TI - Discussion . AB - The present study demonstrates that ESAT-6 modulated the ERK1/2 group of MAP kinase . We also found that this modulation was achieved by inhibition of PHOSphorylation of ERK1/2 in the nucleus . However , the PHOSphorylation of another MAP kinase p38 was not affected by ESAT-6 . Nevertheless , LPS , a general macrophage activator [39-47] triggered PHOSphorylation of ERK1/2 in both cytoplasm and the nucleus . This showed that the limited activation of ERK1/2 in the nucleus was specific for ESAT-6 stimulation . Costimulation of cells with LPS and ESAT-6 dampened the ERK1/2 phosphorylaTION in the nucleus compared to that obtained with LPS alone ; clearly ESAT-6 was exerting a strong inhibitory effect on the PHOSphorylation of ERK1/2 in the nucleus . Our finding that the treatment of cells with Na3VO4 , a tyrosine phosphatase inhibitor [48] along with ESAT-6 caused pERK1/2 to appear in the nucleus indicated that there was some phosphatases activity in the nucleus that was triggered upon stimulation with ESAT-6 . Moreover , when this phosphatase activity was suppressed by Na3VO4 , the pERK1/2 reappeared in the nucleus . The results of kinase assay further corroborated our observations from western blotting that PHOSphorylation of ERK1/2 was concomitant with its activation . Measurement of phosphatase activity associated with ERK1/2 in the nucleus showed that there was an increase in this activity over the given time period ; this finding was consistent with our observation that following treatment with both ESAT-6 and a phosphatase inhibitor (Na3VO4) there was an increase in PHOSphorylation of ERK1/2 . It was already established that ERK1/2 after getting PHOSphorylated in cytoplasm translocates to the nucleus [37] ; therefore at zero minute , we observed little pERK1/2 in the nucleus ( Fig 1C ) . Our findings tend to suggest that although with increase in the ERK1/2 phosphorylaTION in the cytoplasm of the ESAT-6 -stimulated cells , pERK1/2 must have migrated to the nucleus , but increasing phosphatase activity in the nucleus , again associated with ESAT-6 stimulation , dePHOSphorylated the pERK1/2 coming from the cytoplasm ; therefore no pERK1/2 was detectable in the nuclear extract . Since MAP kinases undergo rapid turnover in the nucleus , the levels of total ERK1/2 in the nucleus remained constant over the experimental time period . The c-myc is one of the early response genes that encode a transcription factor c-Myc , which is a key regulator of cell proliferation and apoptosis . Since c-myc expression was reported to occur through Ras/Raf/MEK/ERK pathway [24,49-52] , we studied the effect of ESAT-6 on c-myc expression in RAW264.7 cells . ESAT-6 itself did not have any effect on c-myc expression over the basal level . However the LPS induced c-myc expression was found to be downregulated by ESAT-6 compared to LPS stimulation alone . Again treatment with ESAT-6 along with 1 mM Na3VO4 increased the level of c-myc compared to that observed with ESAT-6 alone while Na3VO4 alone did not have any effect on c-myc levels . These results can be explained by the dampening of LPS -induced ERK1/2 PHOSphorylation in the nucleus by ESAT-6 . As noted above , treatment with Na3VO4 along with ESAT-6 resulted in an increased level of ERK1/2 activation in the nucleus compared to ESAT-6 alone . This differential activation of ERK1/2 pathway resulted in differential c-myc expression . To further confirm the role of ERK1/2 pathway in c-myc expression , we determined c-myc expression in the presence of MEK-1 inhibitor PD98059 [53, 54] and p38 MAP kinase inhibitor SB203580 [55, 56] along with Na3VO4 and ESAT-6 . PD98059 downregulated c-myc levels while SB203580 did not have any effect on c-myc levels . The activation of ERK1/2 pathway in nucleus upon treatment with Na3VO4 and ESAT-6 was abrogated by PD98059 and hence c-myc levels were downregulated . Since SB203580 did not have any effect on c-myc expression , p38 MAP kinase was not involved in the gene expression . It confirmed the earlier observations of p38 PHOSphorylation from western blotting where there was no inhibition in p38 activation in cytoplasm or nucleus by ESAT-6 . Although there are reports that CFP-10 forms a 1 : 1 complex with ESAT-6 [57] ; however other studies [58] have shown that there is discordance between secretion of CFP-10 and ESAT-6 . Okkels and colleagues [59] have shown that there are as many as 8 different forms of ESAT-6 and that the acetylation of ESAT-6 was required for complexation with CFP-10 . Another study has shown that ESAT-6 as well as the CFP-10 : ESAT-6 complex inhibited the PI-3 kinase-Akt signaling , indicating that the active component involved in downregulating the macrophage signaling was the ESAT-6 [60] . Our studies with CFP-10 and CFP-10 : ESAT-6 complex did not show any inhibition of the ERK1/2 PHOSphorylation in cytoplasm or nucleus of the RAW264.7 cells ( see Additional file 1 ) . It has also been shown that ESAT-6 binds to the Toll-like receptor-2 ( TLR-2 ) and not TLR-4 on the surface of RAW264.7 macrophages , and causes inhibition of activation of transcription factors NF-kappaB and Interferon regulatory factors ( IRFs ) through the Akt kinase pathway [60] . Our studies suggest yet another mechanism , viz. , modulation of the ERK arm of the MAP kinase pathway , by which ESAT-6 could bring about deactivation of the host cell .