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 ± 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. β-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 ± 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.
