title: Precisely ordered phosphorylation reactions in the p38 mitogen-activated protein (MAP) kinase cascade.
abstract: The MAP kinase cascades, composed of a MAP3K, a MAP2K, and a MAPK, control switch responses to extracellular stimuli and stress in eukaryotes. The most important feature of these modules is thought to be the two double phosphorylation reactions catalyzed by MAP3Ks and MAP2Ks. We addressed whether the reactions are sequential or random in the p38 MAP kinase module. Mass spectrometry was used to track the phosphorylation of the MAP2K MEK6 by two MAP3Ks, TAO2 and ASK1, and the subsequent phosphorylation of p38α by MEK6/S*T* (where S (Ser) and T (Thr) are the two phosphorylation sites and * denotes phosphorylation). Both double phosphorylation reactions are precisely ordered. MEK6 is phosphorylated first on Thr-211 and then on Ser-207 by both MAP3Ks. This is the first demonstration of a precise reaction order for a MAP2K. p38α is phosphorylated first on Tyr-182 and then on Thr-180, the same reaction order observed previously in ERK2. Thus, intermediates were MEK6/ST* and p38α/TY*. Similarly, the phosphorylation of the p38α transcription factor substrate ATF2 occurs in a precise sequence. Progress curves for the appearance of intermediates were fit to kinetic models. The models confirmed the reaction order, revealed processivity in the phosphorylation of MEK6 by ASK1, and suggested that the order of phosphorylation is dictated by both binding and catalysis rates.
authors.affiliation: Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
authors.forename: John M
authors.initials: JM
authors.lastname: Humphreys
authors.forename: Alexander T
authors.initials: AT
authors.lastname: Piala
authors.forename: Radha
authors.initials: R
authors.lastname: Akella
authors.forename: Haixia
authors.initials: H
authors.lastname: He
authors.forename: Elizabeth J
authors.initials: EJ
authors.lastname: Goldsmith
mesh_terms: D051698:Activating Transcription Factor 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D006801:Humans; D048669:MAP Kinase Kinase 6 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D048848:MAP Kinase Kinase Kinase 5 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020930:MAP Kinase Kinase Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000502:physiology; D048308:Mitogen-Activated Protein Kinase 14 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D008956:Models, Chemical; D010766:Phosphorylation / Q000502:physiology; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D051381:Rats
chemical_list: C497105:ATF2 protein, human; D051698:Activating Transcription Factor 2; C497107:Atf2 protein, rat; D011494:Protein Kinases; C404957:TAOK2 protein, human; D017346:Protein Serine-Threonine Kinases; C122016:Taok2 protein, rat; D048308:Mitogen-Activated Protein Kinase 14; D048848:MAP Kinase Kinase Kinase 5; D020930:MAP Kinase Kinase Kinases; C482858:MAP3K5 protein, human; D048669:MAP Kinase Kinase 6; C482566:MAP2K6 protein, human
keywords: Cascade Signaling; MAP Kinases (MAPKs); Mass Spectrometry (MS); Ordered Phosphorylation; Phosphorylation Enzymes; Protein Kinases; Signal Transduction; p38 MAPK