Regulation of the Yeast Amphiphysin Homologue Rvs167p by Phosphorylation

Friesen, Helena; Murphy, Kelly; Breitkreutz, Ashton; Tyers, Mike; Andrews, Brenda

doi:10.1091/mbc.e02-09-0613

Cited by 33 publications

(48 citation statements)

References 66 publications

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“…The reported localization of these proteins is consisWestern assay (Friesen et al 2003). Thus the interaction of the SH3 domain of Rvs167p with its ligands may be tent with the interactions we describe here.…”

Section: Have Shown That Gyp5psupporting

confidence: 68%

“…The integrity of all PCR products was genesis (Lee and Schekman 2004;Peter et al 2004). in glycine, proline, and alanine (the GPA region) and Affinity chromatography: SH3 domain proteins containing 6-His tags at their carboxy termini were purified from strain is thought to play a role in Rvs regulation since it is GJ1158 (Bhandari and Gowrishankar 1997) using Ni-NTA phosphorylated in vivo (Friesen et al 2003 Uetz et al 2000;Drees et al 2001;Ito et al 2001; Tong 10-l frit made of 150-to 212-m glass beads (Sigma, Oakville, et al 2002;Talarek et al 2005); however, few of these Ontario). Yeast extracts were made and chromatography was done as described by Ho et al (1997) (Colwill et al 1999).…”

Section: Methodsmentioning

confidence: 99%

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Interaction of the Saccharomyces cerevisiae Cortical Actin Patch Protein Rvs167p With Proteins Involved in ER to Golgi Vesicle Trafficking

Friesen

Colwill²,

Robertson³

et al. 2005

Genetics

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We have used affinity chromatography to identify two proteins that bind to the SH3 domain of the actin cytoskeleton protein Rvs167p: Gyp5p and Gyl1p. Gyp5p has been shown to be a GTPase activating protein (GAP) for Ypt1p, a Rab GTPase involved in ER to Golgi trafficking; Gyl1p is a protein that resembles Gyp5p and has recently been shown to colocalize with and belong to the same protein complex as Gyp5p. We show that Gyl1p and Gyp5p interact directly with each other, likely through their carboxyterminal coiled-coil regions. In assays of GAP activity, Gyp5p had GAP activity toward Ypt1p and we found that this activity was stimulated by the addition of Gyl1p. Gyl1p had no GAP activity toward Ypt1p. Genetic experiments suggest a role for Gyp5p and Gyl1p in ER to Golgi trafficking, consistent with their biochemical role. Since Rvs167p has a previously characterized role in endocytosis and we have shown here that it interacts with proteins involved in Golgi vesicle trafficking, we suggest that Rvs167p may have a general role in vesicle trafficking.

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Section: Have Shown That Gyp5psupporting

confidence: 68%

Section: Methodsmentioning

confidence: 99%

Interaction of the Saccharomyces cerevisiae Cortical Actin Patch Protein Rvs167p With Proteins Involved in ER to Golgi Vesicle Trafficking

Friesen

Colwill²,

Robertson³

et al. 2005

Genetics

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“…A BamHI fragment containing full-length RVS167 was subcloned into pAC-GHLT (BD Biosciences PharMingen) that had been digested with BglII to generate a glutathione S-transferase (GST)-His-tagged Rvs167p fusion product as described previously (Friesen et al, 2003). Recombinant baculoviruses containing RVS161 and RVS167 were isolated from Sf9 insect cells and coinfected into Hi5 cells as recommended by the manufacturer (Invitrogen).…”

