Cell Cycle-dependent Phosphorylation and Ubiquitination of a G Protein α Subunit

Torres, Matthew P.; Clement, Sarah T.; Cappell, Steven D.; Dohlman, Henrik G.

doi:10.1074/jbc.m111.239343

Cited by 22 publications

(32 citation statements)

References 58 publications

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“…We previously showed that Elm1 phosphorylates Gpa1, and that phosphorylation is regulated in a cell cycle–dependent manner (6). Elm1 also phosphorylates Snf1, among other substrates; however, in this case, phosphorylation occurs in response to glucose limitation.…”

Section: Resultsmentioning

confidence: 99%

“…In vitro kinase assays were performed by incubating 0.075 to 0.15 pmol of purified TAP kinase (corresponding to a final concentration of 3 to 6 nM) and 12.5 pmol of recombinant Gpa1 (0.5 µM final concentration) in 1× kinase reaction buffer, as described previously for Elm1 (6). Reactions were stopped by the addition of 6× SDS-PAGE loading buffer, and samples were immediately subjected to 10% SDS-PAGE.…”

Section: Methodsmentioning

confidence: 99%

“…With this approach, we identified that the kinase Elm1 phosphorylates Gpa1. Under nutrient-rich conditions, Elm1 is present predominantly during the G 2 -M phase, and this leads to concomitant, cell cycle–dependent phosphorylation of Gpa1 (6). …”

Section: Introductionmentioning

confidence: 99%

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Regulation of Yeast G Protein Signaling by the Kinases That Activate the AMPK Homolog Snf1

2013

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Extracellular signals, such as nutrients and hormones, cue intracellular pathways to produce adaptive responses. Often, cells must coordinate their responses to multiple signals to produce an appropriate outcome. We showed that components of a glucose-sensing pathway acted on components of a heterotrimeric guanine nucleotide–binding protein (G protein)–mediated pheromone signaling pathway in the yeast Saccharomyces cerevisiae. We demonstrated that the G protein α subunit Gpa1 was phosphorylated in response to conditions of reduced glucose availability and that this phosphorylation event contributed to reduced pheromone-dependent stimulation of mitogen-activated protein kinases, gene transcription, cell morphogenesis, and mating efficiency. We found that Elm1, Sak1, and Tos3, the kinases that phosphorylate Snf1, the yeast homolog of adenosine monophosphate–activated protein kinase (AMPK), in response to limited glucose availability, also phosphorylated Gpa1 and contributed to the diminished mating response. Reg1, the regulatory subunit of the phosphatase PP1 that acts on Snf1, was likewise required to reverse the phosphorylation of Gpa1 and to maintain the mating response. Thus, the same kinases and phosphatase that regulate Snf1 also regulate Gpa1. More broadly, these results indicate that the pheromone signaling and glucose-sensing pathways communicate directly to coordinate cell behavior.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Regulation of Yeast G Protein Signaling by the Kinases That Activate the AMPK Homolog Snf1

2013

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“…By precedent, the yeast G␣ subunit is phosphorylated by a non-receptor kinase in response to changes in the cell cycle (48) and is also phosphorylated in response to the limited availability of glucose (49). Also, in the membranes of human leukemia HL-60 cells during activation of G proteins, the G␤ subunit is phosphorylated (50).…”

Section: Discussionmentioning

confidence: 99%

Direct Modulation of Heterotrimeric G Protein-coupled Signaling by a Receptor Kinase Complex

Tunc‐Ozdemir

Urano

Jaiswal

et al. 2016

Journal of Biological Chemistry

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Plants and some protists have heterotrimeric G protein complexes that activate spontaneously without canonical G proteincoupled receptors (GPCRs). In Arabidopsis, the sole 7-transmembrane regulator of G protein signaling 1 (AtRGS1) modulates the G protein complex by keeping it in the resting state (GDP-bound). However, it remains unknown how a myriad of biological responses is achieved with a single G protein modulator. We propose that in complete contrast to G protein activation in animals, plant leucine-rich repeat receptor-like kinases (LRR RLKs), not GPCRs, provide this discrimination through phosphorylation of AtRGS1 in a ligand-dependent manner. G protein signaling is directly activated by the pathogen-associated molecular pattern flagellin peptide 22 through its LRR RLK, FLS2, and co-receptor BAK1.

