Protein Phosphatase 2A Regulates Interleukin-2 Receptor Complex Formation and JAK3/STAT5 Activation

Jeremy A Ross,Hanyin Cheng,Zsuzsanna S Nagy,Jeffrey A Frost,Robert A Kirken

doi:10.1074/jbc.m109.053843

Abstract

Reversible protein phosphorylation plays a key role in interleukin-2 (IL-2) receptor-mediated activation of Janus tyrosine kinase 3 (JAK3) and signal transducer and activator of transcription 5 (STAT5) in lymphocytes. Although the mechanisms governing IL-2-induced tyrosine phosphorylation and activation of JAK3/STAT5 have been extensively studied, the role of serine/threonine phosphorylation in controlling these effectors remains to be elucidated. Using phosphoamino acid analysis, JAK3 and STAT5 were determined to be serine and tyrosine-phosphorylated in response to IL-2 stimulation of the human natural killer-like cell line, YT. IL-2 stimulation also induced serine/threonine phosphorylation of IL-2Rbeta, but not IL-2Rgamma. To investigate the regulation of serine/threonine phosphorylation in IL-2 signaling, the roles of protein phosphatase 1 (PP1) and 2A (PP2A) were examined. Inhibition of phosphatase activity by calyculin A treatment of YT cells resulted in a significant induction of serine phosphorylation of JAK3 and STAT5, and serine/threonine phosphorylation of IL-2Rbeta. Moreover, inhibition of PP2A, but not PP1, diminished IL-2-induced tyrosine phosphorylation of IL-2Rbeta, JAK3, and STAT5, and abolished STAT5 DNA binding activity. Serine/threonine phosphorylation of IL-2Rbeta by a staurosporine-sensitive kinase also blocked its association with JAK3 and IL-2Rgamma in YT cells. Taken together, these data indicate that serine/threonine phosphorylation negatively regulates IL-2 signaling at multiple levels, including receptor complex formation and JAK3/STAT5 activation, and that this regulation is counteracted by PP2A. These findings also suggest that PP2A may serve as a therapeutic target for modulating JAK3/STAT5 activation in human disease.

Highlights

Interleukin-2 (IL-2)3 is a key regulator of normal immune function and acts on a variety of lymphoid cell types including
To determine whether STAT5a/b phosphorylation is regulated by phosphatase types 1 (PP1) or PP2A activity, phosphoamino acid analysis was performed on signal transducer and activator of transcription 5 (STAT5) proteins isolated from YT cells that were metabolically labeled with [32P]orthophosphate and treated without or with the PP1/ PP2A inhibitor calyculin A (CA) for 60 min prior to stimulation with IL-2 for 10 min
The results presented provide direct evidence that serine/threonine phosphorylation functions as an important negative regulator of IL-2 receptor signaling in human lymphocytes and that this is counteracted by the actions of PP2A

Summary

Introduction

Interleukin-2 (IL-2)3 is a key regulator of normal immune function and acts on a variety of lymphoid cell types including. These data indicate that serine/threonine phosphorylation negatively regulates IL-2 signaling at multiple levels, including receptor complex formation and JAK3/STAT5 activation, and that this regulation is counteracted by PP2A.

Results

Conclusion