Modulación de la actividad transcripcional de NFAT1 por las MAP Quinasas p38 y JNK

Abstract

The -Nuclear Factor of Activated T (NFAT) family of transcription factors regulates the transcription of rnany genes that are active in different cell systems including the inmune, cardiovascular and nervous systems. NFAT activity is modulated by the calcium activated phosphatase clacineurin. As such, calcineurin binds to and dephosphorylates the NFAT N-terminal domain when calcium levels rise, leading to its nuclear translocation and enhancing its transcriptional activity. Once the calcium levels are restored, NFAT is exponed to the cytoplasrn by a process dependent on phosphorylation by different protein kinases.

In the present study, we show that the MAPKs, p38 and JNK, phosphorylate NFATI in vitro, and that they interact with NFATI in T lymphocytes in vivo. These two kinases induced opposing effects when activated in HeLa cells cotransfected with either MKK6+p38 or MEKKI+JNK. While p38 counteracts the calcium-induced nuclear accumulation of NFATI (and hence inhibits NFATI mediated transcription). JNK stimulates the transcriptional activity of this transcription factor.

To further analyze these effects, we developed a method based on mass spectrometry and bidimensional phosphopeptide mapping to determine the phosphorylation sites in NFAT1. Through this approach, we detected at least eight residues within the NFATI regulatory domain that are phosphorylated by JNK in vitro: Sil°, T116 , s136 , S 170 . 5219, S 271i , and T332. Moreover. in vivo mass spectrometry analysis of NFATI-transfected cells indicated that at least five residues are phosphorylated under banal conditions: S99, 5136, 5219, 5 223 , T332 , JNK stimulation induced the phosphorylation of an additional residue (TI1()). The transfection of T cells with a chimeric construct encoding the GAL4 DBD-NFATI (4- 385) protein showed that JNK stimulates the transactivation mediated by NFATI, an effect that is inhibited by JNK dominant-negative variants. Likewise, mutation of the phosphorylation sites identified revealed that T'' S 17° were critica] for the transactivation of NFAT1 by JNK. In addition, clustered mutations of the conserved S motifs in NFATI showed that SPI and SP2, but not SP3, were also important in inducing the transactivation of this factor. These findings indicate that the transcriptional activity of NFAT1 is up-regulated by JNK, further suggestin g that the physiological role of JNK mediated phosphorylation of NFATs varíes between different family members.

Altogether, these results demonstrate that the MAPKs p38 and JNK are involved in the rnodulation of NFATI activation, regulating its subcellular localization and its transcriptional activity, respectively.