Noncatalytic function of ERK1/2 can promote Raf/MEK/ERK-mediated growth arrest signaling.
Kinase activity is known as the key biochemical property of MAPKs. Here, we report that ERK1/2 also utilizes its noncatalytic function to mediate certain signal transductions. Sustained activation of the Raf/MEK/ERK pathway induces growth arrest, accompanied by changes in cell cycle regulators (decreased retinoblastoma phosphorylation, E2F1 down-regulation, and/or p21(CIP1) up-regulation) and cell type-specific changes in morphology and expression of c-Myc or RET in the human tumor lines LNCaP, U251, and TT. Ablation of ERK1/2 by RNA interference abrogated all these effects. However, active site-disabled ERK mutants (ERK1-K71R, ERK2-K52R, and ERK2-D147A), which competitively inhibit activation of endogenous ERK1/2, could not block Raf/MEK-induced growth arrest as well as changes in the cell cycle regulators, although they effectively blocked phosphorylation of the ERK1/2 catalytic activity readouts, p90(RSK) and ELK1, as well as the cell type-specific changes. Because this indicated a potential noncatalytic ERK1/2 function, we generated stable lines of the tumor cells in which both ERK1 and ERK2 were significantly knocked down, and we further investigated the possibility using rat-derived kinase-deficient ERK mutants (ERK2-K52R and ERK2-T183A/Y185F) that were not targeted by human small hairpin RNA. Indeed, ERK2-K52R selectively restored Raf-induced growth inhibitory signaling in ERK1/2-depleted cells, as manifested by regained cellular ability to undergo growth arrest and to control the cell cycle regulators without affecting c-Myc and morphology. However, ERK2-T183A/Y185F was less effective, indicating the requirement of TEY site phosphorylation. Our study suggests that functions of ERK1/2 other than its "canonical" kinase activity are also involved in the pathway-mediated growth arrest signaling.
Hong
Seung-Keun
SK
Department of Biochemistry, The Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Yoon
Seunghee
S
Moelling
Cas
C
Arthan
Dumrongkiet
D
Park
Jong-In
JI
D002384:Catalysis; D002453:Cell Cycle; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D034622:RNA Interference; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection; D048490:raf Kinases / Q000235:genetics / Q000378:metabolism
D049452:Green Fluorescent Proteins; D048490:raf Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases