title: Gα12 structural determinants of Hsp90 interaction are necessary for serum response element-mediated transcriptional activation.
abstract: The G12/13 class of heterotrimeric G proteins, comprising the α-subunits Gα12 and Gα13, regulates multiple aspects of cellular behavior, including proliferation and cytoskeletal rearrangements. Although guanine nucleotide exchange factors for the monomeric G protein Rho (RhoGEFs) are well characterized as effectors of this G protein class, a variety of other downstream targets has been reported. To identify Gα12 determinants that mediate specific protein interactions, we used a structural and evolutionary comparison between the G12/13, Gs, Gi, and Gq classes to identify "class-distinctive" residues in Gα12 and Gα13. Mutation of these residues in Gα12 to their deduced ancestral forms revealed a subset necessary for activation of serum response element (SRE)-mediated transcription, a G12/13-stimulated pathway implicated in cell proliferative signaling. Unexpectedly, this subset of Gα12 mutants showed impaired binding to heat-shock protein 90 (Hsp90) while retaining binding to RhoGEFs. Corresponding mutants of Gα13 exhibited robust SRE activation, suggesting a Gα12-specific mechanism, and inhibition of Hsp90 by geldanamycin or small interfering RNA-mediated lowering of Hsp90 levels resulted in greater downregulation of Gα12 than Gα13 signaling in SRE activation experiments. Furthermore, the Drosophila G12/13 homolog Concertina was unable to signal to SRE in mammalian cells, and Gα12:Concertina chimeras revealed Gα12-specific determinants of SRE activation within the switch regions and a C-terminal region. These findings identify Gα12 determinants of SRE activation, implicate Gα12:Hsp90 interaction in this signaling mechanism, and illuminate structural features that arose during evolution of Gα12 and Gα13 to allow bifurcated mechanisms of signaling to a common cell proliferative pathway.
authors.affiliation: Department of Biology, University of North Carolina at Asheville, Asheville, North Carolina (E.R.M., B.K.B., J.W.M., T.P.H., A.C.T., W.C.S., T.E.M.); Departments of Biology (K.A.P., S.L.R., A.M.J.), Biochemistry and Biophysics (B.R.S.T.), Cell Biology and Physiology (C.E.T.), and Pharmacology (A.M.J.), R. L. Juliano Structural Bioinformatics Core Facility (B.R.S.T.), and Carolina Center for Genome Sciences (S.L.R.), University of North Carolina, and the Lineberger Comprehensive Cancer Center, (S.L.R., T.E.M.), Chapel Hill, North Carolina.
authors.forename: Ellyn R
authors.initials: ER
authors.lastname: Montgomery
authors.forename: Brenda R S
authors.initials: BR
authors.lastname: Temple
authors.forename: Kimberly A
authors.initials: KA
authors.lastname: Peters
authors.forename: Caitlin E
authors.initials: CE
authors.lastname: Tolbert
authors.forename: Brandon K
authors.initials: BK
authors.lastname: Booker
authors.forename: Joseph W
authors.initials: JW
authors.lastname: Martin
authors.forename: Tyler P
authors.initials: TP
authors.lastname: Hamilton
authors.forename: Alicia C
authors.initials: AC
authors.lastname: Tagliatela
authors.forename: William C
authors.initials: WC
authors.lastname: Smolski
authors.forename: Stephen L
authors.initials: SL
authors.lastname: Rogers
authors.forename: Alan M
authors.initials: AM
authors.lastname: Jones
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Meigs
mesh_terms: D000818:Animals; D002460:Cell Line; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster / Q000166:cytology; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D010802:Phylogeny; D011485:Protein Binding; D027602:Serum Response Element; D015398:Signal Transduction; D015533:Transcriptional Activation; D020741:rho GTP-Binding Proteins / Q000378:metabolism
chemical_list: D029721:Drosophila Proteins; D018841:HSP90 Heat-Shock Proteins; D044365:GTP-Binding Protein alpha Subunits, G12-G13; C587919:cta protein, Drosophila; D020741:rho GTP-Binding Proteins