Direct Identification of a G Protein Ubiquitination Site by Mass Spectrometry

Marotti, Louis A.; Newitt, Rick; Wang, Yuqi; Aebersold, Ruedi; Dohlman, Henrik G.

doi:10.1021/bi015940q

Cited by 89 publications

(82 citation statements)

References 45 publications

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“…This degradation, although dependent on the N-end enzyme Ubr1, was not targeted by the N-terminal amino acid of Gpa1 (27), a phenomenon believed to be the result of targeting by an internal degron. Recently, the site of ubiquitin modification for Gpa1 was elegantly mapped to residue 165, substitution of which results in moderate pheromone resistance, an observation consistent with its reduced level of ubiquitylation (28). However, given that the ubiquitylation site in Gpa1 is present within a loop absent in mammalian ␣ subunits, should N-end ubiquitin-dependent degradation of mammalian heterotrimeric G protein subunits occur, it is likely to use a different ubiquitylation site and distinct targeting mechanism than that identified thus far for Gpa1 (␣ subunit) in S. cerevisiae.…”

Section: Discussionmentioning

confidence: 89%

γ ₂ subunit of G protein heterotrimer is an N-end rule ubiquitylation substrate

Hamilton

Cook²,

McRackan³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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Heterotrimeric G proteins transduce signals from activated transmembrane G protein-coupled receptors to appropriate downstream effectors within cells. Signaling specificity is achieved in part by the specific ␣, ␤, and ␥ subunits that compose a given heterotrimer. Additional structural and functional diversity in these subunits is generated at the level of posttranslational modification, offering alternate regulatory mechanisms for G protein signaling. Presented here is the identification of a variant of the ␥ 2 subunit of G protein heterotrimer purified from bovine brain and the demonstration that this RDTASIA ␥2 variant, containing unique amino acid sequence at its N terminus, is a substrate for ubiquitylation and degradation via the N-end rule pathway. Although N-end-dependent degradation has been shown to have important functions in peptide import, chromosome segregation, angiogenesis, and cardiovascular development, the identification of cellular substrates in mammalian systems has remained elusive. The isolation of RDTASIA ␥2 from a native tissue represents identification of a mammalian N-end rule substrate from a physiological source, and elucidates a mechanism for the targeting of G protein ␥ subunits for ubiquitylation and degradation.T he G protein heterotrimer, composed of an ␣, ␤, and ␥ subunit, is complexed with the nucleotide GDP in its inactive state. Upon activation, GDP is exchanged for GTP, resulting in subunit dissociation, and the GTP-bound ␣ or the ␤␥ dimer can both subsequently modulate the activity of a variety of downstream effectors (1, 2). The ␥ subunit, although generally associated in the cell with a ␤ subunit, is the smallest and most variable heterotrimeric G protein subunit (3, 4) and has been the least well understood with regard to its specific biological function. Several well established lipid modifications of ␥ subunits include farnesylation or geranylgeranylation of the C terminus (5, 6), and these modifications play an important role in membrane association, receptor coupling, and effector regulation (7-9). Recent work suggests a role for the ␥ subunit of transducin in ubiquitylation and destabilization of G t ␤␥ dimers (10). Although it is unclear whether such observations can be extended to other distinct ␥ isoforms, the crucial question of how a ␥ subunit could be targeted to the ubiquitylation machinery in a regulated fashion also remains unanswered. The amino terminal region of ␥ subunits is the region most divergent between the 12 known ␥ isoforms (11), an observation which suggests that the first 20 amino acids may be crucial for a specific function or subcellular localization. Indeed, presented here is the identification of an N-terminal variant of the G␥ 2 subunit isolated from bovine brain. Our discovery that this RDTASIA ␥ 2 variant undergoes N-end rule degradation highlights regulated degradation as an important function for the ␥ subunit of the G protein heterotrimer, offering a mechanism by which G protein subunits can be targeted to enzymatic components of the...

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Section: Discussionmentioning

confidence: 89%

γ ₂ subunit of G protein heterotrimer is an N-end rule ubiquitylation substrate

Hamilton

Cook²,

McRackan³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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“…In the pheromone response pathway, receptors (37)(38)(39), the G␣ subunit (40,41), and the effector kinase Ste7 (42,43) are known substrates for ubiquitination. Ubiquitination of Ste7 is dependent on prolonged pheromone stimulation, and once ubiquitinated it is rapidly degraded by the proteasome (42,43).…”

Section: Discussionmentioning

confidence: 99%

“…Ubi, ubiquitin; IB, immunoblot; WT, wild type; CHX, cycloheximide. ubiquitination site to be directly mapped by mass spectrometry (41).…”

