Figure 8. Schematic diagram demonstrating how CD98 regu- lates cell proliferation. Overexpression of CD98 results in increased cell proliferation which is dependent on MAPK signaling mediated by the cytoplasmic (CT) domain and increases amino acid transport which is dependent on the extracellular (EC) domain. CD98 overexpression increases mTOR signaling but this has no effect on cell proliferation. doi:10.1371/journal.pone.0040026.g008 acids. It was surprising there was no increase in Akt activation, as it was recently reported that amino acids can activate mTORC2 in a PI3K/Akt-dependent manner [41]. Furthermore, this result effectively rules out the involvement of mTORC2 in the proliferative response of CD98/Lat-1 cells. A marked increase in Erk was present in the amino acid deprived CD98/Lat-1 cells. This is one of the key mechanisms whereby cells can adapt to stress induced by amino acid deprivation as sustained Erk activation increases p21(cip1/waf1) which blocks cell cycle progression and contributes to activation of the GCN2-eIF2a-ATF4 survival pathway [42–44]. Erk phosphorylation was increased when CD98/Lat-1 and IMCD cells were treated with amino acids and this was accompanied by increased p38 MAPK signaling. Inhibiting these pathways individually decreased cell proliferation and there was an additive effect of inhibiting both pathways together, suggesting that they activate proliferation independently from each other. Amino acid-dependent Erk and p38 MAPK activation has been shown to alter signaling of the MAPK- activated protein kinases such as p90 ribosomal S6 kinase (RSK), mitogen-and-stress-activated kinase (MSK) and possibly MAPK- interacting kinases (MNK) [45,46]. Because these multifunctional kinases regulate numerous cellular processes including cell proliferation, this is the likely mechanism whereby CD98- dependent amino acid transport regulates cell proliferation in collecting duct cells. We originally hypothesized that the increased proliferation in CD98/Lat-1 cells, where there was markedly increased amino acid transport, would lead to activation of the mTOR pathway. As expected when starved IMCD cells were treated with serum or amino acids, there was phosphorylation of the two best characterized targets of mTOR, 4EBP1 and S6K1 and this was markedly increased in CD98/Lat-1 cells. These effects were readily reversed by Rapamycin and the mTOR kinase antagonist PP242 a more effective ATP-competitive inhibitor that targets both mTORC1 and mTORC2 [47]. In addition, phosphorylation of both 4EBP1 and S6K1 was inhibited by the PI3K inhibitor LY294002 that is now known to inhibit mTOR as well as class III PI3K hVps34 [48,49]. However, despite marked activation of the mTOR pathway, inhibiting it did not alter cell proliferation. Thus CD98 Regulates Proliferation by Activating MAPKs only the Erk and p38 MAPK pathways are important for the proliferative response of the CD98/Lat-1 IMCD cells. Cyclin D1 was undetectable in amino acid deprived cells but its expression was restored upon amino acid treatment with an earlier recovery in CD98/Lat-1 cells. By contrast, inhibitor studies showed that Cyclin D1 was regulated by all three pathways activated by amino acid treatment. Treatment with Rapamycin, PP242 as well as LY294002 led to drastic inhibition of Cyclin D1 expression, although as mentioned previously, these inhibitors had no effect on cell proliferation following amino acid supplementa- tion. On the other hand, treatment with the MEK inhibitor U0126 and the p38 MAPK inhibitor SB203580 effectively suppressed Cyclin D1 expression as well as inhibiting proliferation. Cyclin D1 is a key target of amino acids in hepatocytes and its overexpression can overcome cell cycle arrest induced by non- essential amino acid deprivation under mitogenic conditions [50]. Several studies have suggested that Cyclin D1 can induce cell growth and protein synthesis in addition to its well-known effects on proliferation [51,52]. In MCF7 cells, 4EBP1 was described as a key modulator of Cyclin D1 therefore coupling its expression to mTORC1 signaling [53]. Although Cyclin D1 is downstream of all three pathways, it does not seem to be a rate limiting factor in the proliferative response to amino acids in our system, suggesting that Erk and p38 MAPK are affecting cell cycle progression and proliferation by controlling expression of cyclin-dependent kinase inhibitors such as p21 and p27 [44,54,55]. In conclusion, our studies demonstrate that overexpression of CD98hc by renal tubular epithelial cells increases cell proliferation by inducing signaling via its cytoplasmic tail and increasing CD98- dependent amino acid transport. These data have ramifications if CD98hc is viewed as a therapeutic target in oncological and immunological diseases. To this end CD98 has clearly been shown to play a role in the pathogenesis of these diseases in mice [27,28,30] and there is strong correlative data linking CD98 expression and tumor metastasis in humans [56–61]. Material and Methods Antibodies and Other Reagents The hybridoma cell line 4F2 (C13) (anti-CD98) was purchased from American Type Culture Collection (ATCC, Manassas, VA) and the anti-CD98 (4F2) antibody was purified from conditioned medium from the hybridoma cell lines by protein A affinity chromatography. We raised a polyclonal antibody to Lat-1 directed against the peptide sequence RHRKPEL found in the N-terminus of human Lat-1. This antibody has been commercial- ized by Capralogics (Hardwick, MA, USA). The specificity of this antibody was verified with another Lat-1 antibody (clone 3B11- 1D, Serotec, Oxford, UK). Antibodies to phosphorylated and total Erk1/2, p38 MAPK, Akt , p70S6K, 4EBP1 and Cyclin D1 were all from Cell Signaling Technology (Beverly, MA). LY294002, U0126, SB203580, rapamycin and PP242 were all purchased from Calbiochem (EMD Biosciences, La Jolla, CA). Culture media were from Mediatech (Manassas, VA) and the selection antibiotics Geneticin and Zeocin were from Invitrogen (Carlsbad, CA). 3H- Isoleucine (Ile) (77 Ci/mM) was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). 3H-Thymidine (6.7 Ci/ mmol) was purchased from MP Biomedicals (Irvine, CA). Plasmids and Cell Lines Inner medullary collecting duct (IMCD) cells, which have been previously described, were used in all experiments [62]. IMCD cells expressing either CD98 or deletion mutants of CD98 (CD98-77, CD98-82 and CD98-87) were previously described PLoS ONE | www.plosone.org 9 June 2012 | Volume 7 | Issue 6 | e40026