CD98 Regulates Proliferation by Activating MAPKs Figure 7. mTOR pathway is activated by amino acids but does not affect amino acid-dependent proliferation. (A) Amino acid treatment increases mTor signaling in CD98/Lat-1 cells. Cells were serum deprived for 24 hours then subjected to amino acid deprivation for an additional two hours (time 0). Cells were then treated with serum free media containing a full complement of amino acids. Lysates were isolated at the time points indicated and subjected to SDS-PAGE. Immunoblots were probed with phos-p70S6K (Thr389) as well as antibodies recognizing the multiple phosphorylated forms of 4EBP1. (B) mTor pathways do not regulate amino acid-dependent proliferation of CD98/Lat-1 cells. Cells were plated onto 96-well plates, serum deprived for 24 hours then deprived of amino acids for an additional 2 hours and pretreated with Rapamycin (100 nM) and PP242 (2.5 mM) prior to addition of 3H-Thymidine in serum free media with a full complement of amino acids for an 8 hour pulse. 3H- Thymidine incorporation was determined as described in Experimental Procedures then. Values are the mean 6 s.e.m. of a representative experiment performed in eight replicates. (C and D) Inhibitors of mTor, MAPK and PI3 kinase were effective in inhibiting amino acid-dependent signaling. (C) Cells were serum deprived for 24 hours then subjected to amino acid deprivation for an additional two hours (time 0) and pretreated with Rapamycin and PP242 . Cells were treated with serum free media containing a full complement of amino acids in the presence of inhibitors. Lysates were isolated after 2hours of amino acid treatment and subjected to SDS-PAGE. Immunoblots were probed with phos-p70S6K (Thr389) as well as an antibody recognizing the hyperphosphorylated forms of 4EBP1. (D) Cells were treated as described in C in the presence of LY294002, SB 203580 and U0126, all at 10 mM. Lysates were isolated after two hours of amino acid treatment in the presence of inhibitors and subjected to SDS-PAGE. doi:10.1371/journal.pone.0040026.g007 regions more distal to the integrin interacting site of the CD98hc tail are required to mediate these effects. We demonstrate that overexpression of CD98hc resulted in increased activation of Erk, p38 MAPK and p70S6 kinase following treatment of cells with medium containing serum; however proliferation was only dependent on activation of Erk and p38 MAPK. These observations contrast with the cell signaling pathways modulated by CD98 following integrin ligation where FAK [18,26], PI3-kinase [18,23,26] and Rac [23] but not Erk [26] were activated and regulated cell adhesion, migration and transformation of different cell types. Thus CD98 controls different pathways in the setting of integrin ligation and growth factor stimulation, which in turn modulate different cell functions. Our data clearly demonstrates that in the setting of overex- pression of CD98hc, cell proliferation is regulated by the transport of amino acids in addition to mechanisms mediated by the cytoplasmic tail which may, at least in part, require the interaction of CD98hc with integrins. This effect was even more obvious when we overexpressed Lat-1, which resulted in more significant amino acid transport than only overexpressing CD98hc. These results suggest that overexpressing CD98hc increases the surface expres- sion of the Lats in IMCD cells, with consequent increased amino acid transport and cell proliferation. This is further emphasized when we co-express both the CD98hc and Lat-1. Our results contrast with studies on lymphocytes and vascular smooth muscle where only the interaction with integrins was required for the increased proliferative response. However they are consistent with multiple other studies where cellular proliferation is affected when the Lat amino acid transporters are overexpressed, decreased or when their function is inhibited [39,40]. A potential reason for the differences in our findings and those in the lymphocytes and vascular smooth muscle cells are that our studies are performed on cells where CD98 is overexpressed, while those are on cells where CD98hc is deleted. Furthermore epithelial cells in the kidney have some of the highest expression levels of CD98 in the body which may potentiate their importance for cell proliferation and survival. Increased Erk and p38 MAPK but not Akt activation was observed in serum starved CD98/Lat-1 cells treated with amino PLoS ONE | www.plosone.org 8 June 2012 | Volume 7 | Issue 6 | e40026