The Proteomics And Function Research On The Overexpression Of The Catalytic C Subunit Of Protein Phosphatase 2A

Protein phosphatase 2A（PP-2A） is one of the most abundant and conserved serine/threonine phosphatase hin eukaryotes.It dephosphorylates a panel of cellular proteins such as kinases and other signal molecules,and participates in many cellular processes including cell division,cell apoptosis,gene regulation,protein synthesis, cytoskeleton organization and so on.The PP-2A holoenzyme exists as a heterotrimer,consisting of a catalytic C subunit,a structural A subunit and a regulatory B subunit.Previous studies have shown that the regulatory B subunit of PP-2A plays a crucial role in cell transformation.In addition,recent studies have also demonstrated that the structural A subunit of PP-2A is also implicated in carcinogenesis.Whether the catalytic C subunits of this phosphatase play a role in cancer remains to be explored.To explore the possible function of the catalytic subunit of PP-2A in cancer,our group has previously established the stable clones overexpressing the catalytic C subunit of PP-2A in both mouse skin cancer,JB6 cells（pCI-PP2Aca-JB6） and human lung cancer,H1299 cells （pCI-PP2Aca-H1299）.Using these stable cell lines and also vector-transfected control cell lines（pCI-Neo-JB6;pCI-Neo-H1299）,we have investigated the possible effects of the overexpressed exogenous PP-2Aca in these cells using proteomics,cDNA microarray and other molecular biology techniques.The differential expression patterns of total proteins between pCI-Neo-JB6 and pCI-PP2Aca-JB6 were analyzed using proteomics which was based on 2-D gel separation coupled with mass spectrum analysis or the samples labeled by ¹⁸O coupled with mass spectrum analysis.At the same time,the differential transcription of the downstream genes affected by PP2Aca overexpression was also examined through cDNA microarray analysis by our collaborators. Although the differential expression patterns of the downstream genes display some inconsistency between the mRNA and protein levels,it is easy to detect the changes in the expression patterns of the downstream genes between vector and PP2Aac-transfected JB6 cells.Our results demonstrate that the proteins involved in oxidative stress,protein metablism,membrane transport,carbohydrate and energy metabolism, protein folding,transcriptional regulation,cell signaling and cytoskeleton dynamics and some other minor aspects are differentially expressed. The anti-oxidative stress system components including catalase,aldehyde dehydrogenase,retinal dehydrogenase-1,glutathione S-transferase Mu1 and Mu2,thioredoxin 2,4 & 6 are all downregulated in pCI-PP2Aca-JB6 cells compared with the control cells（pCI-Neo-JB6）.The downregulation of the above proteins and enzymes likely leads to an attenuated defense against toxic active substance such as active O,N,and aldehyde but enhanced cell transformation through some unknown mechanism.In contrast,factors involved in protein folding such as splicing isoform 1 of small glutamine-rich tetratricopeptide repeat-containing protein A,calreticulin precursor,protein disulfide-isomerase precursor and tetratricopeptide repeat protein are upregulated,which explain the enhanced chaperone activities necessary for cell transformation in pCI-PP2Aca-JB6 cells.The downregulation of the tumor suppression factors including both prohibitin and annexin Al in pCI-PP2Aca-JB6 cells also supports the tumorigenesity function of PP-2Aac.Together,these results suggest that the pCI-PP2Aca-JB6 cells display some characteristics of transformed cancer cells.Through the manipulations of the intracellular levels of PP-2Aac by okadaic acid treatment and overexpression of the exogenous PP-2Aac,we have demonstrated that PP-2A directly regulates p53 activity through its dephosphorylation at Ser-15.Furthermore,we demonstrate that through negative regulation of p53,which directly controls the expression of the Cathepsin gene promoter,Ctsb,PP-2A positively regulates Cathepsin B level to promote cell transformation.This conclusion is also confirmed in lung cancer cell,1299 line.In summary,our results reveal that stable overexpression of PP-2Aca causes significant changes in the protein expression patterns of the JB6 cells,which favor the promotion of cell transformation.Our results also suggest that PP-2A may promote cell transformation via regulation of the n53-cathensin B nathway.