The Effects And Mechanisms Of PAK4 On Cytoskeletal Reorganization And Migration Of Gastric Cancer Cells

Backgroudp21 activated kinases(PAKs),serine/threonine kinases including two groups of members(groupâ… includes PAK1-3 and groupâ…¡includes PAK4-6),was recently found to be key regulators of cancer cell signaling networks.PAK4 was found to be expressed in most tissues examined,with levels highest in the prostate,testis,and colon.More important,PAK4 was found to be overexpressed in cell lines that were derived from many cancer types and is localized to a region of chromosome 19 that is also commol/Lonly amplified in several human colon,ovarian and pancreatic tumors.Two PAK4 genetic mutations were also found among 340 serine/threonine kinases in human colorectal cancers.In addition,an activated allele of PAK4 leads to anchorage-independent cancer-cell growth,whereas a dominant-negative PAK4 mutant(PAK4NE) effectively inhibits Ras-induced transformation.Since PAK4 is newly identified,there are far less reported substrates than PAK1,but PAK4 is the most extensively studied member of the Groupâ…¡PAKs. PAK4 mediates morphological changes through its association with the Rho-family guanine nucleotide exchange factor(GEF),GEF-H1.PAK4 can negatively regulate the activation of Rho through a direct protein-protein interaction with G protein-linked Rho GEFs,dramatically decrease Rho-GTP loading in vivo and the formation of actin stress fibers in response to serum or LPA stimulation.PAK4 interacts with integrinαvβ5 and selectively promotes integrinαvβ5-mediated cell migration.LIMK1 was identified as a substrate for PAK1 and recently PAK4.LIMK1 phosphorylates cofilin,and PAK4-induced cell rounding is dependent on cofilin phosphorylation.This suggests that regulation of LIMK1 activity is required for cell migration,indicating that there is a functional linkage between PAK4,LIMK and cofilin.However,there are still uncovered mechanisms for cancer cell migration induced by PAK4.The DiGeorge critical region 6(DGCR6) gene exists in two highly homologous copies(DGCR6 and DGCR6L) on chromosome 22q11 and is first found deleted in patients with velo-cardio-facial syndrome/DiGeorge syndrome(VCFS/DGS).Both genes share highly conserved intron/exon structures and the deduced proteins,each 220 amino acids in length,are 97%identical at the amino-acid level.DGCR6L was highly expressed in mammary carcinomas and relates to the metastasis,its biochemical activity remained unclear.In this study,we found that the C-terminal kinase domain of PAK4 binds specifically with DGCR6L,which promotes the migration of gastric cancer cells.These findings suggest the existence of a novel biochemical route by which the binding of PAK4 with DGCR6L promotes migration of gastric cancer cells by increasing phosphorylation level of LIMK1.MethodsYeast mating assay for identifying interaction between PAK4 with DGCR6L.DGCR6L full length cDNA was subcloned into pGADT7 vector and using yeast strain Y187 as a host.PAK4N/C was transformed into yeast strain AH109.Then yeast mating performed.GST pull-down assay for binding of PAK4 with DGCR6L or PKN1.In vitro transcription and translation of PAK proteins were performed by using the TNT-coupled transcription and translation system.Using a T7-TNT kit,we translated 1μg of pCDNA3.1 vector in the presents of ³⁵S-methonine.The GST pull-down assay were performed by incubating equal amounts of GST,GST-fusion proteins immol/Lobilized by GST Sepharose Beads with in vitro translated ³⁵S-labeled PAK proteins.Bound proteins were isolated by SDS-PAGE.The bound proteins were then visualized by Fluorography.GST-fused proteins were used to map the regions of two binding proteins.Immol/Lunoprecipitation and western hot were used to identify the interaction of two proteins in mammol/Lalian cells.We determined the cellular localization of proteins by indirect immol/Lunofluorescence.Cells grown on glass coverslips were transfected with GFP-fusion or DsRed-fusion proteins if necessary, then immol/Lnofluorescenced with specific antibodies.Confocal scanning analysis was performed by using a Zeiss Laser Scanning Confocal Microscope in accordance with established methods.Establishment of DGCR6/L stable expression AGS cell lines. pCDNA3.1C-DGCR6/L,which containing selectable marker G418 to facilitate selection of stable transfected cells,were transfected into AGS cells which has a lower DGCR6L protein level.G418 was added and protein expression was detected by western blot after clones were picked.Transwell migration assay was used to assess cell migration in vitro.Kinase assay was used to detect the kinase activity of PKN1 and PAK4.The phosphorylations of PAK4 on DGCR6L,β-actin or PKN1 were detected by in vitro kinase assay;the phosphorylation of PKN1 on PAK4 was detected by in vitro kinase assay.Results1.DGCR6L interacted with PAK4C specifically by yeast mating assay and GST-pull down assay.2.PAK4 interacted with DGCR6L by its 466-572aa region,and the 6G,14G and 115L were critical amino acids for the binding.3.PAK4 interacted and colocalized with DGCR6L in SGC-7901 cells,but direct binding was not found,PAK4 did not phosphorylate DGCR6L. 4.PAK4 formed complex,but did not bind,withβ-actin in SGC-7901 cells,and DGCR6L was necessary for this complex.PAK4 didn't phosphorylateβ-actin.5.PAK4,DGCR6L and F-actin colocalized each two in SGC-7901 cells,and DGCR6L promote the phosphorylation level of LIMK1 and cofilin in a dose dependent manner.6.Stable expressed DGCR6L increased the migration of gastric cancer cells,when DGCR6L was silenced,the potential of migration decreased.7.PAK4 bind to the HR2 domain of PKN1 through its kinase domain.8.PKN1 phosphorylated PAK4,phosphorylation sites was within the region of 1-201aa of PAK4.9.PKN1colocalized with PAK4 in SGC-7901cells and both them colocalized at Golgi apparas.10.The expression of PKN1 induced the localization of PAK4 on F-actin.11.PKN1,PAK4 colocalized with MT in SGC-7901.Conclusions1.DGCR6L and PKN1 are both novel binding protein of PAK4.2.DGCR6L promote PAK4-induced reorgnazition and migration of gastric cancer cells via LIMK1.3.PKN1 phosphorylates PAK4.4.Expression of PKN1 mediates colocalization of both PKN1 and PAK4 with MF.5.PKN1,PAK4 colocalized with MT in gastric cancer cells.