Pak1 Kinase Promotes Activated T Cell Trafficking by Regulating the Expression of L-Selectin and CCR7

Normal function of the adaptive immune system requires trafficking of T cells between the blood and lymphoid organs. Lymphocyte homing to lymph nodes requires that they cross endothelial barriers present in blood vessels and lymphatics. This multi-step process requires a remodeling of the lymphocyte plasma membrane, which is mediated by the dynamic re-arrangement of the actin cytoskeleton. Pak1 plays a central role in cell morphology, adhesion and migration in various cell types. Here we demonstrate that Pak1 is required for activated CD4+ T cell trafficking to lymph nodes. Pak1 deficiency in T cells causes a defect in the transcription of CCR7 and L-selectin, thereby altering lymphocyte trafficking. Additionally, we report an increase in L-selectin shedding in Pak1-deficient T cells, which correlates with a decrease in the recruitment of calmodulin to the cytoplasmic tail of L-selectin during T cell activation. Overall, our findings demonstrate that by regulating the expression of two major lymph node homing molecules, L-selectin and CCR7, Pak1 mediates activated CD4+ T cell trafficking

Sphingosine-1-phosphate activates chemokine-promoted myeloma cell adhesion and migration involving α4β1 integrin function

51 p.-7 fig.-1 tab.-2 fig.supl.Myeloma cell adhesion dependent on α4β1 integrin is crucial for the progression of multiple myeloma (MM). The α4β1-dependent myeloma cell adhesion is up-regulated by the chemokine CXCL12, and pharmacological blockade of the CXCL12 receptor CXCR4 leads to defective myeloma cell homing to bone marrow (BM). Sphingosine-1-phosphate (S1P) regulates immune cell trafficking upon binding to G-protein-coupled receptors. Here we show that myeloma cells express S1P1, a receptor for S1P. We found that S1P up-regulated the α4β1-mediated myeloma cell adhesion and transendothelial migration stimulated by CXCL12. S1P promoted generation of high-affinity α4β1 that efficiently bound the α4β1 ligand VCAM-1, a finding that was associated with S1P-triggered increase in talin-β1 integrin association. Furthermore, S1P cooperated with CXCL12 for enhancement of α4β1-dependent adhesion strengthening and spreading. CXCL12 and S1P activated the DOCK2-Rac1 pathway, which was required for stimulation of myeloma cell adhesion involving α4β1. Moreover, in vivo analyses indicated that S1P contributes to optimizing the interactions of MM cells with the BM microvasculture and for their lodging inside the bone marrow. The regulation of α4β1-dependent adhesion and migration of myeloma cells by CXCL12-S1P combined activities might have important consequences for myeloma disease progressionThis study was supported by the Ministerio de Ciencia e Innovación (Grant Nos SAF2011-24022 to JT, SAF2009-07035 to AGP, SAF2009-11037 to AH, RD06/0020/0011 to JT and AGP, RD06/0020/0006 to NCG and MG and PI081825 to MG), the Comunidad de Madrid (Grant No. P2010/BMD-2314 to AGP, JT and AH) and the Fundación de Investigación Médica Mutua Madrileña (to AGP and MG). AH was also funded by the FP7-People-2009-RG (Grant No. 246655), a Ramón y Cajal Fellowship (Grant No. RYC-2007-00697) and the Pro-CNIC Foundation.Peer reviewe

Regulación por proteínas señalizadoras de la adhesión dependiente de integrinas linfocitarias en respuesta a quimioquinas

154 p.-54 fig.-3 tab.Chemokines stimulate cell migration and activation, and exert their functions upon binding to heterotrimeric guanine nucleotide-binding (G) protein-coupled receptors (GPCR). In the immune system, chemokines promote the migration of immune cells from lymph and blood circulation into lymphoid tissues and sites of inflammation during immune surveillance. For T lymphocytes, this process is achieved after rapid stimulation of α4β1 and αLβ2 integrin activity by chemokines presented on the endothelium. This activation must be rapid to deliver tight cell attachment to resist the blood shear stress, but also transient to allow lymphocyte locomotion on and diapedesis across endothelial layers. Thus, chemokine binding to GPCR induces activation of intracellular effector molecules that lead to integrin-mediated upregulation of lymphocyte adhesion, a process called inside-out signaling. Key inside-out molecules that regulate the activation of α4β1 and αLβ2 include talin and kindlin-3, as well as the Vav1-Rac1 and RAPL-Rap1 pathways, that stimulate the transition to high-affinity α4β1 and αLβ2 conformations. In the present thesis, we havecharacterized the inside-out signaling required for the regulation of chemokine-stimulated T lymphocyte adhesion mediated by α4β1. When T lymphocytes become exposed to chemokines, inside-out signals are generated that finally impinge on integrin cytoplasmic domains, leading to integrin activation and stimulation of cell adhesion. Vav1 is a key component of this signaling because it is required for α4β1 integrin activation. Talin directly interacts with β subunit integrin cytoplasmic domains and regulates integrin activation, thereby representing a main candidate for chemokine-stimulated T lymphocyte adhesion mediated by α4β1. Previous results achieved in our laboratory demonstrated that Vav1 and talin constitutively associate in human resting T lymphocytes and that they complex into an essential signaling platform. Thus, when chemokine-triggered signaling converges on this platform, Vav1 and talin gradually dissociate. This dissociation correlates with an increase in talin-β1 association, which represents a key event for integrin activation and induces an upregulation of cell adhesion. The importance of this platform is based on the fact that silencing Vav1 or talin leads to failure to assemble the complex and to a subsequent blockade of the chemokine CXCL12-stimulated T cell attachment to VCAM-1, the main ligand of α4β1 in endothelial cells.Coincident with the dissociation of Vav1 from talin we observed that the kinase ZAP-70 enters in the Vav1-talin complex, leading to Vav1 tyrosine phosphorilation. The first objective in this thesis consisted in the study of the function of ZAP-70 in the regulation of the dynamics of Vav1, talin and β1 associations, as well as in chemokine stimulated T lymphocyte adhesion mediated by α4β1. Our results indicate that ZAP-70 is involved in α4β1 activation in response to CXCL12, because this activation requires ZAP-70-dependent dissociation of Vav1-talin complexes. ZAP-70 binding to Vav1-talin complexes leads to Vav1 phosphorylation, which possibly weakens Vav1-talin association. Therefore, our data strongly suggest that chemokine promoted, ZAP-70- dependent phosphorylation of Vav1 starts while this protein is still associated with talin in the signaling platform. This conclusion is based on the fact that talin knockdown impairs Vav1-ZAP-70 binding, and inhibition of ZAP-70 activity abolishes chemokine-dependent Vav1 phosphorylation associated with blocking of Vav1-talin disassembly. Furthermore, we show that when ZAP-70 function or expression is inhibited, there is a defect in chemokine-stimulated T lymphocyte adhesion mediated by α4β1 l. This defect is linked with a decrease in integrin activation, as detected with binding of VCAM-1-Fc or HUTS- 21 anti-β1 integrin mAb, a reporter of α4β1 activation. Together, our data suggest that Vav1 functions as a constitutive adaptor of talin that needs to be phosphorylated by ZAP- 70 in order to be released from the Vav1-talin complexes, thus rendering talin available for additional α4β1 integrin activation. We have started preliminary work addressed to determine of TCR-dependent integrin activation following similar pattern as those from chemokine-stimulated activation. Notably, confocal microscopy assays revealed that Vav1-talin and ZAP-70- talin co-localizations decreased following TCR activation. These preliminary data suggest that signaling generated upon chemokine receptor or TCR activation might follow common traits converging in integrin activation.Give the key role of Vav1 in chemokine-stimulated, α4β1-dependent T cell adhesion, we then analyzed the function of Vav1-binding partners in this adhesion process. We focused on SLP-76 and on the non-receptor tyrosine kinase Pyk2. Furthermore, we study the possible implication in this process of ADAP, an adapter that binds SLP-76 SH2 domain. SLP-76 plays a positive regulatory role for T cell development and for T cell activation in response to TCR stimulation. SLP-76 is an adaptor protein with the ability to bind multiple intracellular proteins upon TCR stimulation, and likely acts as a scaffold to recruit these proteins, therefore contributing to integrin activation. ADAP is an adaptor protein with minor roles in lymphocyte differentiation, but with an important involvement in late responses follows TCR activation. ADAP is connected to the cytoskeleton as a result of its binding to the EVH1 domain in Ena and VASP, and it also associates with RIAM, a protein that helps in the recruitment of talin in close proximity to integrin β subunit cytoplasmic domains. Importantly, ADAP plays key roles in TCR-stimulated T cell adhesion mediated by β1 and β2 integrins. Finally, Pyk2 is a protein tyrosine kinase with a high homology with the focal adhesion kinase, FAK. Pyk2 is activated by chemokines and following TCR stimulation, involving both tyrosine autophosphorylation and Src-dependent phosphorylation. Pyk2 regulates cell migration and platelet aggregation. Our results indicated that CXCL12 promotes the association of SLP-76 with ADAP, Vav1, talin and ZAP-70 in T cells following exposure to CXCL12. Furthermore, CXCL12 stimulated Vav1-Pyk2 association, and we found that Pyk2 can also specifically associate with talin. Our data showed that SLP-76, ADAP and Pyk2 function as controllers of chemokine-stimulated T cell adhesion involving α4β1. Thus, adhesion assays under flow conditions revealed that T cells silenced for SLP-76 or ADAP had a reduction in CXCL12-upregulated attachment to VCAM-1 compared with control siRNA transfectants. On the contrary, T cells silenced for Pyk2 expression showed significantly stronger attachment to VCAM-1. Such different effects in adhesion of cells silenced for SLP-76, ADAP or Pyk2 were not due to alterations in generation of α4β1 high-affinity conformation, but to defects in adhesion strengthening and in spreading mediated by α4β1 following stimulation by chemokines. Hence, we found a reduction in the strength of adhesion and in spreading in ADAP-depleted cells, and a development of higher resistance to detachment and larger spreading on VCAM-1 in cells silenced for Pyk2 compared to control siRNA transfectants. We only could detect minor contribution of SLP-76 in α4β1-mediated cell adhesion strengthening, although we observed a reduction in cellular spreading in SLP-76-depleted cells.Notably, in cells silenced for Pyk2, the increase in the strengthening of adhesion and the spreading was associated with higher activation of Rac. Furthermore, expression of a dominant negative form of Rac1 reduced to control levels the chemokine-upregulated adhesion to VCAM-1 that showed Pyk2-silenced T cells, suggesting that Pyk2 regulate the adhesion mediated by α4β1 that is target of chemokine activation by modulating Rac1 activation. Additionally, depletion of Pyk2 led to rescue of adhesion to VCAM-1 due to ADAP silencing, involving Rac function, suggesting that this GTPase represents a Pyk2 and ADAP common mediator of chemokine-activated, α4β1-dependent T cell adhesion. The third objective focused on studying the role of RGS10 in chemokinestimulated T lymphocyte adhesion mediated by α4β1 and αLβ2 integrins. RGS10 belongs to the regulators of G protein signaling (RGS) family, and function as an accelerator of the GTPase activity of Gα subunits in heterotrimeric G proteins. This activity promotes the return of Gα to its inactive form, which leads to faster termination of G proteindependent signaling. Therefore, RGS proteins control the timing and duration of GPCRdependent signaling leading, which could then regulate the adhesive response dependent of integrins on lymphocytes. RGS10 was found to be expressed in T cells, and we show that RGS10 opposes the chemokine-stimulated signaling that is needed for T cell adhesion mediated by α4β1 and αLβ2. Thus, upregulation of adhesion to α4β1 and αLβ2 ligands in response to CXCL12 and CCL21 was significantly stronger in RGS10-depleted cells than in control transfectants. On the contrary, when RGS10 was overexpressed, stimulation of adhesion by these chemokines was limited. RGS10 transiently associated to Gαi in T cells following exposure to CXCL12, and pertussis toxin blocked chemokineupregulated adhesion to VCAM-1 of both control and RGS10-silenced cells, suggesting that RGS10 is inhibiting the adhesion mediated by α4β1 by repressing Gαi-dependent signaling.Flow chamber adhesion experiments revealed that RGS10 functions principally by promoting cell spreading and strengthening of adhesion mediated by α4β1. Additionally, we showed that chemokine-stimulated Rac1 activation, a process taking place during the strengthening and spreading phases of the adhesion after the integrin activation step, was longer sustained and of higher intensity in RGS10-silenced cells, or inhibited in cells overexpressing RGS10. Importantly, expression of constitutively active Rac1 forms in cells overexpressing RGS10 led to the rescue of CXCL12-stimulated adhesion to VCAM- 1 to levels similar to control transfectants. The regulation of Rac1 activation by RGS10 was most likely a consequence of RGS10-dependent control of Vav1 tyrosine phosphorylation, as it was increased or decreased in cells silenced or overexpressing RGS10, respectively. Moreover, flow chamber adhesion experiments, binding of soluble VCAM-1-Fc, and ICAM-1-Fc, and biochemical analysis revealed a minor involvement of RGS10 in the initial integrin activation phases. Therefore, these results strongly suggest that RGS10 actions mostly oppose the Vav1-Rac1-dependent adhesion strengthening and spreading steps of α4β1-mediated T cell adhesion triggered by chemokines. In addition to repress chemokine-upregulated T cell adhesion dependent on α4β1 and αLβ2, RGS10 also inhibited adhesion-independent cell chemotaxis and cdc42 activation in response to CXCL12. Together, these results indicate that chemokine binding to their receptors triggers Gαi-dependent signaling that leads to changes in molecular associations between Vav1, talin and β1 resulting in early α4β1 activation, but also promotes Gαi association with RGS10. While the initial steps of adhesion are not targeted following RGS10-Gαi association, the consequence of the assembly of this complex may well be the gradual termination of Vav1-Rac1 activation that triggers chemokine-upregulated strengthening of the adhesion mediated by α4β1. In conclusion, in this thesis we have studied the functions of signalling and adapter proteins in chemokine-triggered inside-out signalling leading to integrin activation. Three of these proteins, ZAP-70, SLP-76 and ADAP, positively regulated the adhesion mediated by α4β1 in T cells in response to chemokines, whereas RGS10 and Pyk2 are involved in mechanisms opposing or limiting the stimulation of this adhesion.Peer reviewe

