Endosomal targeting of MEK2 requires RAF, MEK kinase activity and clathrin-dependent endocytosis.
To study spatiotemporal regulation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK1/2) signaling cascade in living cells, a HeLa cell line in which MAPK kinase of ERK kinase (MEK) 2 (MAPK kinase) was knocked down by RNA interference and replaced with the green fluorescent protein (GFP)-tagged MEK2 was generated. In these cells, MEK2-GFP was stably expressed at a level similar to that of the endogenous MEK2 in the parental cells. Upon activation of the EGF receptor (EGFR), a pool of MEK2-GFP was found initially translocated to the plasma membrane and then accumulated in a subset of early and late endosomes. However, activated MEK was detected only at the plasma membrane and not in endosomes. Surprisingly, MEK2-GFP endosomes did not contain active EGFR, suggesting that endosomal MEK2-GFP was separated from the upstream signaling complexes. Knockdown of clathrin by small interfering RNA (siRNA) abolished MEK2 recruitment to endosomes but resulted in increased activation of ERK without affecting the activity of MEK2-GFP. The accumulation of MEK2-GFP in endosomes was also blocked by siRNA depletion of RAF kinases and by the MEK1/2 inhibitor, UO126. We propose that the recruitment of MEK2 to endosomes can be a part of the negative feedback regulation of the EGFR-MAPK signaling pathway by endocytosis.
Galperin
Emilia
E
Department of Pharmacology, University of Colorado at Denver and Health Sciences Center, Aurora, CO 80045, USA.
Sorkin
Alexander
A
D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism*; D002966:Clathrin / Q000235:genetics / Q000502:physiology*; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000201:enzymology*; D066246:ErbB Receptors / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D048370:MAP Kinase Kinase 2 / Q000235:genetics / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D048490:raf Kinases / Q000378:metabolism*
D002966:Clathrin; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C482128:MAP2K2 protein, human; D066246:ErbB Receptors; D048490:raf Kinases; D020928:Mitogen-Activated Protein Kinases; D048370:MAP Kinase Kinase 2