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 ASKI 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 autophosphorylation activity. The kinase activity of auto-phosphorylated ASKI (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 phosphoserine and phospho-threonine. This study provides novel mechanistic and kinetic insights into the ASK1-initiated MAPK signal transduction via highly controlled reconstructed protein systems. Published by Elsevier Inc.