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“…(26), polyubiquitination (SCF/Cdc4) (29), deubiquitination (Ubp12) (25), phosphorylation (Elm1, Tos1, Sak3) (30,31,66), and dephosphorylation (Reg1) (66) as well as the seven UBD proteins identified here. We conclude that the ubiquitination domain likely evolved to serve a unique trafficking function and that this function is wholly separate from the regulation of G protein catalytic activity.…”

Section: Discussionmentioning

confidence: 99%

Guanine Nucleotide-binding Protein (Gα) Endocytosis by a Cascade of Ubiquitin Binding Domain Proteins Is Required for Sustained Morphogenesis and Proper Mating in Yeast

Dixit¹,

Baker²,

Sacks³

et al. 2014

Journal of Biological Chemistry

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Background:The yeast G␣ protein contains a unique domain that is monoubiquitinated, leading to vacuolar degradation. Results: A gene deletion screen reveals ubiquitin binding domain proteins necessary for G␣ trafficking. Loss of the ubiquitination domain impedes cellular morphogenesis and mating. Conclusion: Proper endocytosis of G␣ is required for sustained morphogenesis and efficient mating. Significance: G␣ endocytosis promotes signaling. Heterotrimeric G proteins are well known to transmit signals from cell surface receptors to intracellular effector proteins.There is growing appreciation that G proteins are also present at endomembrane compartments, where they can potentially interact with a distinct set of signaling proteins. Here, we examine the cellular trafficking function of the G protein ␣ subunit in yeast, Gpa1. Gpa1 contains a unique 109-amino acid insert within the ␣-helical domain that undergoes a variety of posttranslational modifications. Among these is monoubiquitination, catalyzed by the NEDD4 family ubiquitin ligase Rsp5. Using a newly optimized method for G protein purification together with biophysical measures of structure and function, we show that the ubiquitination domain does not influence enzyme activity. By screening a panel of 39 gene deletion mutants, each lacking a different ubiquitin binding domain protein, we identify seven that are necessary to deliver Gpa1 to the vacuole compartment including four proteins (Ede1, Bul1, Ddi1, and Rup1) previously not known to be involved in this process. Finally, we show that proper endocytosis of the G protein is needed for sustained cellular morphogenesis and mating in response to pheromone stimulation. We conclude that a cascade of ubiquitin-binding proteins serves to deliver the G protein to its final destination within the cell. In this instance and in contrast to the previously characterized visual system, endocytosis from the plasma membrane is needed for proper signal transduction rather than for signal desensitization.G␣ proteins are enzymatic switches that are part of a multicomponent signaling complex. The complex typically consists of a seven-transmembrane G protein-coupled receptor, a guanine nucleotide-binding protein (G␣), and an associated dimer consisting of ␤ and ␥ subunits (G␤␥) (1). Signaling is turned on and off based on receptor activation, which in turn dictates the nucleotide-bound state of the G␣ protein. When G␣ is GDPbound, G␤␥ is sequestered, and signaling pathways are off (1). When G␣ releases GDP and binds GTP, G␤␥ dissociates, and the signaling pathways are turned on. Subsequent GTP hydrolysis is accelerated by regulators of G protein signaling (RGS 3 proteins) (2, 3). Large G␣ proteins contain a Ras-like domain as well as an independently folded ␣-helical domain (1). Within this group of proteins there is a well established role for the Ras-like domain in specifying interactions with G␤␥, effectors, and RGS proteins (1). However, recent evidence has shown that the ␣-helical domain is also important for signa...

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Cell Cycle-dependent Phosphorylation and Ubiquitination of a G Protein α Subunit

Cited by 22 publications

References 58 publications

Regulation of Yeast G Protein Signaling by the Kinases That Activate the AMPK Homolog Snf1

Regulation of Yeast G Protein Signaling by the Kinases That Activate the AMPK Homolog Snf1

Direct Modulation of Heterotrimeric G Protein-coupled Signaling by a Receptor Kinase Complex

Guanine Nucleotide-binding Protein (Gα) Endocytosis by a Cascade of Ubiquitin Binding Domain Proteins Is Required for Sustained Morphogenesis and Proper Mating in Yeast

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