Nuclear factor of activated T3 is a negative regulator of Ras-JNK1/2-AP-1-induced cell transformation

The c-jun-NH₂-kinases (JNK) play a critical role in tumor promoter-induced cell transformation and apoptosis. Here, we showed that the nuclear factor of activated T3 (NFAT3) is phosphorylated by JNK1 or JNK2 at Ser²¹³ and Ser²¹⁷, which are located in the conserved SP motif. The transactivation domain of NFAT3 is found between amino acids (aa) 113 and 260 and includes the phosphorylation targets of JNK1 and JNK2. NFAT3 transactivation activity was suppressed in JNK1^-/- or JNK2 ^-/- mouse embryonic fibroblast (MEF) cells compared with wild-type MEF cells. Moreover, a 3xNFAT-luc reporter gene assay indicated that NFAT3 transcriptional activity was increased in a dose-dependent manner by JNK1 or JNK2. Double mutations at Ser²¹³ and Ser²¹⁷ suppressed NFAT3 transactivation activity; and SP600125, a JNK inhibitor, suppressed NFAT3-induced 3xNFAT-luciferase activity. Knockdown of JNK1 or JNK2 suppressed foci formation in NIH3T3 cells. Importantly, ectopic expression of NFAT3 inhibited AP-1 activity and suppressed foci formation. Furthermore, knockdown of NFAT3 enhanced Ras-JNK1 or JNK2-induced foci formation in NIH3T3 cells. Taken together, these results provided direct evidence for the anti-oncogenic potential of the NFAT3 transcription factor.