Studies On The Molecular Mechanisms Of Stresses-induced Translocation Of P38 Mapk

The specificity of cellular transduction is determined by the characteristics of space and time of the signal.The subcellular localization and translocation of signaling molecules are main features of signal transduction.Under many circumstances,the normal functions of signaling proteins may be achieved by anchoring to adaptor proteins,cytoskeleton,cell membrane structures,or binding to their upstream kinases and downstream substrates.The specific intracellular localizations of signaling molecules are helpful for the cells to perform physiological functions or pathological responses.Thus,the subcellular localization and translocation of signaling proteins have risen large interest in the study of cellular signal transduction.Mitogen-activated protein kinase(MAPK) signal pathway is one of the main signal transduction systems in mammalian cells.It transduces varied signals from cell surface into the nuclei,thus mediates the reactions of cells to stimuli such as regulating gene expression.There exist four subfamilies of MAPK, extracellular-regulated protein kinase(ERK),c-Jun N-terminal kinase(JNK)/ stress-activated protein kinase(SAPK),p38,ERK5/big mitogen-activated protein kinase 1(BMK1).The substrates of MAPK include transcription factors,protein kinases,cytoskeleton-related proteins,and ion channel proteins.MAPKs have relatively specific localization in cells,and translocate into nucleus upon appropriate stimuli,leading to consequent physiological effects.p38 pathway is mainly involved in the inflammatory and stresses-induced responses. Many types of stimuli can induce the activation of p38,including pro-inflammatory cytokines,bacteria components,UV radiation,hyperosmotic,H₂O₂ and heat shock. Our previous studies revealed that p38 dipersed in the whole cell,and tranlocated into the nucleus and induced the expression of TNF-α.However,this nuclear translocation was a transient process,and p38 would return back to the cytosol when the signal was inactivated.The signal transduction was blocked when the nuclear translocation of p38 was inhibited,suggesting that the normal function of p38 requires its translocation.Thus,the elucidation of the mechanisms of localization and translocation of p38 will not only be helpful for us to understand its in vivo functions, but also for the therapy on certain lethal diseases such as endotoxic shock and cardiomyopathy.Owing to the different research methods and the shortcomings of technologies,the conclusions of different researchers on the mechanisms of intracellular localizations and translocations were very different.Many factors may be involved in the regulation of these processes.Thus,it is necessary to demonstrate the main regulation mechanism of p38 localization and translocation under the circumstances of certain diseases,stimuli,and cell types.We first verified the nuclear translocation of p38 upon the stimulation of stresses. Then we found that different stimuli,including NaAsO₂,UV,anisomycin,LPS,H₂O₂, sobitol,induced the nuclear translocation of p38,suggesting that the stimuli of p38 activation are the inducers of p38 translocation as well.Moreover,the nuclear translocation of p38 could be observed in different cell lines,suggesting that this process is a common phenomenon in eukaryotic cells.We also observed the timecourse of the nuclear translocation of p38.When the cells were stimulated with NaAsO₂ transiently,p38 was phosphorylated and translocated into the nuclei,reaching the peak 60 min after the stimulation and then coming back to the basal level.But if the cells were stimulated with NaAsO₂ continuously,the phosphorylation degree of p38 would increase continuously,too,and p38 resided in the nuclei,insteading of nuclear export.These results suggested that the nuclear translocation of p38 depended on its phosphorylation,while its nuclear export depends on its dephoshporylation.The studies with different p38 mutants showed that intracellular localization of the dominant negative mutant of p38,p38(AF),had no change upon stimulation,losing the ability of nuclear translocation,while that of the kinase dead mutant of p38, p38(KM),was not affected,verifying the nuclear translocation of p38 upon stimulation is really phosphorylation-dependent,but not kinase activity-dependent. When the cells were pretreated with different MAPK inhibitors,the nuclear translocation of p38 was blocked by none of them,demonstrating the nuclear translocation of p38 has no relationship with its kinase activity. We further discussed the effects of the upstream kinase and downstream substrates on its intracellular localization.The results showed that the nuclear translocation of p38 was not only dependent on its phosphylation by MKK6,but also on the intracellular localization of MKK6 itself.And,the intracellular localization of p38 downstream substrate,MK2,was also a determining factor of the intracellular localization of p38, probably acting as an anchoring protein.Another p38 downstream substrate,PRAK, had similar effect on the nuclear translocation of p38.These results suggested that stresses-induced nuclear translocation of p38 had close relationships with the intracellular localizations of its upstream kinase and downstream substrates.Our previous studies suggested that the cytoskeleton might be related to the translocaiton of p38.The cells were pretreated with different inhibitors of cytoskeleton,and the translocaiton of p38 was only blocked by the microtubule-destroying agent nocodazole,suggesting that normal microtubule is required for the process,not the microfilament.Moreover,the nuclear translocation of p38 was dynein- and energy-dependent,and myosin-independent.We observed the effects of typical nuclear export inhibitor LMB and nuclear import inhibitor WGA on the nuclear export and nuclear import,and the results suggested that they both have no effect on the nuclear export and import,respectively.Through these studies performed with site-directed mutageneis,fluorescent protein reporter gene,and immunostaining,the conclusions can be drawn as follows:1) The translocation of p38 can be induced by different stimuli;the nuclear translocation of p38 exists in different cell lines;the nuclear translocation of p38 reaches the peak 60 min after the stimulation and then comes back to the basal level;2) The localization of p38 is determined by its phosphorylation,but not its kinase activity;3) The localization of p38 is affected by its upstream kinase MKK6 and downstream substrates MK2 and PRAK;4) The nuclear translocation of p38 is an energy-dependent progress,and can be blocked by destroy of microtubule and inhibition of dynein,while inhibition of microfilament and myosin have no effects;5) The nuclear export of p38 depends on its dephosphorylation;6) Nuclear export inhibitor LMB and nuclear import inhibitor WGA have no effect on the nuclear export and import,respectively. Taken together,we demonstrated that the stresses-induced nuclear translocation of p38 was an energy-dependent process,which was determined by its phosphorylation, and microtubule and dynein were involved in this process,as well as its upstream kinase and downstream substrates.The elucidation of these questions provided us important experimental evidences to understand the molecular mechanisms of nuclear translocaiton upon stimuli.