Studies On The Interaction Between PRAK And SEPTIN 8

As key components of signal transduction systems in organisms, mitogen-activated protein kinases (MAPKs) are considered to be involved in many cellular processes and pathogenesis of many severe diseases. p38, a subfamily of MAPKs, can phosphorylate many different substrates and then exert many different biological functions. The p38 group of kinases has been involved in cell growth and cell apoptosis, in addition, these kinases have been considered to regulate the proinflammtory processes, stress responses activity of transcription factors and cytoskeletal reorgnization, even to play important roles in some disease, such as cardiomyocyte hyperthrophy, ischemia/reperfusion injury, neuronal pathology, infectious diseases, etc.MAP Kinases are activated, in general, by a highly conserved cascade consisting of three kinases (MKKK/MKK/MAPK). p38 MAP kinases are activated in the same way in response to either cellular stresses or binding between receptors and ligands on the cell surface. p38 MAP kinases become activated through phosphorylation on threonine and throsine residued within a Thr-Gly-Tyr dual phosphrylation motif. MKK3 and MKK6 can directly phosphorylate p38 MAP kinases. Both of them are activated by TAK1, ASK1, SPPK and PAK. PRAK, also known as MK5, preliminary study found that, PRAK is subject to the strict regulation of p38 in vitro and in vivo. The sequence analysis of PRAK reveals that PRAK contains 471 amino acid residues, with a molecular mass of 5.4 kD. PRAK distributes in various organizations widely, especially mononuclear-macrophage in the peripheral blood, brain and vascular endothelium, lung, kidney and ovary prostate epithelium. Different interactions of ligands with PRAK can activate different signal transduction pathways. For example, the interaction of PRAK with amphoterin is involved in the neurite outgrowth by Ras and Cdc42; through p38 pathway the interaction between HSP25/27 and PRAK may result in actin assembled, and the formation of stress of fibers during cell oxidative stress and cell stress response. Further studies show that PRAK participates in the occurring and development of many diseases. PRAK play a key role in the pathological process of endotoxin shock and the neurodegenerative diseases; the interaction of PRAK with proteins promotes the growth of neuronal and mediates growth and migration of tumor cells.Because PRAK is correlated with complications of inflammation, cancer and other diseases by different signal transduction pathways, it becomes a focus in the study field of cell signaling transduction. Previously we obtained proteins interacting with PRAK by screening human heart cDNA library using yeast two-hybrid, one of which is SEPTIN 8(Sept8) protein.Human Sept8, a conservative GTP enzyme family, has coding sequence of 1,452 bp with a molecular mass of 55.8 kD. The conserved central core domain of Sept8 is the GTP binding site, with GTP activity; close to the central GTP binding site is relatively conservative and more basic amino acid domain; N terminus of hundreds of amino acids, rich in proline and hydrophobic amino acid residues suggesting possible SH3 binding domain; addition of its C terminal has a coiled-coil structure by the long a helix formed, the domain may mediate protein interaction. There are Sept8 phosphorylation sites, acetylation modification sites and palmitoylation sites.We presume that the interaction between PRAK and Sept8 screened by yeast two-hybrid screening existed, for three reasons. First, PRAK and Sept8 are expressioned in some organizations, such as glioma cells, kidney cells and prostate cells; second, Sept8 participates in signal transduction pathway which is downstream of activation of receptor, such as G protein-mediated signal transduction through the Rho pathways; third, there are some overlaps in the signal transduction pathways involved by PRAK or Sept8, such as the function of Ras, the activation of MAPK (JNK, ERK, etc.) and transcription factor (NF-κB). We can have a hypothesis that after Ras interacting with its ligand, by directly or indirectly actived Raf, Raf-1 can phosphorylate two regulatory serines of MEK1/MEK2 (MAP kinase/ERK kinase), thus activate MEKs. Then the dowenstream kinases pass the signal to PRAK protein in p38 MAPK signaling pathway, with which Sept8 interacts with, and then downstream proteins like p53 or HSP27 protein are actived. The signal transduction result in generation of cellular oxidant stress. Finally, the expression spectrum of cytokine will change, results in phyisiological and pathology functions.If the hypothesis is true, it would make great sense to the functional study of the interaction between PRAK and Sept8 in signal transduction, and we could link the interaction between PRAK and Sept8 with pathogenesis of the diseases. So we identified the interaction