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

Abstract

Spleen tyrosine kinase Syk provides critical transducer functions for a number of immune cell receptors and has been implicated in the generation of several forms of leukemias. Catalytic activity and the ability of Syk to interact with other signaling elements depend on the phosphorylation status of Syk. We have now identified and quantified the full spectrum of phosphoacceptor sites in human Syk as well as the interactome of Syk in resting and activated B cells by high-resolution mass spectrometry. While the majority of inducible phosphorylations occurred on tyrosine residues, one of the most frequently detected phosphosites encompassed serine 297 located within the linker insert distinguishing the long and short isoforms of Syk. Full-length Syk can associate with more than 25 distinct ligands including the 14-3-3c adaptor protein, which binds directly to phosphoserine 297. The latter complex attenuates inducible plasma membrane recruitment of Syk, thereby limiting antigen receptor-proximal signaling pathways. Collectively, the established ligand library provides a basis to understand the complexity of the Syk signaling network.Key words: B-cell activation . Interactome . Spleen tyrosine kinase . 14-3-3 Supporting Information available online IntroductionThe 72 kDa spleen tyrosine kinase Syk provides catalytic activity to hematopoietic cell surface receptors encompassing ITAMs in their signaling subunits [1]. Following ligand-induced receptor aggregation, doubly phosphorylated ITAMs recruit Syk by virtue of its N-terminal Src homology 2 (SH2) domains. Interdomain A of Syk links the two SH2 domains, which are connected to the C-terminal kinase domain by interdomain B. Two Syk isoforms can be generated by alternative splicing, which leads to the presence or absence of 23 amino acids, called the linker insert region, in interdomain B [2,3]. Several mechanisms operate in concert to control Syk activity. The phospho-ITAM/(SH2) 2 interaction leads to allosteric activation most likely by changing the conformation of Syk from a closed inactive form to an open active structure [4,5]. Moreover, phospho-ITAMs act as inducible membrane anchors for cytosolic Syk and the accompanied subcellular relocalization provides Syk with access to key substrates [6]. Phosphorylation of tyrosine residues within the kinase domain or interdomain B boosts the catalytic activity of Syk or generates docking sites for SH2 domain-containing effector proteins, respectively [7]. Termination of Syk activity can be achieved by dephosphorylation through protein tyrosine phosphatases such as SHP1 or proteasomal degradation induced à These authors contributed equally to this work. 1550Frontline by binding of the E3 ubiquitin ligase Cbl to a distinct phosphotyrosine residue in interdomain B [8,9].Syk activation and triggering of downstream effector cascades have been extensively studied in B lymphocytes. In fact, Syk was initially identified as a B-lymphoid tyrosine kinase associated with BCR [10,11]. BCRs comprise membrane-bound Igs of different cla...

β2 integrin–derived signals induce cell survival and proliferation of AML blasts by activating a Syk/STAT signaling axis

“…38 Thus it appears that different posttranslational modification patterns of Syk are 1 reason for differences in the composition of the Syk interactomes in different cell types. We found that the Syk phosphorylation state in AML cells differs from the B-lymphoid one 26 ; this phenomenon of differential cell type-specific phosphorylation might be 1 possible explanation for the existence of the Syk/STAT complex in AML cells. Using a proteomic approach, we identified the b 2 integrin receptor Mac-1 and FcgRI as Syk binding partners.…”

“…26 Interestingly, no interaction between Syk and STAT3 or STAT5 was detected in B cells, although they express STAT3 and STAT5 proteins at significant levels. 26 In contrast, a recent study showed that Syk phosphorylates STAT3 in acute lymphoblastic leukemia cells when they are exposed to oxidative stress.…”

“…26 Interestingly, no interaction between Syk and STAT3 or STAT5 was detected in B cells, although they express STAT3 and STAT5 proteins at significant levels. 26 In contrast, a recent study showed that Syk phosphorylates STAT3 in acute lymphoblastic leukemia cells when they are exposed to oxidative stress. 38 Thus it appears that different posttranslational modification patterns of Syk are 1 reason for differences in the composition of the Syk interactomes in different cell types.…”

“…When we compared the Syk interactomes in AML and B-lymphoid cells, we observed an overlap regarding Vav, phosphatidylinositol 3-kinase, Cbl, dynein, tubulin, and 14-3-3 proteins. 26,27 However, we identified several novel Syk interactors in AML cells that have not been described to interact with Syk in other cell types. Interestingly, we found that in all AML cells tested, the transcription factors STAT3 and STAT5 interacted with Syk.…”

“…The red circle represents p-sites that were identified in AML cells in this study, while the black circle represents the total number of p-sites that were identified in B cells in previous studies. 26 For personal use only. on May 11, 2018.…”