SOS1, ARHGEF1, and DOCK2 rho-GEFs Mediate JAK-Dependent LFA-1 Activation by Chemokines This information is current as Lara Toffali, Alessio Montresor, Michela Mirenda, Giorgio of September 24, 2021. Scita and Carlo Laudanna J Immunol 2017; 198:708-717; Prepublished online 16 December 2016; doi: 10.4049/jimmunol.1600933 http://www.jimmunol.org/content/198/2/708 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2016/12/15/jimmunol.160093 Material 3.DCSupplemental http://www.jimmunol.org/ References This article cites 36 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/198/2/708.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 24, 2021 • No Triage! 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The Journal of Immunology SOS1, ARHGEF1, and DOCK2 rho-GEFs Mediate JAK-Dependent LFA-1 Activation by Chemokines Lara Toffali,*,† Alessio Montresor,*,† Michela Mirenda,* Giorgio Scita,‡,x and Carlo Laudanna*,† JAK-dependent activation of the rho module of integrin affinity triggering mediates chemokine-induced leukocyte adhesion. However, the signaling events linking JAKs to rho small GTPase activation by chemokines is still incompletely described. In this study, we show that son of sevenless 1 (SOS1), rho guanine nucleotide exchange factor (GEF)1 (ARHGEF1), and dedicator of cytokinesis (DOCK)2 GEFs mediate CXCL12-induced LFA-1 activation in human primary T lymphocytes. Downregulated expression of SOS1, ARHGEF1, and DOCK2 impairs LFA-1–mediated rapid T lymphocyte adhesion as well as underflow arrest on ICAM-1 induced by CXCL12. Moreover, LFA-1 affinity triggering by CXCL12 is impaired by SOS1, ARHGEF1, and DOCK2 downregulation. Notably, the three GEFs are all critically involved in chemokine-induced RhoA and Rac1 activation, thus Downloaded from suggesting the occurrence of a SOS1 specificity shift in the context of chemokine signaling. Accordingly, SOS1, ARHGEF1, and DOCK2 are tyrosine phosphorylated upon chemokine signaling with timing coherent with rapid LFA-1 affinity activation. Importantly, chemokine-induced tyrosine phosphorylation of these GEFs is fully mediated by JAK protein tyrosine kinases. Unexpectedly, and differently from VAV1, tyrosine phosphorylation of SOS1, ARHGEF1, and DOCK2 is completely inhibited by pertussis toxin pretreatment, thus suggesting different routes of rho-GEF triggering upon CXCR4 engagement. Taken together, these findings reveal a deeper level of complexity in the rho-signaling module, with at least four different rho-GEFs cooperating in http://www.jimmunol.org/ the regulation of chemokine-induced integrin activation, possibly suggesting the emergence of stochastic concurrency in signaling mechanisms controlling leukocyte trafficking. The Journal of Immunology, 2017, 198: 708–717. eukocyte recruitment is a central homeostatic process in cooperate to increase cell adhesiveness, integrin affinity upregu- the immune response. Although consisting of an ensemble lation plays a prominent role in rapid leukocyte arrest underflow L of continuous, mainly overlapping, cellular and bio- (2–5). Following G protein–coupled receptor engagement by chemical events, involving many different molecules both at the chemoattractants, a complex, still incompletely defined, inside-out qualitative and quantitative level, leukocyte recruitment is never- signaling network is assembled controlling the induction of by guest on September 24, 2021 theless modeled as a sequence of discrete steps, including cap- integrin conformational changes leading to increased affinity for turing, rolling, activation, arrest, crawling to sites of exit, and endothelial ligands. Among many investigated signaling events, transmigration through the barriers of endothelial cells and the Rho (3, 5) and Rap (6, 7) small GTPases are the most studied basement membrane. The transition from rolling to arrest, which is signaling mechanisms linking chemokine-triggered signal trans- the step when leukocytes become fully resistant to the pushing duction to rapid integrin affinity regulation. More recently, we force of the blood flow, is critically controlled by apically presented have demonstrated the critical role of protein tyrosine kinases chemoattractants, such as chemokines, leading to rapid integrin (PTKs) of JAKs, in particular JAK2 and JAK3, as upstream ac- activation (1). Although different modalities of integrin