A global genomic view on LNX siRNA-mediated cell cycle arrest

Dan Zheng ?Shaohua Gu ?Yao Li ?Chaoneng Ji ?Yi Xie ?Yumin Mao

Received:25May 2010/Accepted:8November 2010/Published online:21November 2010óSpringer Science+Business Media B.V.2010

Abstract LNX protein is the ?rst described PDZ domain-containing member of the RING ?nger-type E3ubiquitin ligase family.Studies have approved that LNX could participate in signal transduction,such as Notch pathway,and play an important role in tumorigenesis.In this study,we found that down-regulation of LNX resulted in G0/G1cell cycle arrest in G0/G1phase in HEK293cells.To explore the molecular mechanism of this phenomenon,we employed expression microarray to comparatively analyze the genome-wide expression between the LNX-knockdown cells and the normal cells.We also used quantitative real-time PCR to further con?rm the differential expression patterns of 25transcripts involved in cell http://www.doczj.com/doc/2b440fe958f5f61fb73666d9.htmlbined with known information about genic functions,signal pathways and cell cycle machinery,we analyzed the role of endogenous LNX in cell cycle.The results suggest that down-regulation of LNX could result in cell cycle arrest in G0/G1phase through inhibition of b -catenin,MAPK,NF j B,c-Myc-dependent pathway and activation of p53,TGF-b -dependent pathway.This study provides new per-spectives on LNX’s pleiotropic activities,especially its essential role in cell proliferation and cell cycle.Keywords LNX áUbiquitin ligase áCell cycle áMicroarray áPathway analysis

Introduction

LNX protein is the ?rst described PDZ domain-containing member of the RING ?nger-type E3ubiquitin ligase family.PDZ domains are the most abundant protein–protein interaction modules in the human genome [1],which form PDZ-based multiprotein complexes.In combination with their frequent abililty to multimerize,PDZ proteins thus have the potential to assemble large multiprotein complexes at the cell membrane [2].A variety of signaling pathways connect with each other via the assembly of these PDZ-containing proteins and then form signal transduction networks.

One of the best-studied PDZ-containing proteins is the Drosophila protein INAD,which is composed almost entirely of ?ve PDZ domains.INAD appears to serve as a scaffold to assemble different components of the photo-transduction cascade,including the principal light-acti-vated ion channels [3].

NHERF (Na ?/H ?exchanger regulatory factor)proteins contain two tandem PDZ domains.NHERF interacts with PDGFR [4].As PDGFR,like other growth receptors,is activated through ligand-induced dimerization and trans-phosphorylation of the clustered receptors,NHERF pro-motes PDGFR dimerization in part due to NHERF’s own ability to form dimers.In this manner,NHERF enhances growth factor signaling and activates mitogenic signals transduced by ERKs or MAPKs [5].NHERF also recruits non-membrane proteins such as the c-Yes/YAP-65complex,members of the phospholipase Cb family and the GRK6A protein kinase to apical surface of polarized epithelial cells where they regulate or respond to membrane signals [5].Thus,these PDZ-containing scaffolds are indispensable for signal transductions.More and more studies showed that LNX protein could interact with other proteins via its multi-domain and then take part in regulating several

D.Zheng áS.Gu áY.Li áC.Ji áY.Xie áY.Mao (&)State Key Laboratory of Genetic Engineering,Institute of Genetics,School of Life Science,Fudan University,Shanghai 200433,People’s Republic of China e-mail:ymmao@http://www.doczj.com/doc/2b440fe958f5f61fb73666d9.html

Mol Biol Rep (2011)38:2771–2783DOI 10.1007/s11033-010-0422-6