Kinetic and mechanistic studies of p38α MAP kinase phosphorylation by MKK6

Bistability (coexistence of two stable steady states in a dynamical system) is a key mechanism of cellular decision-making and has been observed in many biochemical reaction networks such as mitogen-activated protein kinase (MAPK) signaling pathways. Theoretical studies have shown that bistability can arise in a single two-site MAPK phosphorylation and dephosphorylation cycle. However, the bistable behavior mostly relies on the kinetic mechanisms and parameters of this two-site modification. In exploring the system-level properties of MAPK regulation, most models to date focus on two limiting reaction regimes, distributive and processive, and are characterized by high levels of parametric uncertainty. Here, we developed a combined kinetic method which applies a continuous spectrophotometric enzyme-coupled assay incorporated with the viscosity approach, to perform detailed kinetic analyses of p38 alpha MAPK dual phosphorylation by MKK6. Almost all kinetic rate constants for the first and second phosphorylation steps in p38 alpha activation have been quantitatively determined, supporting that the phosphorylation occurs randomly in the first step, albeit preferring the tyrosine residue. The release rates of monophosphorylated p38 alpha from MKK6, either as the product in the first modification or as the substrate in the second step, were comparable to the respective adjacent phosphoryl transfer steps. These results indicated that dual phosphorylation of p38 alpha by MKK6 involves a random, partially processive mechanism. Based on the experimentally determined models and parameters, dynamics of the p38 alpha-MKK6-MKP5 system were explored, demonstrating for the first time that bistability can arise with this model at biologically feasible parameter values. Enzymes p38 alpha (); MKK6 ().