Adaptive increase of amino acid transport system A requires ERK1/2 activation

Amino acid starvation markedly stimulates the activity of system A, a widel y distributed transport route for neutral amino acids. The involvement of M APK (mitogen-activated protein kinase) pathways in this adaptive increase o f transport activity was studied in cultured human fibroblasts. In these ce lls, a 3-fold stimulation of system A transport activity required a 6-h ami no acid-free incubation. However, a rapid tyrosine phosphorylation of ERK ( extracellular regulated kinase) 1 and 2, and JNK (Jun N-terminal kinase) 1, but not of p38, was observed after the substitution of complete medium wit h amino acid-free saline solution. ERK1/2 activity was 4-fold enhanced afte r a 15-min amino acid-free incubation and maintained at stimulated values t hereafter. A transient, less evident stimulation of JNK1 activity was also detected, while the activity of p38 was not affected by amino acid deprivat ion. PD98059, an inhibitor of ERK1/2 activation, completely suppressed the adaptive increase of system A transport activity that, conversely, was unaf fected by inhibitors of other transduction pathways, such as rapamycin and wortmannin, as well as by chronic treatment with phorbol esters. In the pre sence of either L-proline or 2-(methylaminoisobutyric) acid, two substrates of system A, the transport increase was prevented and no sustained stimula tion of ERK1/2 was observed. To identify the stimulus that maintains MAPK a ctivation, cell volume was monitored during amino acid-free incubation. It was found that amino acid deprivation caused a progressive cell shrinkage ( 30% after a 6-h starvation). If proline was added to amino acid-starved, sh runken cells, normal values of cell volume were rapidly restored. However, proline-dependent volume rescue was hampered if cells were pretreated with PD98059. It is concluded that (a) the triggering of adaptive increase of sy stem A activity requires a prolonged activation of ERK1 and 2 and that (b) cell volume changes, caused by the depletion of intracellular amino acid po ol, may underlie the activation of MAPKs.