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PMID:21139582

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Citation

Oliver, AW, He, X, Borthwick, K, Donne, AJ, Hampson, L and Hampson, IN (2011) The HPV16 E6 binding protein Tip-1 interacts with ARHGEF16, which activates Cdc42. Br. J. Cancer 104:324-31

Abstract

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.

Links

PubMed PMC3031889 Online version:10.1038/sj.bjc.6606026

Keywords

Base Sequence; Blotting, Western; DNA Primers; Gene Silencing; Guanine Nucleotide Exchange Factors/metabolism; Intracellular Signaling Peptides and Proteins/metabolism; Mutagenesis, Site-Directed; Oncogene Proteins, Viral/metabolism; Polymerase Chain Reaction; Protein Binding; RNA, Small Interfering; Repressor Proteins/metabolism; Reverse Transcriptase Polymerase Chain Reaction; Two-Hybrid System Techniques; cdc42 GTP-Binding Protein/metabolism

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

HUMAN:TX1B3

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:P03126

F

Seeded From UniProt

complete

HUMAN:TX1B3

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:Q5VV41

F

Seeded From UniProt

complete

HUMAN:ARHGG

GO:0017147: Wnt-protein binding

ECO:0000021:

UniProtKB:O14907 UniProtKB:Q5VV41


F

The introduction of this paper states that Tip-I has been shown to be a protein involved in the wnt(Reynaud et al,2000; Kanamori et al,2003)

Experimental evidence in this paper show the interaction od TipI and GEF 16. Figure 1 Interactions, regulation and stability of GEF16. (A) Yeast mating assay confirming that Tip-1 binds to short carboxyl terminal GEF16 fragments isolated from the primary Tip-1 yeast two-hybrid screen. (B) Glutathione transferase pull-down of Tip-1 with IVTT full-length wildtype and mutant GEF16 (wtGEF16/mutGEF16). The wtGEF16 contains the PDZ-binding domain ETDV, whereas mutGEF16 has the TDV residues deleted. Tip-1-GST fusion protein and GST control protein were bound to glutathione-sepharose beads. These were mixed with equal amounts of the IVTT wtGEF16 or mutGEF16 proteins, and then washed with binding buffer. Bound proteins were separated by SDS–PAGE and visualised by western immunoblotting with anti-GEF16. The GST-Tip-1 protein bound to the wtGEF16, but did not bind to the mutant form, whereas the GST control did not bind either product.

complete

HUMAN:ARHGG

GO:0043088:

ECO:0000314:

Fluorescent kinetic analysis of Cdc42 activation by GEF16 in the presence of high-risk HPV16 E6 and Tip-1. Different combinations of 2 mM concentrations of the recombinant proteins GEF16, Tip-1 and HPV16 E6 were added after 4 min of initial readings being taken with Cdc42 and assay buffers alone. Each combination was carried out in triplicate. It can be seen that Cdc42 activation by GEF16 is enhanced in the presence of Tip-1 and E6. HPV16 E6, Tip-1, ARHGEF16 and Cdc42 activation.

complete

HUMAN:ARHGG

enables

GO:0030165: PDZ domain binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:O14907

F

Seeded From UniProt

complete

HUMAN:ARHGG

involved_in

GO:0090630: activation of GTPase activity

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

HUMAN:TX1B3

involved_in

GO:0090630: activation of GTPase activity

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

HUMAN:TX1B3

GO:0043088: regulation of Cdc42 GTPase activity

ECO:0000314:

P

Fluorescent kinetic analysis of Cdc42 activation by GEF16 in the presence of high-risk HPV16 E6 and Tip-1. Different combinations of 2 mM concentrations of the recombinant proteins GEF16, Tip-1 and HPV16 E6 were added after 4 min of initial readings being taken with Cdc42 and assay buffers alone. Each combination was carried out in triplicate. It can be seen that Cdc42 activation by GEF16 is enhanced in the presence of Tip-1 and E6. HPV16 E6, Tip-1, ARHGEF16 and Cdc42 activation.

complete

HPV16:VE6

GO:0030165 : PDZ domain binding

ECO:0000021:

UniProtKB:O14907 UniProtKB:P03126


F

Binding of HPV E6 and Tip-I’s PDZ domain is shown in: Figure 1 (A) Yeast mating assay confirming that HPV16 E6 binds to short carboxyl terminal of Tip-I’ PDZ domain. The abstract and introduction explain in more detail and make reference to the original paper for these findings (Hampson et al, 2004)

complete

HPV16:VE6

GO:0043088: regulation of Cdc42 GTPase activity

ECO:0000314:

P

Fluorescent kinetic analysis of Cdc42 activation by GEF16 in the presence of high-risk HPV16 E6 and Tip-1. Different combinations of 2 mM concentrations of the recombinant proteins GEF16, Tip-1 and HPV16 E6 were added after 4 min of initial readings being taken with Cdc42 and assay buffers alone. Each combination was carried out in triplicate. It can be seen that Cdc42 activation by GEF16 is enhanced in the presence of Tip-1 and E6. HPV16 E6, Tip-1, ARHGEF16 and Cdc42 activation

complete

HPV16:VE6

GO:0035023: regulation of Rho protein signal transduction

ECO:0000314:

P

Figure 3 Expression of HPV16 E6 is associated with the upregulation of GEF16 and higher levels of activated Cdc42 in non-transformed cells.Fig3(C) Western immunoblot analysis of GTP-activated Cdc42 in HPV16 E6- and HPV6 E6-transfected hTert cells. hTertT16 E6 cells showed increased levels of activated Cdc42. The addition of GTP to the PAK1 Cdc42 assay shows constitutive GTP activation of the available Cdc42.

complete

HPV16:VE6

enables

GO:0030165: PDZ domain binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:O14907

F

Seeded From UniProt

complete

HPV16:VE6

involved_in

GO:0090630: activation of GTPase activity

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

HPV16:VE6

involved_in

GO:0045944: positive regulation of transcription by RNA polymerase II

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete


See also

References

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