Section: Expression and Purification Of Rvs167p And Rvs161p From Insementioning

confidence: 99%

“…This Rvs167p SH3 mutant protein does not interact with the proline-rich region of Abp1 in a two-hybrid assay, but it does bind other proteins that interact with Rvs167p outside the SH3 domain, indicating that this mutant specifically disrupts the function of the SH3 domain (Colwill et al, 1999). We also tested for complementation using a plasmid with RVS167 encoding a protein with alanine substitutions at all four of the residues that are phosphorylated in vivo (Rvs167p-4A; Friesen et al, 2003). The rvs167⌬ xxx⌬ mutants were complemented equally efficiently by wild-type RVS167 and by RVS167-P473L and by RVS167-4A ( Figure 4B).…”

mentioning

confidence: 99%

Characterization of the Yeast Amphiphysins Rvs161p and Rvs167p Reveals Roles for the Rvs Heterodimer In Vivo

Friesen

Humphries

et al. 2006

MBoC

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We have used comprehensive synthetic lethal screens and biochemical assays to examine the biological role of the yeast amphiphysin homologues Rvs161p and Rvs167p, two proteins that play a role in regulation of the actin cytoskeleton, endocytosis, and sporulation. We found that unlike some forms of amphiphysin, Rvs161p-Rvs167p acts as an obligate heterodimer during vegetative growth and neither Rvs161p nor Rvs167p forms a homodimer in vivo. RVS161 and RVS167 have an identical set of 49 synthetic lethal interactions, revealing functions for the Rvs proteins in cell polarity, cell wall synthesis, and vesicle trafficking as well as a shared role in mating. Consistent with these roles, we show that the Rvs167p-Rvs161p heterodimer, like its amphiphysin homologues, can bind to phospholipid membranes in vitro, suggesting a role in vesicle formation and/or fusion. Our genetic screens also reveal that the interaction between Abp1p and the Rvs167p Src homology 3 (SH3) domain may be important under certain conditions, providing the first genetic evidence for a role for the SH3 domain of Rvs167p. Our studies implicate heterodimerization of amphiphysin family proteins in various functions related to cell polarity, cell integrity, and vesicle trafficking during vegetative growth and the mating response. INTRODUCTIONRVS161 and RVS167, which encode closely related proteins in Saccharomyces cerevisiae, were first identified in a screen for mutants that exhibited reduced viability upon starvation (Bauer et al., 1993). Mutation of RVS161 or RVS167 causes a phenotype consistent with a role for the Rvs proteins in cortical actin cytoskeleton organization and endocytosis: loss of viability and unusual cell morphology in poor growth medium or salt-containing medium, delocalized actin distribution under suboptimal growth conditions, abnormal (random) budding in diploids, and defects in endocytosis and sporulation (Bauer et al., 1993). Ultrastructural studies have revealed that rvs mutants accumulate late secretory vesicles at sites of membrane and cell wall construction (Breton et al., 2001), suggesting an additional role for Rvs161p and Rvs167p in vesicle trafficking.Rvs161p and Rvs167p are members of the BAR-domain family of proteins, which includes Bin1, Amphiphysin, and Rvs proteins (Sivadon et al., 1997). Amphiphysins are enriched in the mammalian brain and seem to function in synaptic vesicle endocytosis (for review, see Zhang and Zelhof, 2002). Bin1 is a splice isoform of amphiphysin 2 that has features of a tumor suppressor (Sakamuro et al., 1996). Proteins in this family are characterized by the presence of a conserved BAR domain, and it is through their BAR domains that Rvs161p interacts with Rvs167p (Navarro et al., 1997;Sivadon et al., 1997;Colwill et al., 1999). The crystal structure of the BAR domain of Drosophila amphiphysin has recently been solved (Peter et al., 2004). It is a crescentshaped dimer, in which each monomer forms a coiled coil. The curved shape is only revealed upon dimerization of the two slightly kinked ...

