Specific outcomes upon activation of the c-Jun N-terminal kinase (JNK) pathway 
critically depend on the intensity and duration of signal transmission. 
Dual-specificity phosphatases (DUSPs) play a very important role in these events 
by modulating the extent of JNK phosphorylation and activation and thus 
regulating cellular responses to stress. M3/6 (DUSP8) is one of the 
dual-specificity protein phosphatases with distinct specificity towards JNK. It 
has been shown that M3/6 itself is phosphorylated by JNK upon stimulation with 
arsenite, but the role of this phosphorylation has not been investigated. In 
this study, we mapped JNK-induced phosphorylation sites on M3/6 using mass 
spectrometry. Phosphorylated residues Ser 515, Thr 518 and Ser 520 were 
identified and site-directed mutagenesis was employed to investigate their role. 
Upon arsenite stimulation, M3/6 mutated at these sites exhibited decreased 
phosphorylation compared to the wild-type protein. No difference was observed in 
terms of the enzyme's in vitro phosphatase activity, its substrate specificity 
towards JNK isoforms, its interactions with JNK and the scaffold family of 
JNK-interacting proteins (JIPs), its stability or its subcellular localization. 
Interestingly, expression of M3/6 phosphorylation mutants delayed the 
time-course of JNK phosphorylation and activation by arsenite. We propose that 
phosphorylation of the M3/6 phosphatase by JNK in response to stress stimuli 
results in attenuation of phosphatase activity and acceleration of JNK 
activation.