The Adaptor Protein HSH2 Attenuates Apoptosis in Response to Ligation of the B Cell Antigen Receptor Complex on the B Lymphoma Cell Line, WEHI-231

Brantley R Herrin,Alison L Groeger,Louis B Justement

doi:10.1074/jbc.m407690200

Brantley R Herrin, Alison L Groeger + Show 1 more

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https://doi.org/10.1074/jbc.m407690200

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Abstract

Signals transduced by the B cell antigen receptor (BCR) play a central role in regulating the functional response of the cell to antigen. Depending on the nature of the antigenic signal and the developmental or differentiation state of the B cell, antigen receptor signaling can promote either apoptosis or survival and activation. Understanding the molecular mechanisms underlying BCR-mediated apoptosis constitutes an important area of research because aberrations in programmed cell death can result in the development of autoimmunity or cancer. Expression of the adaptor protein hematopoietic Src homology 2 (HSH2) was found to significantly decrease BCR-mediated apoptosis in the murine WEHI-231 cell line. Analysis of signal transduction pathways activated in response to BCR ligation revealed that HSH2 does not significantly alter total protein tyrosine phosphorylation or Ca2+ mobilization. HSH2 does not potentiate the activation-dependent phosphorylation of AKT either. With respect to MAPK activation, HSH2 was not observed to alter the activation of ERK or p38 in response to BCR ligation, but it does significantly potentiate JNK activation. Analysis of processes directly associated with apoptosis revealed that HSH2 inhibits mitochondrial depolarization to a significant degree, whereas it has only a slight effect on caspase activation and poly ADP-ribose polymerase cleavage. BCR-induced apoptosis of WEHI-231 cells is associated with the loss of endogenous HSH2 expression within 12 h, whereas inhibition of apoptosis in response to CD40-mediated signaling leads to stabilization of HSH2 expression. Thus, endogenous HSH2 expression correlates directly with survival of WEHI-231 cells, which supports the hypothesis that HSH2 modulates the apoptotic response through its ability to directly or indirectly promote mitochondrial stability.

Highlights

Normal B lymphocyte homeostasis and immune function are critically dependent on regulatory pathways that control programmed cell death [1,2,3,4]
To confirm that hematopoietic Src homology 2 (HSH2) is capable of inhibiting apoptosis initiated by antigen receptor cross-linking, WEHI-231 cells transduced with empty vector, HSH2, or Bcl-xL were stimulated with polyclonal anti-IgM F(abЈ)2 Ab and assayed for apoptosis by annexin V (ANV) and propidium iodide (PI) staining
To determine whether HSH2 expression decreases the magnitude of inducible protein tyrosine phosphorylation initiated by cross-linking of the B cell antigen receptor (BCR), WEHI231 cells transduced with empty vector or HSH2 were stimulated with polyclonal anti-IgM F(abЈ)2 Ab, after which whole cell lysates were probed with anti-phosphotyrosine (4G10) mAb (Fig. 2A)

Summary

Introduction

Normal B lymphocyte homeostasis and immune function are critically dependent on regulatory pathways that control programmed cell death (apoptosis) [1,2,3,4]. To determine whether HSH2 expression alters the apoptotic response of WEHI-231 cells to stimulation through the BCR, cells expressing empty vector, HSH2, or Bcl-xL were stimulated with polyclonal anti-IgM F(abЈ)2 Ab. Cells were assayed for the induction of apoptosis based on PI staining (Fig. 1A).

Results

Conclusion