MAPK3 (ERK1) [Untagged] Kinase

MAPK3 (ERK1) [untagged] Kinase

Alternate Names: Extracellular Signal-Regulated Kinase 1, p44ERK1, p44MAPK

Cat. No. 66-0026-050 Quantity: 50 µg Lot. No. 30305 Storage: -70˚C

FOR RESEARCH USE ONLY NOT FOR USE IN HUMANS CERTIFICATE OF ANALYSIS Page 1 of 2

Background Physical Characteristics

Protein ubiquitylation and protein Species: human Protein Sequence: Please see page 2 phosphorylation are the two major mechanisms that regulate the func- Source: E. coli tions of proteins in eukaryotic cells. Quantity: 50 μg However, these different posttrans- lational modifications do not operate Concentration: 0.28 mg/ml independently of one another, but are frequently interlinked to enable bio- Formulation: 50 mM Tris/HCl pH7.5, 0.1 mM EGTA, 150 mM NaCl, 0.1% ß-Mercap- logical processes to be controlled in a toethanol, 270 mM sucrose, 0.03% Brij-35, more complex and sophisticated man- 1 mM Benzamidine, 0.2 mM PMSF ner. Studying how protein phosphorylation events control the ubiquitin sys- Molecular Weight: ~43.2 kDa tem and how ubiquitylation regulates Purity: >95% by InstantBlue™ SDS-PAGE protein phosphorylation has become a focal point of the study of cell regula- Stability/Storage: 12 months at -70˚C; tion and human disease. MAP kinas- aliquot as required es are serine, threonine, and tyrosine specific protein kinases that regulate Quality Assurance proliferation, gene expression, differ- entiation, mitosis, cell survival, and Purity: Protein Identification: apoptosis in response to stimuli, such 4-12% gradient SDS-PAGE Confirmed by mass spectrometry. as mitogens, osmotic stress, heat InstantBlue™ staining shock and pro-inflammatory cytok- Lane 1: MW markers Activity Assay: ines. Cloning of human Mitogen Acti- Lane 2: 2.5 µg GST-MAPK3 The specific activity of MAPK3 was determined using the method described by Hastie et al. (2006) with vated Protein kinase 3 (MAPK3) was the enzyme being assayed at several concentrations. first described by Charestet al. (1993) MAPK3 was incubated for 10 minutes at 30˚C in kinase and Garcia et al. (1998). Activation of reaction buffer in the presence of MBP substrate (0.333 MAPK3 requires both tyrosine and mg/ml) and [γ-32P]ATP (100 μM). Duplicate reactions threonine phosphorylated that is me- were stopped by spotting the assay mixture onto What- diated by Map/Erk Kinase Kinase 1 man P81 paper – capturing the phosphorylated sub- (MEKK1) (Cobb, et al., 1994). MAPK3 strate.The radioactivity incorporated was measured on is ubiquitously distributed in tissues a scintillation counter and the enzyme’s mean specific with the highest expression in heart, activity was calculated. brain and spinal cord (Boulton, et al., MAPK3 specific activity: 1991). MAPK3 translocates to the nu- 1299 Units/mg (364 Units/ml) cleus resulting in the phosphorylation of several transcription factors such as 1 Unit = 1 nmole of phosphate incorporated into the sub- Elk-1, c-Myc, c-Jun, c-Fos, and C/EBP strate in 1 minute beta. MAPK3 has also been shown to interact with several proteins includ- Substrate: Myelin Basic Protein (MBP)

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Alternate Names: Extracellular Signal-Regulated Kinase 1, p44ERK1, p44MAPK

Cat. No. 66-0026-050 Quantity: 50 µg Lot. No. 30305 Storage: -70˚C

FOR RESEARCH USE ONLY NOT FOR USE IN HUMANS CERTIFICATE OF ANALYSIS Page 2 of 2

Background Physical Characteristics

Continued from page 1 Continued from page 1 ing DUSP3, HDAC4, MAP2K1 and Protein Sequence: MAP2K2 (Marti, et al., 1997; Todd, et LGSAAAAAQGGGGGEPRRTEGVGPGVPGEVEM al., 1999; Zhou, et al., 2000). VKGQPFDVGPRYTQLQYIGEGAYGMVSSAYDH VRKTRVAIKKISPFEHQTYCQRTLREIQILL References: RFRHENVIGIRDILRASTLEAMRDVYIVQDL METDLYKLLKSQQLSNDHICYFLYQILRGLKY Boulton TG et al. (1991) ERKs: a family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in IHSANVLHRDLKPSNLLSNTTCDLKICDF response to insulin and NGF. Cell 65, 663-675. GLARIADPEHDHTGFLTEYVATRWYRAPEIM Charest DL et al. (1993) Molecular cloning, expression, and LNSKGYTKSIDIWSVGCILAEMLSNRPIFPG characterization of the human mitogen-activated protein kinase KHYLDQLNHILGILGSPSQEDLNCIINMKAR p44erk1. Mol Cell Biol 13, 4679-4690. NYLQSLPSKTKVAWAKLFPKSDSKALDLLDRM Cobb MH et al. (1994) The mitogen-activated protein kinases, ERK1 and ERK2. Semin Cancer Biol 5, 261-268. LTFNPNKRITVEEALAHPYLEQYYDPTDEP VAEEPFTFAMELDDLPKERLKELIFQETAR Garcia F et al. (1998) Molecular cloning and characterization of the human p44 mitogen-activated protein kinase gene. Genomics FQPGVLEAP 50, 69-78.

Hastie CJ, McLauchlan HJ, Cohen P (2006) Assay of protein The residues underlined remain after cleavage and removal of kinases using radiolabeled ATP: a protocol. Nat Protoc 1, 968- the purification tag. 71. MAPK3 (regular text): Start bold italics (amino acid residues 2-379). Marti A et al. (1997) Actin-binding protein-280 binds the stress- activated protein kinase (SAPK) activator SEK-1 and is required Accession number: CAA42744.1 for tumor necrosis factor-alpha activation of SAPK in melanoma cells, J Biol Chem 272, 2620-2628.

Todd JL, Tanner KG and Denu JM (1999) Extracellular regulated kinases (ERK) 1 and ERK2 are authentic substrates for the dual- specificity protein-tyrosine phosphatase VHR. A novel role in down- regulating the ERK pathway. J Biol Chem 274, 13271-13280.

Zhou X et al. (2000) Histone deacetylase 4 associates with extracellular signal-regulated kinases 1 and 2, and its cellular localiza- tion is regulated by oncogenic Ras. Proc Natl Acad Sci U S A 97, 14329-14333.