TI - RESULTS DUSP6/MKP-3 is an FGF-inducible protein in NIH 3T3 cells . AB - Studies in vertebrate embryos have provided compelling evidence for the involvement of FGFR -mediated signalling in the induction of DUSP6/MKP-3 mRNA expression during early development . However , it is uncertain exactly how this occurs with studies invoking essential roles for both ERK and PI3K in mediating this response . To dissect the molecular mechanisms underlying FGF-inducible DUSP6/MKP-3 expression in a highly tractable system , we screened a panel of mammalian cell lines for expression of the protein . Detectable levels of DUSP6/MKP-3 were seen in HepG2 ( human hepatoma ) cells and mouse NIH 3T3 fibroblasts . In contrast , DUSP6/MKP-3 was absent in Cos-1 , HeLa and HEK-293T ( human embryonic kidney ) cells ( results not shown ) . Because NIH 3T3 cells have been widely used in studies of inducible gene expression , we determined whether levels of DUSP6/MKP-3 are increased following FGF treatment (Figure 1A) . Elevated levels of DUSP6/MKP-3 protein were detected following exposure to FGF2 , FGF4 and FGF8 , and this correlated with increased PHOSphorylation of ERK2 . In contrast , none of the FGF treatments led to PHOSphorylation of either p38 or JNK . We note that DUSP6/MKP-3 is resolved as a doublet in these immunoblots . This reflects the use of alternative translational start sites ( at codons 1 and 14 ) within the DUSP6/MKP-3 mRNA [27] . Exposure of mouse fibroblasts to FGF was shown previously to result in activation of both ERK1/2 and PI3K [28] . Consistent with this , we observed increased PHOSphorylation of both ERK1/2 and the Akt protein kinase , a downstream target of the PI3K pathway , in response to FGF2 . PHOSphorylation of both ERK and Akt was apparent 30 min after addition of FGF2 , reached peak levels after 1 h and declined thereafter ( Figure 1B , upper panels ) . As expected , Akt PHOSphorylation was abolished by LY294002 ( 10 muM ) , a specific inhibitor of PI3K signalling ( Figure 1B , lower panel ) . Elevated levels of DUSP6/MKP-3 protein were detected approx. 1 h after exposure to FGF2 and persisted for atleast 8 h ( Figure 1B , upper panel ) while DUSP6/MKP-3 mRNA levels were increased within 30 min of FGF exposure , reached maximum levels after approx. 90 min and declined thereafter ( Figure 1C ) .