Doi: /electroph.61.9 Phos-tag SDS-PAGE MEK1 * SUMMARY MEK1, a chief component of the mitogen-activated protein

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1 Doi: /electroph.61.9 Phos-tag SDS-PAGE MEK1 * SUMMARY MEK1, a chief component of the mitogen-activated protein kinase (MAPK) pathway, is phosphorylated during activation of the pathway; 12 phosphorylation sites have been identified in human MEK1 by MS-based phosphoproteomic methods. By using Phos-tag SDS-PAGE, we found that multiple variants of MEK1 with different phosphorylation states are constitutively present in typical human cell lines. The Phos-tag-based strategy, which makes effective use of existing information on the location of phosphorylation sites (phosphoproteomic data), permits quantitative time-course profiling of MEK1 phosphospecies in their respective phosphorylation states. By subsequent immunoblotting with an anti-halotag antibody, we analyzed a HaloTag-fused MEK1 protein and 12 potential phosphorylation-site-directed mutants of the protein transiently expressed in HEK 293 cells. This strategy revealed that MEK1 is constitutively and mainly phosphorylated at the Thr-292, Ser-298, Thr-386, and Thr-388 residues in vivo, and that combinations of phosphorylations at these four residues produce at least six phosphorylated variants of MEK1. Like the levels of phosphorylation of the Ser-218 and Ser-222 residues by RAF1, which have been well studied, the phosphorylation statuses of Thr-292, Ser-298, Thr-386, and Thr-388 residues vary widely during activation and deactivation of the MAPK pathway. The Phos-tag-based strategy, which effectively uses phosphoproteomic data, permits qualitative and quantitative time-course profiling of intact MEK1 phosphospecies molecules in their respective phosphorylation states without loss of information about the molecular masses of the protein species. This combined study involving a Phos-tag-gel-based approach and existing phosphoproteomic data previously obtained by MS-based phosphoproteomic methods has shed new light on the phosphorylation dynamics of MEK1. Key words: HaloTag, MAPK pathway, MEK1, PhosphoSitePlus, Phos-tag SDS-PAGE 1 Phos-tag Phos-tag SDS-PAGE 2 5 Phos-tag SDS-PAGE methodology that effectively uses phosphoproteomic data for profiling the phosphorylation dynamics of MEK1 Eiji Kinoshita, Emiko Kinoshita-Kikuta, Tohru Koike Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University * Corresponding author: Eiji Kinoshita; Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Kasumi 1-2-3, Hiroshima , Japan kinoeiji@hiroshima-u.ac.jp 67 10

2 mitogen-activated protein kinase MAPK MEK1 PhosphoSitePlus, org/homeaction.do MEK1 12 Phos-tag SDS-PAGE 6 1 Phos-tag SDS-PAGE Phos-tag SDS-PAGE 357 mm Bis-Tris HCl ph w/v 25 μm Phos-tag 50 μm ZnCl mm Bis-Tris HCl ph w/v TEMED 100 mm Tris 100 mm MOPS 0.1 w/v SDS 5 mm NaHSO μg 1 40 ma 85 PVDF 2 MEK1 A431 HeLa HEK293 EGF, 100 ng/ml SDS-PAGE Phos-tag SDS-PAGE MEK1 pmek1/2 S218/S222 pmek1 pt292, ps298, pt386 3 Flexi HaloTag MEK1 Flexi HaloTag MEK1 pfn21ae0668 Promega Cell Signaling Technology CST PhosphoSitePlus MEK1 12 S72 S212 S218 S222 S231 T286 T292 S298 Y300 S385 T386 T388pFN21AE Agilent Pfu turbo DNA polymerase 4 HaloTag MEK1 HEK293 pfn21ae0668 Promega FuGENE HD Transfection reagent HEK SDS-PAGE SDS-PAGE HaloTag HaloTag MEK1 Phos-tag SDS-PAGE HaloTag MEK1 1 MEK1 A431 HeLa HEK293 MEK1 Phos-tag SDS-PAGE 6 Fig. 1A 0 min #1 7 EGF S218/S222 MEK1 7, 8 EGF #8 9 pmek1/2 S218/S222Fig. 1A 0 min EGF MEK1 S218/S222 Phos-tag SDS-PAGE Phos-tag SDS-PAGE Phos-tag-free SDS-PAGE EGF MEK1 MEK1 Fig. 1B

(A) The time course of MEK1 phosphorylation after stimulation with EGF (100 ng/ml) for 0 300 min was analyzed by using Zn 2+ Phos-tag SDS-PAGE (6.

