The MEK1 proline-rich insert is required for efficient activation of the mitogen-activated protein kinases ERK1 and ERK2 in mammalian cells.
MEK1 and MEK2 contain a proline-rich insert not present in any other known MEK (MAP (mitogen-activated protein)/ERK (extracellular signal-regulated kinase) kinase) family members. We examined the effect of removing the MEK1 polyproline insert on MEK activity, its binding to Raf, and its ability to activate ERKs in cells. Deletion of the insert had no effect on either the activity of MEK1 or on its ability to bind to Raf-1. Both wild type and constitutively active MEK1 coimmunoprecipitated with Raf-1 whether or not the insert was present. Deletion of the insert did not reduce activation of MEK1 by EGF or activated Raf in cells. The proline-rich insert enhanced the ability of an otherwise equally active MEK1 protein to regulate endogenous ERKs in mammalian cells. Overexpression of either constitutively active MEK1 lacking the insert or ERK2 compensates for the weaker in vivo activity of the MEK1 deletion mutant. Expression of the insert in cells reduced activation of ERKs by EGF. We conclude that the proline-rich insert is not the site of the MEK-Raf interaction and that the polyproline insert is required for its efficient activation of downstream ERKs in cells.
University of Texas Southwestern Medical Center, Department of Pharmacology, Dallas, Texas 75235-9041, USA.
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Dang
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JA
Frost
M H
MH
Cobb
D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000235:genetics; D004815:Epidermal Growth Factor / Q000502:physiology; D006801:Humans; D048369:MAP Kinase Kinase 1; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D010455:Peptides / Q000502:physiology; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry*; D011505:Protein-Tyrosine Kinases / Q000737:chemistry*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics
D010455:Peptides; C011083:polyproline; D004815:Epidermal Growth Factor; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases