Negative regulation of chemokine receptor signaling and B-cell chemotaxis by p66Shc

L Patrussi,N Capitani,F Finetti,P G Pelicci,O M Lucherini,C T Baldari,E Cannizzaro

doi:10.1038/cddis.2014.44

Abstract

Shc (Src homology 2 domain containing) adaptors are ubiquitous components of the signaling pathways triggered by tyrosine kinase-coupled receptors. In lymphocytes, similar to other cell types, the p52 and p66 isoforms of ShcA/Shc participate in a self-limiting loop where p52Shc acts as a positive regulator of antigen receptor signaling by promoting Ras activation, whereas p66Shc limits this activity by competitively inhibiting p52Shc. Based on the fact that many signaling mediators are shared by antigen and chemokine receptors, including p52Shc, we have assessed the potential implication of p66Shc in the regulation of B-cell responses to chemokines, focusing on the homing receptors CXCR4 (C-X-C chemokine receptor type 4) and CXCR5 (C-X-C chemokine receptor type 5). The results identify p66Shc as a negative regulator of the chemotactic responses triggered by these receptors, including adhesion, polarization and migration. We also provide evidence that this function is dependent on the ability of p66Shc to interact with the chemokine receptors and promote the assembly of an inhibitory complex, which includes the phosphatases SHP-1 (Src homology phosphatase-1) and SHIP-1 (SH2 domain-containing inositol 5'-phosphatase-1), that results in impaired Vav-dependent reorganization of the actin cytoskeleton. This function maps to the phosphorylatable tyrosine residues in the collagen homology 1 (CH1) domain. The results identify p66Shc as a negative regulator of B-cell chemotaxis and suggest a role for this adaptor in the control of B-cell homing.

Highlights

Chemokine receptors orchestrate the sequential steps of lymphocyte homing, that is, arrest, polarization and transendothelial migration, both by triggering the conversion of integrins to their high-affinity conformation for their ligands on endothelial cells (ECs), which results in firm adhesion, and by promoting the cytoskeletal rearrangements required for polarization and migration.[4]
The integrin high-affinity conformational shift triggered by CXCR4 involves Ras-proximate-1 (Rap1), protein tyrosine kinase 2 (Pyk2) and focal adhesion kinase (FAK) activation.[26]
CXCR4 triggers the Lyn, Syk- and PI3K-dependent activation of Rho GTPases through a cascade involving Bruton’s tyrosine kinase (Btk) and phospholipase C, g (PLCg).[27]

Summary

Results

P66Shc inhibits CXCR4- and CXCR5-dependent B-cell adhesion and polarization. B-cell adhesion to ECs is regulated by the integrins lymphocyte function-associated antigen-1 (LFA-1), which interacts with intercellular adhesion molecule-1 (ICAM-1), and very late antigen-4 (VLA-4), which interacts with both VCAM-1 and the ECM component, fibronectin (FN). The ability of p66Shc to affect B-cell polarization and migration in response to CXCL12/CXCL13 suggests that it may be implicated in coupling CXCR4/ CXCR5 to actin polymerization This issue was addressed by confocal microscopy of control and p66Shc-expressing MEC cells plated on ICAM-1/FN in the presence or absence of CXCL12 or CXCL13. The results obtained with the p66Shc mutants suggests that p66Shc may interfere with CXCR4/CXCR5 coupling to Vav by acting as an adaptor to recruit negative regulators of the TK-dependent or the PI3-K-dependent pathways triggered by these receptors To address this issue, p66Shc/p66Shc3F was immunoprecipitated from MEC transfectants expressing the GFP-tagged proteins and probed for the presence of CXCR4 and CXCR5. A basal interaction, which did not increase in response to chemokine stimulation, was observed in cells expressing p66Shc3F (Figure 7a), indicating that p66Shc inhibition of TK signaling involves SHP-1 recruitment to CXCR4/CXCR5 through YYY239/240/Y317

Discussion

Materials and Methods