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“…Cdc28p and the mating MAPKs are both proline-dependent kinases with potential for overlap if they recognize minimal S/TP sites in Ste5p, although their idealized consensus recognition sites are likely to be different. Overlap in recognition sites has been noted for the Fus3p and Pcl/Pho85 class of cyclin-dependent kinases (45). Possible levels of regulation of Ste5p include influencing the binding properties of the kinases or other components or influencing the stability of the Ste5p signaling complex at the plasma membrane.…”

Section: Hyperactivation Of the Mating Mapk Cascade In The Absence Ofmentioning

confidence: 99%

Localized Feedback Phosphorylation of Ste5p Scaffold by Associated MAPK Cascade

Flotho¹,

Simpson

et al. 2004

Journal of Biological Chemistry

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Scaffold proteins play pivotal roles during signal transduction. In Saccharomyces cerevisiae, the Ste5p scaffold protein is required for activation of the mating MAPK cascade in response to mating pheromone and assembles a G protein-MAPK cascade complex at the plasma membrane. To serve this function, Ste5p undergoes a regulated localization event involving nuclear shuttling and recruitment to the cell cortex. Here, we show that Ste5p is also subject to two types of phosphorylation and increases in abundance as a result of MAPK activation. During vegetative growth, Ste5p is basally phosphorylated through a process regulated by the CDK Cdc28p. During mating pheromone signaling, Ste5p undergoes increased phosphorylation by the mating MAPK cascade. Multiple kinases of the mating MAPK cascade contribute to pheromone-induced phosphorylation of Ste5p, with the mating MAPKs contributing the most. Pheromone induction or overexpression of the Ste4p G␤ subunit increases the abundance of Ste5p at a post-translational step, as long as the mating MAPKs are present. Increasing the level of MAPK activation increases the amount of Ste5p at the cell cortex. Analysis of Ste5p localization mutants reveals a strict requirement for Ste5p recruitment to the plasma membrane for the pheromone-induced phosphorylation. These results suggest that the pool of Ste5p that is recruited to the plasma membrane selectively undergoes feedback phosphorylation by the associated MAPKs, leading to an increased pool of Ste5p at the site of polarized growth. These findings provide evidence of a spatially regulated mechanism for post-activation control of a signaling scaffold that potentiates pathway activation.Cells employ complex signal transduction networks to properly adapt to environmental stimuli and integrate different external cues with the physiological state of the cell. Scaffold and adapter proteins play crucial roles in mediating the temporal and spatial organization of the networks of signal transduction enzymes that mediate responses to stimuli (1-8). To fulfill these tasks, scaffold and adapter proteins are regulated at the level of phosphorylation, oligomerization, and cellular distribution, which may influence the activity and location of the scaffold and its binding partners (9 -14).The mating response of haploid Saccharomyces cerevisiae cells provides one of the best studied examples of a signaling pathway that is regulated by a scaffold protein (15-17). Upon binding of mating pheromone to a G protein-coupled receptor of the serpentine family, the G␤␥ (Ste4p/Ste18p) dimer of the G protein is released from an inhibitory G␣ subunit (Gpa1p) and activates a mitogen-activated protein kinase (MAPK) 1 cascade. The MAPK cascade consists of a MAPKKK Ste11p, a MAPKK Ste7p, and two MAPKs, Fus3p and Kss1p, of which Fus3p is the major MAPK. The relay of the signal through the MAPK cascade is achieved through sequential phosphorylation of each kinase. Previous work has established that the Ste5p scaffold is essential for this signal relay and plays ...

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Regulation of the Yeast Amphiphysin Homologue Rvs167p by Phosphorylation

Cited by 33 publications

References 66 publications

Interaction of the Saccharomyces cerevisiae Cortical Actin Patch Protein Rvs167p With Proteins Involved in ER to Golgi Vesicle Trafficking

Interaction of the Saccharomyces cerevisiae Cortical Actin Patch Protein Rvs167p With Proteins Involved in ER to Golgi Vesicle Trafficking

Characterization of the Yeast Amphiphysins Rvs161p and Rvs167p Reveals Roles for the Rvs Heterodimer In Vivo

Localized Feedback Phosphorylation of Ste5p Scaffold by Associated MAPK Cascade

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