A Study On The Interaction Between Metastasis Suppressor Nm23-H1 And N-terminal Mutant Of The Kinase Suppressor Of Ras KSR Scaffold In Ras-to-MAPK Pathway

Background and object Morbidity and mortality of lung cancer has been continuously risen since 1960s. Lung cancer has becomes the biggest malignant tumor threatening human health and life in the world. Invasion and metastasis is not only the malignant characteristic of lung cancer, but also the main cause of failure to cure and death. It has been proved that invasion and metastasis in lung cancer is a developing process with multiple genes and steps involved in alteration of several signal pathways; It is the reason that key genetic structure or dysfunction in some cell-signal pathway activate or trapping related-signal transduction pathway, which results in expression of phenotypic invasion and metastasis in lung cancer. Nm23-H1 was identified as a tumor metastasis suppressor gene, which the abnormality of its structure and function was closely related to the invasive and metastatic ability of lung cancer and play a critical role in MAPK downstream signaling cascade. Ras-to-MAPK signal transduction pathway widespreadly resides in many creature from yeast to mammals, which plays a critical role in the transmission of many growth and developmental signals, and a major route by which Ras transmits signals is through the sequential activation of the cytoplasmic kinases, Raf, MEK and MAP kinase(MAPK). Kinase suppressor of Ras (KSR) is a novel gene which was discovered in 1995 in Ras-to-MAPK pathway. KSR provides a scaffold that facilitates the phosphorylation reactions that are required for executing critical signal transduction steps downstream of Ras and thus accelerates the signal transduction of MAPK pathway. Previous data has shown that nm23-H1 has a histinde kinase activity. The phosphorylated state of KSR will influence its function in MAPK signaling pathway. Our previous study has explored the relationship between nm23-H1 and KSR. And confirmed KSR acts as a scaffolding protein and nm23-H1 as a histidine kinase can change the phosphorylation state of KSR, especially in amino terminus of KSR. Up to now, It has not been confirmed that molecule target of KSR regulated by nm23-H1 and little has been known about the exact relationship between nm23-H1 and KSR mutant in lung cancer's metastasis. MAPK pathway may be the downstream signaling cascade that involved in the process of nm23-H1 on suppressing lung cancer's metastasis. Site-directed mutant technique can correctly change the base sequence and get mutant proteins to explore the relationship between the functional and structural change of genes. In order to elucidate the association between mutant KSR and nm23-H1, KSR mutant genes will be constructed by site-directed mutagensis and gene recombinant technique in this study. The eventual aim is to provide rationale and experimental evidence for manifesting the molecular mechanism of nm23-H1 regulating metastasis of lung cancer.Meterials and Methods (1). Expression and purification of the prokaryotic expressing vectors of wild pET28a-nm23-H1 was determined by metal chelate affinity resin and identification its biochemical activity and function was determined by autoradiography and Western blot. (2). Eukaryotic expression vectors of pCMV-Tag2b-N-296-KSR was constructed was determined by gene recombinant technique and recombinant plasmid pCMV-Tag2b-KSR,pCMV-Tag2b-N-KSR,pCMV-Tag2b-N-296-KSR, pCMV-Tag2b-C-KSR, have been transient trasfected into 293Tcell, Then fusion proteins are identified by western blot. The phosphorylated interaction between wild nm23-H1 prokaryotic proteins and KSR, N-KSR,N-296-KSR, C-KSR eukaryotic proteins was examined by in vitro kinase activity assay. (3). The desired nine mutations pCMV-Tag2b-KSR ^S297A,pCMV-Tag2b-KSR ^S392A,pCMV-Tag2b-KSR ^S431A,pCMV-Tag2b-KSR ^S434A,pCMV-Tag2b-KSR ^S435A,pCMV-Tag2b-KSR ^S438A,pCMV-Tag2b-KSR ^S439A, pCMV-Tag2b-KSR ^S442A and pCMV-Tag2b-KSR ^S443A weresuccessgully introduced into pCMV-Tag2b-KSRby using the modifed QuickChange Site-directed Mutagenesis kit method .Then the sequence of the KSR mutant genes were identified by DNA sequencing and transfected into 293 T cell line expressing mutants identified by Western blot. (4). Mutant proteins were purified by anti-FLAG M2 Affinity Gel and examined by in vitro kinase activity assay in order to elucidate the potential phosphorylated site of KSR by nm23-H1. Results The results in this study firstly showed in the world as follows: (1) prokaryotic expressing vectors pET28a-nm23-H1 constructed by our prevoius research could express a great amount of target proteins after transforming the plasmids into BL21 (DE3). These proteins can be identificated by Westem blot and autoradiography. (2) pCMV-Tag2b-N-296-KSR eukaryotic expression vectors was successfully constructed from pCMV-Tag2b-KSR vector. The four eukaryotic expression vectors were successfully transfected into 293T cells and got fusion proteins of FLAG-KSR, FLAG-N-KSR, FLAG-N-296-KSR and FLAG-C-KSR. These proteins were identificated by Western blot. (3) The desired nine mutations gene pCMV-Tag2b-KSR^S297A, pCMV-Tag2b-KSR^S392A, pCMV-Tag2b-KSR^S431A, pCMV-Tag2b-KSR^S434A, pCMV-Tag2b-KSR^S435A, pCMV-Tag2b-KSR^S438A, pCMV-Tag2b-KSR^S439A, pCMV-Tag2b-KSR^S442A and pCMV-Tag2b-KSR^S443Awere successfully constructed using the modifed QuickChange Site-directed Mutagenesis kit method. Then the sequence of the KSR mutant genes were identified by DNA sequencing and transfected into 293 T cell line expressing mutants. Identified by Western blot. (4) Autophosphorylation nm23-H1 can not phosphorylate N-296-KSR and carboxyl terminus of KSR in vitro kinase activity assay. Based on these results we presume that nm23-H1 phosphorylate KSR between N-296 and N-538,mainly in CA3and CA4. (5) Mutant proteins were examined by in vitro kinase activity assay in order to elucidate the exactly phosphorylated site of KSR by nm23-H1. Autophosphorylation nm23-H1 interact with KSR mutant proteins. Phosphorylation strap of FLAG-KSR^S297A, FLAG-KSR^S392A, FLAG-KSR^S431A, FLAG-KSR^S435A, FLAG-KSR^S438A, FLAG-KSR^S442Aand FLAG-KSR^S443Aemerged in autoradiogram,but not in mutant proteins FLAG-KSR^S439A,FLAG-KSR^S434A, FLAG-KSR^SZ97A can be phosphorlated, but phosphorylation straps were very weak, presentation of results has show:different mutant KSR genes serin 297, 392,431,435,438,442,443 were not phosphorylation site of nm23-H1 and the ability of nm23-H1 phosphorylating mutant KSR are different. Mutant KSR 439 was phosphorylation site ofnm23-H1.Conclusion (1) The pET28a-nm23-H1 prokaryotic expressing vectors are successfully constructed, which can express wild nm23-H1 proteins. (2) pCMV-Tag2b-N-296-KSR eukaryotic expression vectors are successfully constructed, The pCMV-Tag2b-KSR, pCMV-Tag2b-N-296-KSR, Tag2b-N-KSR and pCMV-Tag2b-C-KSR eukaryotic expression vectors are successfully transient transfected into 293T cell and finally get the fusion proteins of FLAG-KSR, FLAG-N-KSR, FLAG-N-296-KSR and FLAG-C-KSR. (3) There is a close interaction between nm23-H1 and KSR. The interaction is closely related to the N-termini of KSR, but not the fragment of N-296 and C-KSR. It confirmed that nm23-H1 phosphorylate KSR between N-296 and N-538,mainly in KSR CA3and CA4 domain. (4) The interaction between nm23-H1 and KSR is major the phosphorylated relationship, and N-termini of KSR is the targeted site regulated by nm23-H1, phosphorylation nm23-H1 has different effects on different point mutation of KSR. Mutant KSR 439 is phosphorylation site of nm23-H1.The mechanism of nm23-H1 on suppressing lung cancer's metastasis might be related to the state of KSR's phosphorylation.