CD98 Regulates Proliferation by Activating MAPKs 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) compared to IMCD cells. (**) denotes a statistically significant difference between CD98 and CD98/Lat-1. (E) Cells expressing CD98 deletion mutants and Lat-1 proliferated less than CD98/Lat-1 cells. IMCD cells overexpressing Lat-1 were transfected with either CD98 or CD98 deletion mutant constructs described in Fig.1. Thymidine incorporation was determined as described above. Values are the mean 6 s.e.m. of a representative experiment performed in eight replicates. (*)indicates statistically significant differences (p,0.05) between double transfectants and cells transfected with CD98 or deletion mutants only. doi:10.1371/journal.pone.0040026.g005 integrin interactions are sufficient for CD98 to regulate prolifer- ation in these cells because they do express b3 integrins (data not shown). A more likely explanation for the requirement of CD98hc for IMCD cell survival is that CD98 also facilitates critical aspects of amino acid transport. This is consistent with the observation that mice expressing CD98hc that lacks an extracellular domain die between E7.5 and E 9.5 [38]. CD98 is required for proliferation of B and T lymphocytes [27,28], vascular smooth muscle cells [29] and intestinal epithelial cells [30] in the setting of different in vivo injury models. In the lymphocyte and vascular smooth muscle studies, an interaction between integrins and CD98hc were shown to be required for the proliferative response. Our deletion mutant results demonstrate that the CD98hc cytoplasmic tail is required for these proliferative responses and the critical amino acids required for signaling are distal to the membrane proximal 5 amino acids. We previously demonstrated that an interaction between CD98hc and b integrins requires either the transmembrane domain or the proximal part of the cytoplasmic tail of CD98hc [26]. Thus although an integrin/ CD98hc interaction may be necessary to promote proliferation, Figure 6. CD98hc/Lat-1-dependent amino acids transport increases cell proliferation mediated by Erk- and p38 MAPK. (A) CD98hc/ Lat-1-dependent amino acids transport increases cell proliferation. Cells were plated onto 96-well plates, serum deprived for 24 hours then deprived of amino acids for 2 hours. 3H-Thymidine was then added 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. Values are the mean 6 s.e.m. of a representative experiment performed in eight replicates. (*)indicates statistically significant differences (p,0.05) compared to IMCD cells. (**) denotes a statistically significant difference between CD98 and CD98/Lat-1. (B, C) treatment of CD98/Lat-1 cells with amino acids increases cyclin D1 expression and MAPK activation. 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 and lysates were isolated at the time points indicated. Lysates were analyzed by SDS- PAGE and immunoblots were performed with the antibodies against cyclin D1 (B) and Erk, Akt and p38 MAPK. b-Actin is a loading control (C). (D) MAPK increases CD98/Lat-1 dependent amino acid induced proliferation. 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 inhibitors 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. Values are the mean 6 s.e.m. of a representative experiment performed in eight replicates. (*) indicates statistically significant differences (p,0.05) compared to untreated cells. U+SB denotes concomitant treatment with the MEK inhibitor U0126 (U) the p38 MAPK inhibitor SB203580 (SB). (**) denotes statistically significant differences between U+SB versus either inhibitor alone. doi:10.1371/journal.pone.0040026.g006 PLoS ONE | www.plosone.org 7 June 2012 | Volume 7 | Issue 6 | e40026