Pleinis John M, Davis Cameron W, Cantrell Caleb B, Qiu David Y, Zhan Xuanzhi
Department of Chemistry, Tennessee Technological University, Cookeville, TN 38505, USA.
Department of Chemistry, Tennessee Technological University, Cookeville, TN 38505, USA.
Protein Expr Purif. 2017 Apr;132:34-43. doi: 10.1016/j.pep.2017.01.002. Epub 2017 Jan 7.
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.
凋亡信号调节激酶1（ASK1）是一种丝裂原活化蛋白激酶激酶激酶（MAP3K），可激活来自两个MAP激酶级联反应的下游MAP激酶激酶（MKK）：c-Jun氨基末端激酶（JNK）和p38。ASK1的重要生理功能已引起广泛关注。然而，我们对ASK1的分子机制，包括ASK1的激活机制和ASK1介导的MKK磷酸化的催化机制，仍不清楚。缺乏纯化的ASK1蛋白阻碍了对ASK1启动的信号转导机制的阐明。在此，我们报告了一种从大肠杆菌中表达和纯化功能性全长ASK1的一步层析方法。纯化的ASK1表现出自磷酸化活性。还在来自JNK级联反应的两个MKK底物MKK4和7上评估了自磷酸化ASK1（pASK1）的激酶活性。我们的结果表明，pASK1对MKK7的磷酸化作用比对MKK4更有效。稳态动力学分析表明，MKK7是比MKK4更好的ASK1底物。直接下拉试验进一步证实了这些观察结果，该试验表明ASK1与MKK7的结合明显强于MKK4。此外，除了磷酸丝氨酸和磷酸苏氨酸外，在pASK1对MKK4的磷酸化过程中还观察到了强烈的磷酸酪氨酸信号。这项研究通过高度可控的重组蛋白系统，为ASK1启动的MAPK信号转导提供了新的机制和动力学见解。