3 Fig. 1 Endogenous MEK1 phosphorylation in A431, HeLa, or HEK293 cells stimulated with EGF, analyzed by using Phos-tag SDS- PAGE or conventional (Phos-tag-free) SDS-PAGE followed by immunoblotting. (A) The time course of MEK1 phosphorylation after stimulation with EGF (100 ng/ml) for min was analyzed by using Zn 2+ Phos-tag SDS-PAGE (6.0% w/v polyacrylamide and 25 μm Zn 2+ Phos-tag) followed by immunoblotting with anti-mek1 antibody (left panels) or anti-pmek1/2 (Ser-218/Ser-222) antibody (right panels). (B) The same samples were analyzed by using Phos-tag-free SDS-PAGE followed by immunoblotting with anti-mek1 antibody. Reprinted with permission from Ref. 6 (2016) Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. 2 HaloTag MEK1 MEK1 MEK1 CST PhosphoSitePlus MEK1 12 Phos-tag SDS-PAGE MEK1 HaloTag pfn21ae0668 HaloTag HaloTag MEK1 7 Fig. 2A WT MEK1

2017 61 12 Fig. 2 Profiling of HaloTag-fused MEK1 phosphorylation expressed in HEK 293 cells using Phos-tag SDS-PAGE followed by immunoblotting.

5% polyacrylamide w/v and 25 μm Zn 2+ Phos-tag) followed by immunoblotting with anti-halotag antibody. (B) Phosphorylation sites responsible for each band are summarized.

4 Fig. 2 Profiling of HaloTag-fused MEK1 phosphorylation expressed in HEK 293 cells using Phos-tag SDS-PAGE followed by immunoblotting. (A) A HaloTag-fused MEK1 protein (WT) and 23 mutants expressed in HEK 293 cells were analyzed by using Zn 2+ Phostag SDS-PAGE (5.5% polyacrylamide w/v and 25 μm Zn 2+ Phos-tag) followed by immunoblotting with anti-halotag antibody. (B) Phosphorylation sites responsible for each band are summarized. Reprinted with permission from Ref. 6 (2016) Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. Fig. 1A 0 min T292A S298A T386A T388A WT 7 4 MEK1 4 T292 S298 T386 T Fig. 2A 6 #2 7 Fig. 2B 3 MEK1 Phos-tag SDS-PAGE HaloTag MEK1 7 MEK1 MEK1 T292 S298 T386 T MEK1 pt292, ps298, pt386 Phos-tag SDS-PAGE MEK1 Fig. 3 pt292 MEK1 #3 4 7 HaloTag MEK1 ps298 #5 6 7 pt386 #2 4 6 HaloTag MEK1 MEK1

2017 61 13 T292 S298 T386 T388 4 6 #8 9 2 T292 S298 T386 3 EGF MEK1 S218/S222 6 Phos-tag SDS-PAGE Fig. 3 Phos-tag-free SDS-PAGE EGF MEK1 4 MEK1 6 MEK1 2 Fig. 1A EGF 9 MEK1 Fig.

3 Mapping of phosphorylation sites in the respective upshifted migration bands of endogenous MEK1 by immunoblotting with site-specific phosphorylated MEK1 antibodies.

5 T292 S298 T386 T #8 9 2 T292 S298 T386 3 EGF MEK1 S218/S222 6 Phos-tag SDS-PAGE Fig. 3 Phos-tag-free SDS-PAGE EGF MEK1 4 MEK1 6 MEK1 2 Fig. 1A EGF 9 MEK1 Fig. 4 2 EGF 10 3 #5 S298 S298 MEK1 9, 10 #5 MEK1 RAF1 T386 #2 T386 T292 MEK1 ERK 11, 12 MEK1 EGF #3 T292 MEK1 1 MEK1 S218/S222 Phos-tag SDS- PAGE Fig. 3 Mapping of phosphorylation sites in the respective upshifted migration bands of endogenous MEK1 by immunoblotting with site-specific phosphorylated MEK1 antibodies. The time course of MEK1 phosphorylation in HEK 293 cells after stimulation with EGF (100 ng/ml) for 0 10 min was analyzed by using Zn 2+ Phos-tag SDS-PAGE (6.0% w/v polyacrylamide and 25 μm Zn 2+ Phostag) followed by immunoblotting with the anti-mek1 antibody and site-specific phosphorylated MEK1 antibodies against the phosphorylated Thr-292 residue (pt292), phosphorylated Ser-298 residue (ps298), and phosphorylated Thr-386 residue (pt386). We also confirmed that changes occurred in the phosphorylation levels of the three residues of MEK1 in the same samples by using Phos-tag-free SDS-PAGE followed by immunoblotting with the three site-specific phosphorylated MEK1 antibodies. Reprinted with permission from Ref. 6 (2016) Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