RGS10 restricts upregulation by chemokines of T cell adhesion mediated by α4β1 and αLβ2 Integrins.

9 páginas, 6 figuras -- PAGS nros. 1264-1272Chemokines rapidly and transiently upregulate α4β1 and αLβ2 integrin-mediated adhesion during T lymphocyte extravasation by activating Gα-dependent inside-out signaling. To limit and terminate Gα-mediated signaling, cells can use several mechanisms, including the action of regulator of G protein signaling (RGS) proteins, which accelerate the GTPase activity of Gα subunits. Using human T cells silenced for or overexpressing RGS10, we show in this article that RGS10 functions as an inhibitor of Gαi-dependent, chemokine-upregulated T cell adhesion mediated by α4β1 and αLβ2. Shear stress-dependent detachment and cell spreading analyses revealed that RGS10 action mainly targets the adhesion strengthening and spreading phases of α4β1-mediated cell attachment. Associated with these observations, chemokine-stimulated Vav1–Rac1 activation was longer sustained and of higher intensity in RGS10-silenced T cells, or inhibited in cells overexpressing RGS10. Of importance, expression of constitutively activated Rac1 forms in cells overexpressing RGS10 led to the rescue of CXCL12-stimulated adhesion to VCAM-1 to levels similar to those in control transfectants. Instead, adhesion under flow conditions, soluble binding experiment, flow cytometry, and biochemical analyses revealed that the earlier chemokine-triggered integrin activation step was mostly independent of RGS10 actions. The data strongly suggest that RGS10 opposes activation by chemokines of the Vav1–Rac1 pathway in T cells, leading to repression of adhesion strengthening mediated by α4β1. In addition to control chemokine-upregulated T cell attachment, RGS10 also limited adhesion-independent cell chemotaxis and activation of cdc42. These results identify RGS10 as a key molecule that contributes to the termination of Gα-dependent signaling during chemokine-activated α4β1- and αLβ2-dependent T cell adhesionThis work was supported by Grant SAF2008-00479 from Ministerio de Ciencia e Innovación (to J.T.)Peer reviewe

Chemokine-Induced Zap70 Kinase-Mediated Dissociation of the Vav1-Talin Complex Activates α4β1 Integrin for T Cell Adhesion