Section: Discussionmentioning

confidence: 99%

Regulators of G Protein Signaling and Transient Activation of Signaling

Hao

Yıldırım

Wang

et al. 2003

Journal of Biological Chemistry

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Cellular responses to hormones and neurotransmitters are necessarily transient. The mating pheromone signal in yeast is typical. Signal initiation requires cell surface receptors, a G protein heterotrimer, and downstream effectors. Signal inactivation requires Sst2, a regulator of G protein signaling (RGS) protein that accelerates GTPase activity. We conducted a quantitative analysis of RGS and G protein expression and devised computational models that describe their activity in vivo. These results indicated that pheromone-dependent transcriptional induction of the RGS protein constitutes a negative feedback loop that leads to desensitization. Modeling also suggested the presence of a positive feedback loop leading to resensitization of the pathway. In confirmation of the model, we found that the RGS protein is ubiquitinated and degraded in response to pheromone stimulation. We identified and quantitated these positive and negative feedback loops, which account for the transient response to external signals observed in vivo.One measure of our understanding of biological systems is our ability to predict their behavior in detail. One aspect of this endeavor is to model signal transduction events, defined here as the dynamic changes that occur within a cell in response to an external stimulus (1-3). Such models can help us to understand how small changes outside a cell produce strongly amplified changes within a cell, how graded signals are converted to all-or-none responses (4), or how activators of one pathway influence the function of a second pathway (5). A second goal, and the focus of this work, is to understand how transient external signals are prevented from being propagated indefinitely within the cell. Here we describe the molecular basis for signal activation, desensitization, and eventual resensitization of G proteins by receptors and RGS 1 proteins. The experimentally observed behavior is described mechanistically by computational modeling of the pathway.For these studies we investigated the mating pheromone signaling pathway in yeast Saccharomyces cerevisiae. The yeast mating response is arguably the best characterized signal transduction pathway of any eukaryote, and it has long served as a prototype for hormone, neurotransmitter, and sensory response systems in humans (6). Disruption or activation of pathway components leads to highly specific changes that can be easily quantified. Finally, because it is a unicellular eukaryote, every cell in a population is genetically and phenotypically identical (all cells are "typical").Mating in yeast is the fusion of a and ␣ haploid cell types to form an a/␣ diploid. The events leading to fusion are initiated by specific pheromones: ␣-type cells secrete ␣-factor pheromone, which binds to a specific receptor (Ste2) on a-cells, while a-cells secrete a-factor that binds to receptors (Ste3) on ␣-cells. Upon pheromone binding to its receptor, the G protein ␣ subunit (Gpa1) releases GDP, binds to GTP, and liberates the G protein ␤␥ subunits (Ste4/Ste18). Sustained...

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“…Therefore, desensitization to pheromone still occurs with respect to G protein activation. Further, a lysine to arginine substitution mutation in G␣-Gpa1 that prevents its ubiquitin-dependent degradation causes an enhanced adaptation response (52). It remains possible that other components of the pathway upstream from Fus3 might be subject to ubiquitindependent degradation and would also contribute to attenuation of Fus3 activation.…”

Section: Discussionmentioning

confidence: 99%

Pheromone induction promotes Ste11 degradation through a MAPK feedback and ubiquitin-dependent mechanism

Esch

Errede

2002

Proc. Natl. Acad. Sci. U.S.A.

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Ste11 is the mitogen-activated protein kinase (MAPK) kinase kinase in the MAPK cascades that mediate mating, high osmolarity glycerol, and filamentous growth responses in Saccharomyces cerevisiae. We show stimulation of the mating pathway by pheromone promotes an accelerated turnover of Ste11 through a MAPK feedback and ubiquitin-dependent mechanism. This degradation is pathway specific, because Ste11 is stable during activation of the high osmolarity glycerol pathway. Because the steady-state amount of Ste11 does not change significantly during pheromone induction, we infer that maintenance of MAPK activation involves repeated cycles in which naïve Ste11 is activated and then targeted for degradation. This model predicts that elimination of active Ste11 would rapidly curtail MAPK activation upon attenuation of the upstream signal. This prediction is confirmed by the finding that blocking ubiquitin-dependent Ste11 degradation during pheromone induction abolishes the characteristic attenuation profile for MAPK activation.

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Direct Identification of a G Protein Ubiquitination Site by Mass Spectrometry

Cited by 89 publications

References 45 publications

γ ₂ subunit of G protein heterotrimer is an N-end rule ubiquitylation substrate

γ ₂ subunit of G protein heterotrimer is an N-end rule ubiquitylation substrate

Regulators of G Protein Signaling and Transient Activation of Signaling

Pheromone induction promotes Ste11 degradation through a MAPK feedback and ubiquitin-dependent mechanism

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Direct Identification of a G Protein Ubiquitination Site by Mass Spectrometry

Cited by 89 publications

References 45 publications

γ 2 subunit of G protein heterotrimer is an N-end rule ubiquitylation substrate

γ 2 subunit of G protein heterotrimer is an N-end rule ubiquitylation substrate

Regulators of G Protein Signaling and Transient Activation of Signaling

Pheromone induction promotes Ste11 degradation through a MAPK feedback and ubiquitin-dependent mechanism

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γ ₂ subunit of G protein heterotrimer is an N-end rule ubiquitylation substrate

γ ₂ subunit of G protein heterotrimer is an N-end rule ubiquitylation substrate