2017 61 14 Fig. 4 Changes in relative abundance ratios of multiple endogenous MEK1 species before and after EGF stimulation. Densitometric analyses of migration bands #1 to #9 shown in Fig.

6 Fig. 4 Changes in relative abundance ratios of multiple endogenous MEK1 species before and after EGF stimulation. Densitometric analyses of migration bands #1 to #9 shown in Fig. 1 were performed. The ratios (%) of bands at given incubation times are represented as averages of three independent experiments using the same sample. Reprinted with permission from Ref. 6 (2016) Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

7 MEK1 Phos-tag SDS-PAGE Phos-tag SDS-PAGE 28 1) Kimura Y, Toda T, Hirano H. ModProt: a database for integrating laboratory and literature data about protein posttranslational modifications. J Electrophoresis. 2016;60:1 4. 2) Kinoshita E, Kinoshita-Kikuta E, Takiyama K, et al. Phosphate-binding tag, a new tool to visualize phosphorylated proteins. Mol Cell Proteomics. 2006;5: ) Kinoshita-Kikuta E, Aoki Y, Kinoshita E, et al. Label-free kinase profiling using phosphate affinity polyacrylamide gel electrophoresis. Mol Cell Proteomics. 2007;6: ) Kinoshita E, Kinoshita-Kikuta E. Improved Phos-tag SDS- PAGE under neutral ph conditions for advanced protein phosphorylation profiling. Proteomics. 2011;11: ) Kinoshita E, Kinoshita-Kikuta E, Koike T. Phos-tag SDS- PAGE systems for phosphorylation profiling of proteins with a wide range of molecular masses under neutral ph conditions. Proteomics. 2012;12: ) Kinoshita E, Kinoshita-Kikuta E, Kubota Y, et al. A Phos-tag SDS-PAGE method that effectively uses phosphoproteomic data for profiling the phosphorylation dynamics of MEK1. Proteomics. 2016;16: ) Alessi DR, Saito Y, Campbell DG, et al. Identification of the sites in MAP kinase kinase-1 phosphorylated by p74raf-1. EMBO J. 1994;13: ) Zheng CF, Guan KL. Activation of MEK family kinases requires phosphorylation of two conserved Ser/Thr residues. EMBO J. 1994;13: ) Coles LC, Shaw PE. PAK1 primes MEK1 for phosphorylation by Raf-1 kinase during cross-cascade activation of the ERK pathway. Oncogene. 2002;21: ) Eblen ST, Slack JK, Weber MJ, et al. Rac-PAK signaling stimulates extracellular signal-regulated kinase (ERK) activation by regulating formation of MEK1-ERK complexes. Mol Cell Biol. 2002;22: ) Xu B-e, Wilsbacher JL, Collisson T, et al. The N-terminal ERK-binding site of MEK1 is required for efficient feedback phosphorylation by ERK2 in vitro and ERK activation in vivo. J Biol Chem. 1999;274: ) Elben ST, Slack-Davis JK, Tarcsafalvi A, et al. Mitogenactivated protein kinase feedback phosphorylation regulates MEK1 complex formation and activation during cellular adhesion. Mol Cell Biol. 2004;24:

平成１４年度本態性多種化学物質過敏状態の調査研究研究報告書

平成１４年度本態性多種化学物質過敏状態の調査研究研究報告書 226 227 µ 228 Ω 229 230 0 ppb 2000ppb DG subthreshold (µa) 91 ± 59 86 ± 68 max stim. (µa) 755 ± 287 n=37 678 ± 300 max PS (mv) 8.7 ± 3.9 7.7 ± 4.3 n=61 CA1 subthreshold (µa) 36 ± 26 32 ± 13 max stim. (µa)