title: The HPV16 E6 binding protein Tip-1 interacts with ARHGEF16, which activates Cdc42.
abstract: BACKGROUND
  Guanidine exchange factor (GEF)-catalysed activation of Rho proteins such as Cdc42 has been shown to have a crucial role in cellular transformation, malignant progression and invasion. We have previously shown that the HPV16 E6 oncoprotein binds to the PDZ domain protein Tax-interacting-protein 1 (Tip-1) and we now report identification and functional analysis of a novel Tip-1 binding GEF.
  
  
  METHODS
  Yeast two-hybrid, in vitro pull-down, site-directed mutagenesis, semiquantitative PCR, co-immunoprecipitation and western blotting were used to identify/confirm novel Tip-1 binding partners and analyse cellular expression levels. In vitro kinetic analyses of recombinant proteins, siRNA gene silencing and in cell assays were used to measure Rho protein activation.
  
  
  RESULTS
  Tax-interacting-protein 1 was shown to interact with ARHGEF16 by its carboxyl PDZ binding motif. Levels of ARHGEF16 were increased in transformed and immortalised cells expressing ectopic HPV16 E6 and Cdc42 was co-immunoprecipitated by ARHGEF16 in the presence of high-risk HPV E6. In vitro kinetic analysis confirmed that recombinant ARHGEF16 activates Cdc42 and this was increased by the addition of recombinant Tip-1 and E6. Cells expressing HPV16 E6 had higher levels of Cdc42 activation, which was decreased by siRNA silencing of either Tip-1 or ARHGEF16.
  
  
  CONCLUSIONS
  These data suggest that HPV16 E6, Tip-1 and ARHGEF16 may cooperate to activate Cdc42 and support a potential link between the expression of HPV16 E6 and Cdc42 activation.
authors.affiliation: University of Manchester Gynaecological Oncology Laboratories, School of Cancer and Enabling Sciences, St Mary's Hospital, Oxford Road, Manchester M13 9WL, UK.
authors.forename: A W
authors.initials: AW
authors.lastname: Oliver
authors.forename: X
authors.initials: X
authors.lastname: He
authors.forename: K
authors.initials: K
authors.lastname: Borthwick
authors.forename: A J
authors.initials: AJ
authors.lastname: Donne
authors.forename: L
authors.initials: L
authors.lastname: Hampson
authors.forename: I N
authors.initials: IN
authors.lastname: Hampson
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D017931:DNA Primers; D020868:Gene Silencing; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009856:Oncogene Proteins, Viral / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D034741:RNA, Small Interfering; D012097:Repressor Proteins / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020798:Two-Hybrid System Techniques; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*
chemical_list: C556035:ARHGEF16 protein, human; D017931:DNA Primers; C068651:E6 protein, Human papillomavirus type 16; D020662:Guanine Nucleotide Exchange Factors; D047908:Intracellular Signaling Peptides and Proteins; D009856:Oncogene Proteins, Viral; D034741:RNA, Small Interfering; D012097:Repressor Proteins; C421249:TAX1BP3 protein, human; D020764:cdc42 GTP-Binding Protein