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G?12 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 ?-subunits G?12 and G?13, 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 G?12 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 G?12 and G?13. Mutation of these residues in G?12 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 G?12 mutants showed impaired binding to heat-shock protein 90 (Hsp90) while retaining binding to RhoGEFs. Corresponding mutants of G?13 exhibited robust SRE activation, suggesting a G?12-specific mechanism, and inhibition of Hsp90 by geldanamycin or small interfering RNA-mediated lowering of Hsp90 levels resulted in greater downregulation of G?12 than G?13 signaling in SRE activation experiments. Furthermore, the Drosophila G12/13 homolog Concertina was unable to signal to SRE in mammalian cells, and G?12:Concertina chimeras revealed G?12-specific determinants of SRE activation within the switch regions and a C-terminal region. These findings identify G?12 determinants of SRE activation, implicate G?12:Hsp90 interaction in this signaling mechanism, and illuminate structural features that arose during evolution of G?12 and G?13 to allow bifurcated mechanisms of signaling to a common cell proliferative pathway.

SUBMITTER: Montgomery ER

PROVIDER: S-EPMC3965892 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Gα12 structural determinants of Hsp90 interaction are necessary for serum response element-mediated transcriptional activation.

Montgomery Ellyn R ER Temple Brenda R S BR Peters Kimberly A KA Tolbert Caitlin E CE Booker Brandon K BK Martin Joseph W JW Hamilton Tyler P TP Tagliatela Alicia C AC Smolski William C WC Rogers Stephen L SL Jones Alan M AM Meigs Thomas E TE

Molecular pharmacology 20140116 4

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 ev ...[more]

PMID: 24435554

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Project description:UnlabelledMany bacteria contain large cyclic diguanylate (c-di-GMP) signaling networks made of diguanylate cyclases (DGCs) and phosphodiesterases that can direct cellular activities sensitive to c-di-GMP levels. While DGCs synthesize c-di-GMP, many DGCs also contain an autoinhibitory site (I-site) that binds c-di-GMP to halt excess production of this small molecule, thus controlling the amount of c-di-GMP available to bind to target proteins in the cell. Many DGCs studied to date have also been found to signal for a specific c-di-GMP-related process, and although recent studies have suggested that physical interaction between DGCs and target proteins may provide this signaling fidelity, the importance of the I-site has not yet been incorporated into this model. Our results from Pseudomonas fluorescens indicate that mutation of residues at the I-site of a DGC disrupts the interaction with its target receptor. By creating various substitutions to a DGC's I-site, we show that signaling between a DGC (GcbC) and its target protein (LapD) is a combined function of the I-site-dependent protein-protein interaction and the level of c-di-GMP production. The dual role of the I-site in modulating DGC activity as well as participating in protein-protein interactions suggests caution in interpreting the function of the I-site as only a means to negatively regulate a cyclase. These results implicate the I-site as an important positive and negative regulatory element of DGCs that may contribute to signaling specificity.ImportanceSome bacteria contain several dozen diguanylate cyclases (DGCs), nearly all of which signal to specific receptors using the same small molecule, c-di-GMP. Signaling specificity in these networks may be partially driven by physical interactions between DGCs and their receptors, in addition to the autoinhibitory site of DGCs preventing the overproduction of c-di-GMP. In this study, we show that disruption of the autoinhibitory site of a DGC in Pseudomonas fluorescens can result in the loss of interactions with its target receptor and reduced biofilm formation, despite increased production of c-di-GMP. Our findings implicate the autoinhibitory site as both an important feature for signaling specificity through the regulation of c-di-GMP production and a necessary element for the physical interaction between a diguanylate cyclase and its receptor.

Dataset Information

G?12 structural determinants of Hsp90 interaction are necessary for serum response element-mediated transcriptional activation.

Publications

Gα12 structural determinants of Hsp90 interaction are necessary for serum response element-mediated transcriptional activation.

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