title: Purification, auto-activation and kinetic characterization of apoptosis signal-regulating kinase I.
abstract: Apoptosis signal-regulating kinase I (ASK1) is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates the downstream MAP kinase kinases (MKKs) from two MAP kinase cascades: c-Jun N-terminal kinase (JNK) and p38. The essential physiological functions of ASK1 have attracted extensive attention. However, our understanding of the molecular mechanisms of ASK1, including the activation mechanism of ASK1 and the catalytic mechanism of ASK1-mediated MKK phosphorylation, remain unclear. The lack of purified ASK1 protein has hindered the elucidation of ASK1-initiated signal transduction mechanisms. Here, we report a one-step chromatography method for the expression and purification of functional full-length ASK1 from Escherichia coli. The purified ASK1 demonstrates auto-phosphorylation activity. The kinase activity of auto-phosphorylated ASK1 (pASK1) was also evaluated on two MKK substrates, MKK4 and 7, from the JNK cascades. Our results show that MKK7 can be phosphorylated by pASK1 more effectively than MKK4. The steady-state kinetic analysis demonstrates that MKK7 is a better ASK1 substrate than MKK4. These observations are further confirmed by direct pull-down assays which shows ASK1 binds MKK7 significantly stronger than MKK4. Furthermore, robust phospho-tyrosine signal is observed in MKK4 phosphorylation by pASK1 in addition to the phospho-serine and phospho-threonine. This study provides novel mechanistic and kinetic insights into the ASK1-initiated MAPK signal transduction via highly controlled reconstructed protein systems.
authors.affiliation: Department of Chemistry, Tennessee Technological University, Cookeville, TN 38505, USA.
authors.forename: John M
authors.initials: JM
authors.lastname: Pleinis
authors.affiliation: Department of Chemistry, Tennessee Technological University, Cookeville, TN 38505, USA.
authors.forename: Cameron W
authors.initials: CW
authors.lastname: Davis
authors.affiliation: Department of Chemistry, Tennessee Technological University, Cookeville, TN 38505, USA.
authors.forename: Caleb B
authors.initials: CB
authors.lastname: Cantrell
authors.affiliation: Department of Chemistry, Tennessee Technological University, Cookeville, TN 38505, USA.
authors.forename: David Y
authors.initials: DY
authors.lastname: Qiu
authors.affiliation: Department of Chemistry, Tennessee Technological University, Cookeville, TN 38505, USA. Electronic address: xzhan@tntech.edu.
authors.forename: Xuanzhi
authors.initials: X
authors.lastname: Zhan
mesh_terms: D004789:Enzyme Activation; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D048670:MAP Kinase Kinase 4 / Q000737:chemistry; D048688:MAP Kinase Kinase 7 / Q000737:chemistry; D048848:MAP Kinase Kinase Kinase 5 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D048848:MAP Kinase Kinase Kinase 5; C482858:MAP3K5 protein, human; D048670:MAP Kinase Kinase 4; D048688:MAP Kinase Kinase 7; C482559:MAP2K4 protein, human; C482569:MAP2K7 protein, human
keywords: Apoptosis signal-regulating kinase I (ASK1); Autophosphorylation; Mitogen activated protein kinase (MAPK); Mitogen activated protein kinase kinase 4 (MKK4); Mitogen activated protein kinase kinase 7 (MKK7); Signaling specificity