Lymphocyte integrins mediate cell arrest on endothelium during immune surveillance after activation by chemokine-stimulated inside-out signals. Here we show that a Vav1-talin complex in T cells is a key target for chemokine-triggered inside-out signaling leading to integrin α4β1 activation. Thus, Vav1 dissociation from talin was required to generate high-affinity α4β1 conformations. Assembly of the Vav1-talin complex required PtdIns(4,5)P2, which was provided by talin-bound phosphatidylinositol phosphate kinase Iγ. Chemokine-promoted Vav1 dissociation from talin followed an initial increase in talin binding to α4β1. This process was dependent on ZAP-70, which binds to and phosphorylates Vav1 in the complex, leading to further α4β1 activation and cell adhesion strengthening. Moreover, Vav1-talin dissociation was needed for Rac1 activation, thus indicating that α4β1 and Rac1 activation can be coupled by chemokine-stimulated ZAP-70 function. Our data suggest that Vav1 might function as a repressive adaptor of talin that must dissociate from α4β1-talin complexes for efficient integrin activation.This work was supported by grant SAF2005-02119 and SAF2008-00479 from Ministerio de Ciencia e Innovación.Peer reviewe

The Cish SH2 domain is essential for PLC-γ1 regulation in TCR stimulated CD8+ T cells

Abstract Cish, participates within a multi-molecular E3 ubiquitin ligase complex, which ubiquitinates target proteins. It has an inhibitory effect on T cell activation mediated by PLC-γ1 regulation, and it functions as a potent checkpoint in CD8+ T cell tumor immunotherapy. To study the structural and functional relationships between Cish and PLC-γ1 during CD8+ T cell activation, we tested mutants of the Cish-SH2 (R107K) and D/BC (L222Q, C226Q) domains. We confirmed that Cish-SH2-specific binding was essential for PLC-γ1 ubiquitination and degradation. This domain was essential for the Cish-mediated inhibition of Ca2+ release upon TCR stimulation. No effect on inhibition of cytokine release was observed with SH2 or D/BC mutants, although the absence of Cish led to an increased release of IFN-γ and TNF-α. Using imaging we showed that Cish was expressed mostly in the cytoplasm and we did not see any Cish clustering at the plasma membrane upon stimulation. We conclude that the Cish-SH2 domain is essential for PLC-γ1 regulation in TCR-stimulated CD8+ T cells

Positive and negative regulation by SLP-76/ADAP and Pyk2 of chemokine-stimulated T-lymphocyte adhesion mediated by integrin α4β1

Stimulation by chemokines of integrin α4β1-dependent T-lymphocyte adhesion is a crucial step for lymphocyte trafficking. The adaptor Vav1 is required for chemokine-activated T-cell adhesion mediated by α4β1. Conceivably, proteins associating with Vav1 could potentially modulate this adhesion. Correlating with activation by the chemokine CXCL12 of T-lymphocyte attachment to α4β1 ligands, a transient stimulation in the association of Vav1 with SLP-76, Pyk2, and ADAP was observed. Using T-cells depleted for SLP-76, ADAP, or Pyk2, or expressing Pyk2 kinase-inactive forms, we show that SLP-76 and ADAP stimulate chemokine-activated, α4β1-mediated adhesion, whereas Pyk2 opposes T-cell attachment. While CXCL12-promoted generation of high-affinity α4β1 is independent of SLP-76, ADAP, and Pyk2, the strength of α4β1-VCAM-1 interaction and cell spreading on VCAM-1 are targets of regulation by these three proteins. GTPase assays, expression of activated or dominant-negative Rac1, or combined ADAP and Pyk2 silencing indicated that Rac1 activation by CXCL12 is a common mediator response in SLP-76-, ADAP-, and Pyk2-regulated cell adhesion involving α4β1. Our data strongly suggest that chemokine-stimulated associations between Vav1, SLP-76, and ADAP facilitate Rac1 activation and α4β1-mediated adhesion, whereas Pyk2 opposes this adhesion by limiting Rac1 activation.This work was supported by grants SAF2011-24022 from Ministerio de Economía y Competitividad, RD12/0036/0061, and S2010/BMD-2314 from Comunidad de Madrid to J.T.Peer Reviewe