Immunoinhibitory Adapter Protein Src Homology Domain 3 Lymphocyte Protein 2 (SLy2) Regulates Actin Dynamics and B Cell Spreading

Max Von Holleben,Antje Gohla,Klaus-Peter Janssen,Brian M Iritani,Sandra Beer-Hammer

doi:10.1074/jbc.m110.155184

Abstract

Appropriate B cell activation is essential for adaptive immunity. In contrast to the molecular mechanisms that regulate positive signaling in immune responses, the counterbalancing negative regulatory pathways remain insufficiently understood. The Src homology domain 3 (SH3)-containing adapter protein SH3 lymphocyte protein 2 (SLy2, also known as hematopoietic adapter-containing SH3 and sterile α-motif (SAM) domains 1; HACS1) is strongly up-regulated upon B cell activation and functions as an endogenous immunoinhibitor in vivo, but the underlying molecular mechanisms of SLy2 function have been elusive. We have generated transgenic mice overexpressing SLy2 in B and T cells and have studied the biological effects of elevated SLy2 levels in Jurkat and HeLa cells. Our results demonstrate that SLy2 induces Rac1-dependent membrane ruffle formation and regulates cell spreading and polarization and that the SLy2 SH3 domain is essential for these effects. Using immunoprecipitation and confocal microscopy, we provide evidence that the actin nucleation-promoting factor cortactin is an SH3 domain-directed interaction partner of SLy2. Consistent with an important role of SLy2 for actin cytoskeletal reorganization, we further show that SLy2-transgenic B cells are severely defective in cell spreading. Together, our findings extend our mechanistic understanding of the immunoinhibitory roles of SLy2 in vivo and suggest that the physiological up-regulation of SLy2 observed upon B cell activation functions to counteract excessive B cell spreading.

Highlights

Because commercial antibodies capable of detecting endogenous SLy2 protein are not available, SLy2 was fused to an HA tag to allow for its detection by immunofluorescence and immunoblotting
Appropriate B cell activation, proliferation, and differentiation are essential for adaptive immunity
The recognition of membrane-bound antigen by B cells leads to the formation of an immunological synapse (IS) [29], which is preceded by extensive cytoskeletal rearrangements that mediate cell spreading and subsequent contraction, facilitating antigen gathering by B cells [2]

Summary

Introduction

These results indicate that increased SLy2 expression reduces cell proliferation in an SH3-domain dependent manner and suggest that impaired cell division by SLy2 is a general cellular response because this is observed in both B cells and the HeLa epithelial cell line. The cooperativity of SLy2 and Rac1 signaling in F-actin dynamics was further supported by the enhanced ruffling phenotype of HeLa cells expressing SLy2-WT together with the constitutively active Rac1 point mutant Rac1-V12.

Results

Conclusion