CD98 Regulates Proliferation by Activating MAPKs Figure 3. CD98 regulates serum dependent cell proliferation by increasing Erk and p38 MAPK signaling. (A, C) CD98hc expression increases MAPK, Akt and mTor signaling in response to serum. Subconfluent cells were serum deprived for 24 hours then treated with complete medium. Lysates were prepared at the time points indicated and analyzed by western blot with the antibodies indicated. (B, D) MAPK regulates CD98hc-dependent IMCD cell proliferation. Cells seeded onto 96-well plates were serum deprived for 24 hours, pretreated with inhibitors prior to addition of 3H-Thymidine in complete medium as described in Experimental Procedures. U+SB denotes concomitant treatment with the MEK inhibitor U0126 (U) and the p38 MAPK inhibitor SB203580 (SB). (*) indicates statistically significant differences (p,0.05) between treated and untreated cells. (**) denotes statistically significant differences between U+SB versus either inhibitor alone. doi:10.1371/journal.pone.0040026.g003 (Figure 5A) and we were able to generate multiple clones of IMCD cells (3 shown) which overexpressed Lat-1. We subsequently overexpressed CD98 or the truncation mutants of CD98-77, CD98-82 and CD98-87 into the Lat-1 overexpressing cells and sorted for equal expression by flow cytometry (data not shown). To verify that Lat-1 heterodimerized with CD98hc or the mutants, we immunopreciptated CD98hc from the different IMCD cell populations and immunoblotted the precipitates with antibodies directed against either CD98hc (Figure 5B, upper panel) or Flag (Figure 5B, lower panel). It is clear that Lat-1 was part of the HAT complex in non-reducing conditions and that it became disasso- ciated from CD98hc under reducing conditions. Thus, the HAT complex was formed by CD98hc and all the mutants. We initially examined the effect of overexpressing Lat-1 in the IMCD cells. Like CD98 cells, the Lat-1 expressing cells had a small but significant (40%) increase in isoleucine transport when compared to IMCD cells, while expressing both CD98 and Lat-1 resulted in an 2 fold increase in isoleucine transport (Figure 5C). Consistent with this increase in amino acid transport there was a 40% increase in proliferation of IMCD/Lat-1 cells relative to wildtype IMCD cells in low serum medium. Furthermore, cells overexpressing Lat-1 increased CD98 cell proliferation by an additional 38% such that these cells proliferated 2.4 fold more than IMCD cells (Figure 5D). Similar to CD98 cells, expressing the Lat-1 transporter increased cell proliferation of cells of CD98- 77, CD98-82 and CD98-87 cells by 40% in low serum medium (Figure 5E). These studies further confirm that CD98-dependent amino acid transport increased proliferation by a mechanism that does not require the CD98hc cytoplasmic tail. Increased Transport of Amino Acids by CD98 Induces Activation of Erk and p38 MAPK Pathways which Enhance Cell Proliferation Due to the observation that overexpression of CD98 increased proliferation of cells grown in complete medium with serum by increasing amino acid transport, we developed conditions (see methods) to define the mechanisms whereby CD98 regulates proliferation in the absence of serum. When CD98 cells were placed in DMEM that did not contain serum they proliferated 60% more than IMCD cells as measured by thymidine PLoS ONE | www.plosone.org 4 June 2012 | Volume 7 | Issue 6 | e40026