Protein Kinase Cζ Phosphorylates Nuclear Factor of Activated T Cells and Regulates Its Transactivating Activity

Manuel Fresno,Miguel A. Íñiguez,Belén San-Antonio

doi:10.1074/jbc.m106983200

Abstract

Although several isoforms of protein kinase C (PKC) have been implicated in T lymphocyte activation events, little is known about their mode of action. To address the role of PKCzeta in T cell activation, we have generated Jurkat T cell transfectants expressing either the wild type (J-PKCzeta) or "kinase-dead" mutant (J-PKCzeta(mut)) versions of this protein. Expression of PKCzeta but not PKCzeta(mut) increased transcriptional activation mediated by the NF-kappaB or nuclear factor of activated T cells (NFAT). PKCzeta cooperates with calcium ionophore and with NFAT1 or NFAT2 proteins to enhance transcriptional activation of a NFAT reporter construct. However, neither NFAT nuclear translocation nor DNA binding were in J-PKCzeta cells. Our results show that PKCzeta enhanced transcriptional activity mediated by Gal4-NFAT1 fusion proteins containing the N-terminal transactivation domain of human NFAT1. Interestingly, PKCzeta synergizes with calcineurin to induce transcriptional activation driven by the NFAT1 transactivation domain. Co-precipitation experiments showed physical interaction between PKCzeta and NFAT1 or NFAT2 isoforms. Even more, PKCzeta was able to phosphorylate recombinant glutathione S-transferase-NFAT1 (1-385) protein. These data reveal a new role of PKCzeta in T cells through the control of NFAT function by modulating the activity of its transactivation domain.

Highlights

The serine/threonine protein kinase C (PKC)1 family is constituted by several isoenzymes that have been grouped into three subfamilies according to their activation profile and their regulatory properties [1, 2]
We have recently described the involvement of PKC␨ in the regulation of NF-␬B transactivation in T cells [24], but little is known about the effect of this atypical PKC on the function of other transcription factors known to be essential for T cell activation as the nuclear factor of activated T cells (NFAT)
We describe for the first time the regulation of NFAT activity by PKC␨ in T lymphocytes through a novel mechanism

Summary

Introduction

The serine/threonine protein kinase C (PKC) family is constituted by several isoenzymes that have been grouped into three subfamilies according to their activation profile and their regulatory properties [1, 2]. PKC␨ has been involved in the regulation of several critical pathways for cell survival, proliferation, and differentiation, especially those involving NF-␬B and activating protein-1 (AP-1) transcription factors [13,14,15], and it is likely one of the most important mediators of phosphoinositide 3-kinasedependent responses. We have recently described the involvement of PKC␨ in the regulation of NF-␬B transactivation in T cells [24], but little is known about the effect of this atypical PKC on the function of other transcription factors known to be essential for T cell activation as the nuclear factor of activated T cells (NFAT) This transcription factor plays a prominent role in T cell activation by regulating the expression of a large number of inducible genes encoding cytokines and cell surface receptors that are essential for a productive immune response [31, 32]. We propose that phosphorylation of the N-terminal transactivation domain of NFAT by PKC␨ might mediate modulation of gene expression

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Results

Conclusion