between PRAK and Sept8 in vivo and in vitro and the function of the interaction between PRAK and Sept8.Then, human glioma cells cDNA library was constructed and Sept8 gene was amplified by specific primers from the library, and was cloned into pET14b and pcDNA3 vector respectively. In the in vitro binding experiment, GST-PRAK and His-Sept8 were expressed in BL21 (DE3), and then purified with Glutathione affinity resin and Ni2+-NTA resin, respectively. When two purified protein were accessible, the in vitro binding was performed. The results showed that GST-PRAK could be co-precipitated by His-Sept8, which pre-bound to the Ni-NTA resin, and the GST protein alone could not in the same condition, which suggested that Sept8 could bind to PRAK in vitro.In the in vivo binding experiment, HA-PRAK and FLAG-Sept8 plasmids were co-transfected into HEK293 cell, then the cells were lysed and the supernatant was used in co-immunoprecipitation. The co-immunoprecipitation results through anti-HA antibody magnetic beads showed that Sept8 and PRAK with a small amount of binding, but the binding intensity of these two proteins gradually increased upon stimulation of arsenite. The co-immunoprecipitated results through anti-FLAG antibody beads showed the same results. The result showed that the interaction between PRAK and Sept8 had stimulation responsibility, suggesting the binding between theses two proteins may play an important role in the cell reactions against stresses. As NaAsO2 can strongly activate the p38 pathway, thus, we have initially to consider such interactions may be related to p38 pathway activation.In determining the Sept8 and PRAK in the combination of increased after stimulation by arsenite, we have stimulated with different time, and then time course of the binding between PRAK and Sept8 was studied. We set up 8 groups that were stimulated by 200μmol/L NaAsO2 for 0 min,10 min,15 min,30 min,60 min,90 min, 120 min and 240 min, The results showed that the binding intensity of these two proteins obviously increased upon stimulation when stimulated 10 min approximately, and then decreased gradually in 240 min.The influential issues on the binding between Sept8 and PRAK were then studied. We studied which signaling pathway to work, and the relationship of PRAK activation and the interaction between them. Firstly, HEK293 cells were pretreated with PD98059, the specific inhibitor of ERK1/2 pathway, and SP600125, the specific inhibitor of JNK pathway, and it is shown that these two inhibitors had no effect on the binding. But pretreated with SB203580, the specific inhibitor of p38 pathway, and then treated with NaAsO2 for 60 min, the results showed that the binding intensity of these two proteins gradually increased upon stimulation. Then, PRAK (182A), the dominant negative mutant of PRAK, PRAK (182D), the dominant positive mutant of PRAK, and PRAK (KM), lose the mutant with active ATP, were co-transfected with FLAG-Sept8 respectively. The responsive pattern of PRAK (182D) was similar to that of wild type PRAK. But PRAK (182A) and PRAK (KM) were found to bind to Sept8 intensively and the bindings were markedly decreased after stimulation. The results suggested that p38 pathway specific inhibitor maybe have inextricably lined to the binding of PRAK and Sept8.Finally, we have also carried out immunofluorescence assay to observe the co-localization of the endogenous expression of PRAK and Sept8 in NIH/3T3 cells. The results showed that endogenous PRAK and endogenous Sept8 lied in the cytoplasm when cells were not stimulated, and were locally co-locolizated; after stimulation of cells, the co-localization phenomenon of PRAK and Sept8 was dramatically enhanced and spot-like structures were formed, the biological significance should still be furtherly investigated.Through these studies, we draw conclusions as follows:1. PRAK interacted with Sept8 in vitro and in vivo.2. The interaction between PRAK and Sept8 had stimulation responsibility, and the stress stimuli of NaAsO2 could promote the binding, suggesting the binding between these two proteins may play an important role in the cell reactions against stresses.3.ERK pathway specific inhibitor PD98095, JNK pathway specific inhibitor SP600125, microtubule-depolymerizing and reagent Nocodazole all had no effect on the binging between Sept8 and PRAK. On the contrary, the binding would be markedly inhibited by p38 pathway specific inhibitor SB203580.4. The dominant negative mutant and the mutant of PRAK lack of ATP activity could bind to Sept8, but after stimulation the interaction between the mutant of PRAK lack of ATP activity and Sept8 can’t be dramatically enhanced.5. after stimulation, which may be caused by theses treatments interference the binding between Sept8 and PRAK in the normal circumstances.6. Under normal circumstances, endogenous Sept8 and PRAK are co-localization in NIH/3T3 cells.