The Function Analysis Of STK11' Sinteracting Protein LOH12CR1

Peutz-Jeghers syndrome(PJS)is a dominantly inherited disorder characterized by multiple gastrointestinal hamartomatous polyposis and mucocutaneous melanin pigmentation.It's also known for predisposition to benign and malignant tumors in multiple organ system.The locus responsible for PJS was mapped genetically to the STK11 gene(also known as LKB1) which is defined as a tumor suppressor.The protein STK11 is a 433aa serine/threonine protein kinase that located both in cytoplasm and nucleus.During the function research of STK11,some interacting proteins with known functions were found and thus provided more important clues for people to explore the detailed mechanisms in STK11's function or the high tumor-predisposition.STK11 has physical interaction with P53.STK11 can translocate to mitochondrial membrane,and then induce apoptosis through the mitochondrial pathway,also STK11 can arrest cell growth in G1 by induce the expression of P21,both of those are P53-dependent.By forming the heterotrimer with STRAD and MO25,STK11's kinase activity was remarkablely promoted.The activated STK11 phosphorate its substrate AMPK,which in turn was activated and then initiate the katabolic metabolism. Other AMPK family proteins such as MAPK3/PAR1A can also be activated by STK11,through the relationship with those protein,STK11 was found to play important roles in the regulation network of cell polarity.There are more interacting proteins such as Brg-1,LIP-1,FLIP-1,AGS3 were found one by one,each of them provided evidences that STK11 can control cell growth,apoptosis,energy metabolism and cell polarity through multiple signal pathway.By using the method of GST-pull down with mass chromatographic analysis and co-immunoprecipitation,our research team has already screened and defined a new STK11 interacting protein—LOH12CR in L02 cell.We wonder whether the unknown-function but high reserved protein LOH12CR1 have important biologic functions or not.So we perform the bioinformatic analysis of LOH12CR1,observe its subcellular localization by the use of GFP tag,detect its express level in tumor cell lines by Realtime-PCR,clone its CDS region and reconstruct its eukaryotic express vector and then investiagate its effect in cell proliferation and growth in a overexpress manner.Our research have found that:①The conserved "orphan" protein LOH12CR1 may have some post-translational modification sites: LOH12CR1 can't be classified to any known protein family;there are no regions have high similarity with known protein functional domain or motif in its protein sequence.However,its may have some post-translational modification sites which include 1 N-myristoylation sites,2 N-glycosylation sites and a Casein kinaseⅡphosphorylation site.②LOH12CR1 is predominantly localized in cytoplasm:after overexpress the reconstructed protein GFP-LOH12CR1 in L02(STK11 pozitive express)and HeLa(STK11 negative express)cells,we observe both cells under the laser confocal microscopy and found that most of the bulky-spot-like green fluorescence are scattered in cytoplasm,few is located in the nucleous.The same location patterns of LOH12CR1 in both cells also imply that STK11 expression won't change its subcellular location;③LOH12CR1 express in some tumor cell lines: By using Realtime-PCR,We can detect the expression of LOH12CR1 in L02,COS7,Be17402,HMC,Huh-7,Hela,Hela-STK11~+,HL60,HT-29 and HCT116 cell lines,and LOH12CR1 has the highest express level in L02 cell while the lowest in COS7.The express level of LOH12CR1 in Hela-STK11~+ cell is higher than that in Hela cell,which imply that STK11 may have negative impact on LOH12CRI's expression;④LOH12CR1 can suppress the cell growth and proliferation:the overexpress of LOH12CR1 in Hela and Hct116 cell both led to less cell clone formation and down-regulated cytoactive,the result imply that LOH12CR1 may have some negative effects in cell growth and proliferation.With those research work,we have cloned the CDS of LOH12CR1 and reconstructed its eukaryotic express vector at the first time,also we find that LOH12CR1 is predominantly localized in cytoplasm,what's more important is that we find the overexpressed LOH12CR1 can suppress the cell proliferation and growth.All of those recognition have provide basic clues and directions for the on-going research of LOH12CR1's function.