Molecular Regulation Mechanism Of MKPs Toward MAPKs In The MAPK Pathway

The mitogen-activated protein kinase?MAPK?cascades are conserved signal transduction pathways,which are activated in response to a diverse array of extracellular stimuli and can mediate both physiological and pathological responses in mammalian cells and tissues,including cell proliferation,differentiation and transformation,stress responses,inflammation,growth arrest and apoptosis.The core MAPK signalling module consists of a three-tier kinase cascade leading to the dual-phosphorylation and activation of MAPKs in the final tier.In mammalian cells,the down-regulation of MAPK is mediated by the differential expression and activities of a family of 10 dual-specificity MAPK phosphatases?MKPs?.In this thesis,a combination of approaches including biochemical experiment,molecular dynamics simulation,and mathematical model building,together with new quantitative assays that we developed here,were employed to study the molecular regulation mechanism between MKPs and MAPKs.Firstly,kinetic experiments were designed to study the molecular mechanism of dephosphorylation of MAPK by MKPs.We have developed a mechanistic approach to study the colvalent modification?or de-modification?of functioning enzymes.Using this method,we studied the mechanism of p38?dephosphorylation by MKP3,which dephosphorylates only the pTyr residue,and by MKP7,which dephosphorylates both phosphorylated residues in the activation loop.It was found that the substrate and MKP3 binds to p38?in a non-competitive way and that the two reactions could coexist.Our results also show that MKP7 dephosphorylate p38?in two catalytic steps instead of one.We also performed molecular dynamics?MD?simulations for the catalytic domain of MKPs in both active and inactive conformations.The simulations show that the catalytic loops in MKP have distinct dynamic properties from the classical protein tyrosine phosphatases?PTPs?,despite the fact that they come from the same PTP superfamily and share similar catalytic mechanisms.We further summarized the similarities and differences for the dynamic features of intrinsically active and inducible active MKPs,providing a basis for the understanding of difference in their catalytic activities.Next,the molecular mechanism of allosteric activation of MKP3 by ERK2 was investigated.Mutational and kinetic study shows that the ³³⁴FNFM³³⁷ motif in the MKP3 catalytic domain is essential for MKP3-mediated ERK2 inactivation and responsible for ERK2-mediated MKP3 activation.The long-term MD simulations further reveal a complete dynamic process,in which the catalytic domain of MKP3gradually changes to a conformation that resembles an active MKP catalytic domain over the timescale of the simulation,providing a direct time-dependent observation of allosteric signal transmission in ERK2 induced MKP3 activation.Finally,for the induced dimerization phenomena that are widely existed in the signal transduction pathways,we built an exact mathematical model that shows how to calculate the amount of any possible species present at equilibrium.The theory is subsequently confirmed in vitro using FRET experiments.As the approach only requires the measurement of the dimerized proteins,which could be detected for cell-based assays,this approach provides the potential to analyze induced dimerization inside cells in a quantitative aspect.