activation tivators of the rho module of LFA-1 affinity triggering in human T lymphocytes (8). In this context, we also started the character- ization of the biochemical link between JAKs and rho small *Department of Medicine, University of Verona, Verona 37134, Italy; †Center for Biomedical Computing, University of Verona, Verona 37134, Italy; GTPases demonstrating the involvement of VAV1 rho guanine ‡Italian Foundation for Cancer Research Institute for Molecular Oncology, Milan nucleotide exchange factor (GEF) in JAK-mediated rho module 20139, Italy; and xDepartment of Oncology and Hemato-Oncology, Italian Database of Operational Losses, University of Milan, Milan 20142, Italy activation by CXCL12. Small GTPase activity is tightly regulated by the rapid turnover Received for publication May 31, 2016. Accepted for publication November 16, 2016. between GDP/GTP-bound states, promoted by GTPase-specific This work was supported by Italian Association for Cancer Research Grant IG16797, GEFs. GEF activity is controlled by different mechanisms, in- Ministero dell’Istruzione, dell’Universita` e della Ricerca Grant PRIN 2009, Nano- cluding protein–protein interactions, protein–lipid interaction, medicine project funding from the University of Verona and the Fondazione Cariverona (to C.L.), Italian Association for Cancer Research Grant IG10168, European Research binding to second messengers, and posttranslational modifications Council Advanced Grant ERC 268836, and by Worldwide Cancer Research Grants (9). Tyrosine phosphorylation is one of the best characterized AICR-09-0582 and 14-0335 (to G.S.). mechanisms of GEF activation (10). For instance, phosphorylation Address correspondence and reprint requests to Prof. Carlo Laudanna, Department of of tyrosine 174 is crucial to VAV1 GEF activity (11). Moreover, Medicine, University of Verona, Strada le Grazie 8, Verona 37134, Italy. E-mail address: [email protected] son of sevenless 1 (SOS1), a ras-specific GEF, manifests a spec- The online version of this article contains supplemental material. ificity switch upon tyrosine phosphorylation, becoming capable of promoting GDP/GTP exchange on Rac1 (12, 13). Notably, VAV1 Abbreviations used in this article: ARHGEF1, rho guanine nucleotide exchange factor 1; DOCK, dedicator of cytokinesis; GEF, guanine nucleotide exchange factor; gene silencing causes only a partial inhibition of LFA-1 affinity P1, Penetratin 1; PTK, protein tyrosine kinase; PTx, pertussis toxin; siRNA, small triggering, and of RhoA and Rac1 activation, suggesting that other interfering RNA; SOS1, son of sevenless 1; TKIP, tyrosine kinase inhibitor peptide. rho-GEFs are at play in CXCL12-induced LFA-1 affinity upreg- Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 ulation. Consistent with the latter notion, dedicator of cytokinesis www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600933 The Journal of Immunology 709 (DOCK)2 has been proposed to participate in VLA-4–mediated Human primary cells integrin activation (14). Moreover, JAK-mediated phosphorylation Human primary CD3+ lymphocytes were isolated from whole blood of of rho-GEF 1 (ARHGEF1) has been reported in an angiotensin II– healthy donors by Ficoll and Percoll gradients. Purity of T lymphocyte dependent murine vascular model of hypertension (15). preparation was evaluated by flow cytometry after staining with FITC- conjugated anti-CD3 Ab and was .95%. Isolated T lymphocytes were In this study, we pursued a deeper characterization of the upstream kept at 37˚C in standard adhesion buffer (PBS, 1 mM CaCl2, 1 mM MgCl2, signaling mechanisms linking JAK PTKs to integrin activation and 10% FBS [pH 7.2]) and used within 1 h. The University of Verona Ethics investigated the role of SOS1, ARHGEF1, and DOCK2 as media- Committee approved experimentation involving human primary cells. tors of chemokine-triggered JAK-dependent rho module activa- Static adhesion assay tion leading to LFA-1 affinity upregulation in human primary T lymphocytes. We show that SOS1, ARHGEF1, and DOCK2 are Human primary T lymphocytes were suspended at 3 3 106 cells/ml in phosphorylated on tyrosine by the CXCR4 ligand CXCL12 in a standard adhesion buffer. Adhesion assays were done on 18-well glass slides coated with human 1 mg/ml ICAM-1 in PBS; 20 ml of cell sus- time frame coherent with rapid integrin triggering. By using JAK-