The Vav binding site of the non–receptor tyrosine kinase Syk at Tyr 348 is critical for β2 integrin (CD11/CD18)–mediated neutrophil migration

Abstract

IntroductionPolymorphonuclear neutrophils (PMNs) play an important role in host defense and inflammation. During the acute inflammatory response, PMNs extravasate from the blood into the tissue, where they migrate toward the sites of lesion by following a gradient of chemoattractants. 1,2 Site-directed migration (chemotaxis) requires 3 different processes 3 : (1) the periodical formation of lamellipodia; (2) the establishment of cell polarity that is characterized by the formation of a posterior pole (uropod) and an anterior pole (leading edge) that forms the lamellipodium with enhanced sensitivity for chemoattractants; and (3) the translation of the external chemotactic gradient into an internal signaling gradient. This process of directional sensing is due to an asymmetric distribution of signaling components within the cell. 3 Accordingly, neutrophil-like differentiated cells, an established model for PMNs, orient their polarity in response to a chemoattractant gradient and perform site-directed migration. 4 Chemoattractants signal via G-protein-coupled receptors that are responsible for directional sensing and the establishment of the polarized sensitivity within the cell. 3 However, ordered leukocyte polarization not only requires the presence of chemoattractants, but also depends on adhesive cell-matrix interactions that are mediated mainly by the leukocyte adhesion molecules of the integrin family. 5 ␤ 2 integrins (CD11/CD18) especially are critically involved in firm adhesion and migration of PMNs. 6 Recently, we and others have shown that the non-receptor tyrosine kinase Syk is constitutively associated with CD18, the ␤ subunit of the ␤ 2 integrins in human PMNs and in HL-60 cells. [7][8][9] Syk is widely expressed in hematopoietic cells and fulfills essential roles in hematopoietic cell functions like Fc receptor 10 and cytokine receptor signaling, 11 phagocytosis, 12 lymphocyte development, 13,14 and platelet activation. 15,16 Syk and ZAP-70, the other member of this protein family, contain 2 Src homology 2 (SH2) domains in a tandem repeat that are separated by the interdomain A. The C-terminal kinase domain is separated from the 2 SH2 domains by the interdomain B. 17 Outside-in signaling of Mac-1 (CD11b/CD18), the most abundant ␤ 2 integrin on PMNs, occurs upon ligand binding to immobilized fibrinogen or the intercellular adhesion molecules-1 and -2. 18 Mac-1-mediated adhesion induces tyrosine phosphorylation of different signaling components, including Syk. 7 A physiologic role for Syk has been demonstrated previously for multiple ␤ 2 integrin-mediated functions of murine PMNs, including the respiratory burst, degranulation, and spreading. 19 Recently, we have shown that endogenous Syk is enriched at the leading edge of both murine PMNs and HL-60 cells, where Syk plays a role in the stabilization of the leading edge of the polarized cell and colocalizes with CD18. 19,20 Syk has several downstream targets, including Pyk2, and Vav. 17,21 Vav is a guanine exchange factor for the Rho family GTPases Ra...

Phosphoproteomic analysis of Syk kinase signaling in human cancer cells reveals its role in cell–cell adhesion

“…Kinase-negative DsRed-Syk (K402R) also localizes at MCF7 cell-cell junctions but does not exhibit pTyr-525-526 staining (Figure 5a). Observations in hematopoietic cells demonstrate, nevertheless, that a kinase-dead EGFP Syk fusion protein may exhibit a different spatiotemporal regulation at the plasma membrane as compared to wild-type Syk (Ma et al, 2001;Schymeinsky et al, 2006). We finally analysed the phosphorylation status of E-cadherin in MCF7 cells transiently expressing wild-type or kinase-negative (K402R) DsRed-Syk ( Figure 5b).…”

The Mammalian Actin-Binding Protein 1 Is Critical for Spreading and Intraluminal Crawling of Neutrophils under Flow Conditions

“…Murine PMN were isolated, as described previously (28). The human myeloid HL-60 cells (ACC 3) and the murine WEHI-3B cells (ACC 26) were obtained from the German Resource Centre for Biological Material (Braunschweig, Germany).…”

“…Chemotactic migration of isolated murine PMN in response to a gradient of 10 mM fMLP was analyzed on immobilized murine fibrinogen (50 mg/ ml) or murine ICAM-1 (12.5 mg/ml) in Zigmond chambers (45) using a Zeiss Axiovert 200M microscope equipped with Plan-Apochromat original magnification 310/0.25 or Plan-Apochromat original magnification 320/0.75 objectives, an AxioCam HR digital camera, and the AxioVision 4 software (Zeiss), as described previously (28). Tracking of PMN migration was carried out, as described above.…”

“…Previous studies have demonstrated a crucial role of Syk for different b 2 integrin-mediated PMN functions, including slow leukocyte rolling and firm adhesion during inflammation in vivo, as well as the respiratory burst, cell spreading, migration, and phagocytosis (4,16,(18)(19)(20)(21)(22)(23)(24). Signaling downstream of Syk involves several molecules, including phospholipase C-g, the guanine nucleotide exchange factor Vav, the Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa, the adaptor protein Cbl, and the regulatory subunit p85 of the PI3K class I A (18,22,(25)(26)(27)(28)(29)(30). In addition, the mammalian actin-binding protein 1 (mAbp1; HIP-55, SH3P7, debrin-like protein) has been shown to become phosphorylated by Src and Syk family kinases in fibroblasts and in lymphocytes (31)(32)(33).…”

Spleen tyrosine kinase inhibition in the treatment of autoimmune, allergic and autoinflammatory diseases

“…In addition, Syk inhibition reduced synovial fluid cytokine levels and cartilage oligomeric matrix protein in serum in these animals [38]. R406 was also found to limit an Arthus reaction in mice [37] and rats [38] and reverse passive Arthus reaction in murine chimeras with Syk-deficient hematopoietic cells [43,44]. This effect is probably due to the suppression of immune-complex-mediated inflammation by inhibiting the Fc receptor signaling.…”