CD98 Regulates Proliferation by Activating MAPKs Figure 2. The cytoplasmic tail of CD98 is required for serum-dependent cell proliferation. (A) Schematic representation of the CD98 constructs used for CD cell transfection. CD, cytoplasmic domain; TM, transmembrane domain; EC, extracellular domain. (B) Full-length CD98hc and its truncation mutants are expressed in membrane fractions of IMCD cells. IMCD cells expressing CD98hc or the different truncation mutants were fractionated into cytoplasmic (Cyt) Triton X-100–soluble and – insoluble membrane fractions (MF). These fractions were subjected to SDS-PAGE and immunoblotted with an antibody directed against human CD98hc. (C) CD98hc induces cell proliferation in serum. IMCD were plated onto 96-well plates, serum deprived for 24 hours then treated for 16 hours with 3H-Thymidine in media containing 1% serum. 3H-Thymidine incorporation was determined as described in Experimental Procedures. Values are the mean 6 s.e.m. of a representative experiment performed in eight replicates. (*) indicates statistically significant differences (p,0.05) between CD98 and deletion mutants. (**) denotes a statistically significant difference between CD98 and IMCD. (D) Time course of Cyclin D1 expression. Sub-confluent cells were serum deprived for 24 hours and then treated with complete medium. Lysates were isolated at the time points indicated and analyzed by SDS-PAGE. doi:10.1371/journal.pone.0040026.g002 activated [18,26], there was no change in p-Akt signaling between the wild type and CD98 cells. There was however earlier and more pronounced Erk and p38 MAPK activation in the CD98 cells (Figure 3A). To define whether this increased signaling altered cell proliferation, thymidine incorporation assays were performed in low serum medium (1%) in the presence and absence of the PI3K inhibitor LY294002, the MEK inhibitor U0126 and the P38 MAPK inhibitor SB203580 (Figure 3B). IMCD cell proliferation was decreased by 40% with the MEK inhibitor; however CD98 cell proliferation was significantly decreased by both the MEK (40%) and p38 MAPK (25%) either added alone or in combination. Because overexpression of CD98 increases amino acid transport and amino acids are well known to stimulate the mTor pathway, we defined the role of this pathway in modulating proliferation of CD98 cells. We assessed whether the mTor pathway was activated by defining the phosphorylation state of p70S6K and 4EBP1, both of which are downstream of mTorc1. Adding complete medium with 10% serum stimulated phosphorylation of p70S6K only in CD98 cells and, although the phosphorylation pattern of 4EBP1 was similar in both cell lines, there was a more robust signal in CD98 cells 30 minutes after stimulation (Figure 3C). Despite increased mTOR downstream signaling in CD98 cells, addition of the mTor inhibitors Rapamycin or PP242 did not alter cell proliferation (figure 3D). These results suggest that proliferation of IMCD cells, which is increased in cells overexpressing CD98, is dependent on Erk and p38 MAPK but not mTor or PI3K activation. IMCD Cell Proliferation is Increased in Response to CD98- dependent Enhanced Amino Acid Transport CD98hc interacts with the Lat transporters via its extracellular domain and increases CD98-dependent amino acid transport. To define the role of amino acid transport by CD98 on cell proliferation we generated IMCD cells that overexpressed Cless, a previously defined mutant of CD98hc where the two cysteines in the extracellular domain are mutated making it unable to interact with the HAT light chains [36]. This population of cells expressed Cless at the same level as CD98 as verified by flow cytometry (data not shown). As expected, overexpressing CD98 alone caused a 40% increase in isoleucine transport relative to IMCD cells which was not seen in the Cless cells (Figure 4A). When proliferation in complete medium with 1% serum was determined in the Cless IMCD cells, there was only a 50% increase, while there was over a 2 fold increase in the CD98 cells compared to IMCD cells (Figure 4B). These data suggest that the extracellular domain of CD98 increased proliferation in the CD98 cells by inducing additional amino acid transport. To further explore the effects of amino acid transport of HATs on IMCD cell proliferation, we generated IMCD cells that overexpressed the amino acid transporter Lat-1 in conjunction with CD98hc. We utilized a Flag-tagged Lat-1 for these over- expression studies. Lat-1 is endogenously expressed in IMCD cells PLoS ONE | www.plosone.org 3 June 2012 | Volume 7 | Issue 6 | e40026