Kinetic and mechanistic studies of p38α MAP kinase phosphorylation by MKK6

Abstract

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 p38α MAPK dual phosphorylation by MKK6. Almost all kinetic rate constants for the first and second phosphorylation steps in p38α 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 p38α 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 p38α by MKK6 involves a random, partially processive mechanism. Based on the experimentally determined models and parameters, dynamics of the p38α‐MKK6‐MKP5 system were explored, demonstrating for the first time that bistability can arise with this model at biologically feasible parameter values. Enzymes p38α (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/7/11/24.html); MKK6 (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/7/12/2.html).

Architecture of the MKK6-p38α complex defines the basis of MAPK specificity and activation

“…Kinetic data have shown that the dual-phosphorylation of p38α by MKK6 involves a partially processive mechanism in vitro, in which the monophosphorylated intermediates can either dissociate from the enzyme or proceed to the next catalytic step. Wang et al (51) and others (52) proposed that both p38α monophosphorylated forms can be produced by MKK6 catalysis with a preference for Y182, whose phosphorylation is four-fold faster than that of T180. With respect to the second catalytic step, the experimentally measured kinetic rates indicate that phosphorylation at Y182 in the first step enhances the catalytic efficiency of MKK6 phosphorylation at T180 in the second step.…”

Architecture of the MKK6-p38α complex defines the basis of MAPK specificity and activation

“…Wang et al . ( 61 ) and others ( 62 ) propose that both p38α monophosphorylated forms can be produced by MKK6 catalysis with a preference for Y182, whose phosphorylation is four times as fast as that of T180. With respect to the second catalytic step, the experimentally measured kinetic rates indicate that phosphorylation at Y182 in the first step enhances the catalytic efficiency of MKK6 phosphorylation at T180 in the second step.…”

“…Kinetic data have shown that the dual phosphorylation of p38a by MKK6 involves a partially processive mechanism in vitro, in which the monophosphorylated intermediates can either dissociate from the enzyme or proceed to the next catalytic step. Wang et al (61) and others (62) propose that both p38a monophosphorylated forms can be produced by MKK6 catalysis with a preference for Y182, whose phosphorylation is four times as fast as that of T180. With respect to the second catalytic step, the experimentally measured kinetic rates indicate that phosphorylation at Y182 in the first step enhances the catalytic efficiency of MKK6 phosphorylation at T180 in the second step.…”

Positive feedback induces switch between distributive and processive phosphorylation of Hog1

“…Our findings on the processivity of Hog1 phosphorylation may have important implications to explain dynamic properties of MAPK pathways in mammalian systems. For example, the mammalian Hog1 homolog p38 was recently shown to be phosphorylated in a semi-processive manner 47 , which may be best explained by a mixed activation model. Likewise, mammalian ERK shows context-specific differences in its ratio of single and double phosphorylated species 48 .…”