these authors found reduced activation of transcription factors nuclear factor of activated T cells and Elk-1 in BCR-stimulated DT40 B cells expressing the S291A mutant of murine Syk.…”

“…Interestingly, the short Syk isoform that is predominantly expressed in breast cancer cells [46] lacks the linker insert encompassing serine 297. It is thus tempting to speculate that the absence of the inhibitory 14-3-3 module is involved in Syk-related pathogenicity.While this manuscript was in preparation, Paris et al reported that protein kinase C phosphorylates murine Syk at serine 291, which corresponds to S297 in human Syk [47]. Using luciferasebased reporter gene assays these authors found reduced activation of transcription factors nuclear factor of activated T cells and Elk-1 in BCR-stimulated DT40 B cells expressing the S291A mutant of murine Syk.…”

Prohibitin in Adipose and Immune Functions

“…In a separate study, PHB was identified as a highly expressed protein during phorbol ester-induced maturation of human B lymphocytes, suggesting a role of PHB in B cell maturation [29]. Subsequent studies have identified PHB as a phosphoprotein in B cell receptor signaling [30,31]. For example, Syk tyrosine kinase, which is an important signaling intermediate in B cell antigen receptor signaling, has been found to interact with PHB in a phosphorylationdependent manner [31].…”

“…Subsequent studies have identified PHB as a phosphoprotein in B cell receptor signaling [30,31]. For example, Syk tyrosine kinase, which is an important signaling intermediate in B cell antigen receptor signaling, has been found to interact with PHB in a phosphorylationdependent manner [31]. In addition to B lymphocytes, a number of studies have identified prohibitins as a membrane-associated protein in T lymphocytes and have been shown to have a role in T cell receptor signaling [32,33].…”