Oligomerization Regulates the Localization of Cdc24, the Cdc42 Activator in Saccharomyces cerevisiae

Mionnet, Cyril; Bogliolo, Stéphanie; Arkowitz, Robert A.

doi:10.1074/jbc.m800305200

Cited by 22 publications

(20 citation statements)

References 50 publications

(54 reference statements)

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“…For example, binding of Bud1 and Bem1 to Cdc24 has been proposed to allosterically activate Cdc24 by promoting an open conformation and/or be instrumental in bringing Cdc24 to the plasma membrane [14], [15], [19], [20]. Oligomerization of Cdc24 has also been proposed to regulate its activity [41]. Furthermore, two studies have characterized the phosphorylation of Cdc42 GTPase activating proteins (GAPs) and suggested that Cdc42 activation at the initiation of budding may be primarily regulated through downregulation of GAP activities through phosphorylation [42], [43].…”

Section: Discussionmentioning

confidence: 99%

Multisite Phosphorylation of the Guanine Nucleotide Exchange Factor Cdc24 during Yeast Cell Polarization

Wai

Gerber

2009

PLoS ONE

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BackgroundCell polarization is essential for processes such as cell migration and asymmetric cell division. A common regulator of cell polarization in most eukaryotic cells is the conserved Rho GTPase, Cdc42. In budding yeast, Cdc42 is activated by a single guanine nucleotide exchange factor, Cdc24. The mechanistic details of Cdc24 activation at the onset of yeast cell polarization are unclear. Previous studies have suggested an important role for phosphorylation of Cdc24, which may regulate activity or function of the protein, representing a key step in the symmetry breaking process.Methodology/Principal FindingsHere, we directly ask whether multisite phosphorylation of Cdc24 plays a role in its regulation. We identify through mass spectrometry analysis over thirty putative in vivo phosphorylation sites. We first focus on sites matching consensus sequences for cyclin-dependent and p21-activated kinases, two kinase families that have been previously shown to phosphorylate Cdc24. Through site-directed mutagenesis, yeast genetics, and light and fluorescence microscopy, we show that nonphosphorylatable mutations of these consensus sites do not lead to any detectable consequences on growth rate, morphology, kinetics of polarization, or localization of the mutant protein. We do, however, observe a change in the mobility shift of mutant Cdc24 proteins on SDS-PAGE, suggesting that we have indeed perturbed its phosphorylation. Finally, we show that mutation of all identified phosphorylation sites does not cause observable defects in growth rate or morphology.Conclusions/SignificanceWe conclude that lack of phosphorylation on Cdc24 has no overt functional consequences in budding yeast. Yeast cell polarization may be more tightly regulated by inactivation of Cdc42 by GTPase activating proteins or by alternative methods of Cdc24 regulation, such as conformational changes or oligomerization.

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Section: Discussionmentioning

confidence: 99%

Multisite Phosphorylation of the Guanine Nucleotide Exchange Factor Cdc24 during Yeast Cell Polarization

Wai

Gerber

2009

PLoS ONE

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“…Localization and activity of Cdc24p are regulated at different levels (Gulli and Peter, 2001). Cdc24p is subject to cell cycle–dependent nuclear sequestration (Toenjes et al , 1999; Nern and Arkowitz, 2000; Shimada et al , 2000), oligomerization (Mionnet et al , 2008), and autoinhibition (Shimada et al , 2004). In addition, Cdk- and Cla4-dependent phosphorylation of Cdc24 has been observed, but the biological function of these modifications is still elusive (Gulli et al , 2000; Bose et al , 2001; Moffat and Andrews, 2004; Cole et al , 2009; Wai et al , 2009).…”

Section: Introductionmentioning

confidence: 99%

Cla4 kinase triggers destruction of the Rac1-GEF Cdc24 during polarized growth inUstilago maydis

Frieser

Hlubek

Sandrock

et al. 2011

MBoC

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“…It was suggested that oligomerization of onco-Dbl is essential for cellular transformation (Zhu et al 2001). Oligomerization of the yeast RhoGEF, Cdc24, was shown to control its localization to the bud tip and nucleus, and the oligomer dissociation was necessary for nuclear export (Mionnet et al 2008). Site-specific mutagenesis revealed that the conserved region 2 of Cdc24 had a major role in these processes.…”

Section: Discussionmentioning

confidence: 99%

N-terminal Dbl domain of the RhoGEF, Kalirin

et al. 2012

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Guanine nucleotide exchange factors (GEF) promote the release of GDP from GTPases, thus allowing the free GTPase molecule to bind the more abundant GTP molecule. In the GTPbound state, the GTPase elicits signal transduction by acting on its effector proteins. Spontaneous release of GDP is a slow process and the catalysis of the GDP release by a GEF is generally a prerequisite for efficient signaling (Vetter and Wittinghofer 2001). The structurally related GEFs form subfamilies that regulate a specific family of GTPase proteins. GEFs that activate Rho GTPases have been implicated in cancer and mental retardation. RhoGEFs are a relatively large family, and many of the ~69 human RhoGEFs were discovered based on their oncogenic activation in cancer and cancer models. The catalytic components of RhoGEFs are referred to as Dbl homology domains, after the screen that identified the protein Dbl encoded by the diffuse B-cell lymphoma (dbl) oncogene (Eva and Aaronson 1985). Thus the RhoGEF family is a potential target for treating tumors and cancer.RhoGEFs contain either the Dbl-homology (DH) domain followed by a pleckstrinhomology (PH) domain or the structurally unrelated domain in the DOCK (dedicator of cytokinesis) family. A preliminary mechanism of GEF action on GTPase was proposed based mainly on available X-ray structures of the complexes GEF/GTPase or GDP/GTPase, but the biophysical basis for GEF activity remains elusive (Vetter and Wittinghofer 2001). Crystallographic studies have shown that the binding site of RhoGEFs for GTPases is located on the DH domain. Upon binding, the DH domain displaces the switch 1 and 2 loops, and the P loop of a RhoGTPase from the orientation required to hold GDP in the catalytic pocket. As a result of these conformational changes in the RhoGTPase, the affinity for a nucleotide decreases and GDP dissociates (Vetter and Wittinghofer 2001). However, in some cases the presence of an adjacent PH domain facilitates, or is even essential for full catalytic activity of the DH domain (Rossman et al. 2005).In this work we describe studies on a DH domain from rat Kalirin, a large multifunctional protein found primarily in the nervous system (Ma et al. 2001;Rabiner et al. 2005). Kalirin is involved in axon and dendritic spine formation, regulation of neuropeptide formation and induction of nitric-oxide synthase (iNOS) activity. Several isoforms of Kalirin have been found that differ in the C-terminus by the presence of additional domains: the second DH/ * Corresponding author. hoch@uchc.edu. Database accession numbersThe chemical shift assignments, NOEs, RDCs and the coordinates of the Kalirin DH1 domain have been deposited to BMRB and RCSB, entries ID 16632 and 2KR9, respectively. et al. 2006), and propose a mechanism of action for CPEPD based on our observations. NIH Public Access Methods and results Protein expression and purificationIsotopically 15 N-and 15 N, 13 C-labeled rat Kalirin DH1 domains and unlabeled Rac1 were overexpressed in E. coli. as GST fusion proteins. Purification was perf...

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Oligomerization Regulates the Localization of Cdc24, the Cdc42 Activator in Saccharomyces cerevisiae

Cited by 22 publications

References 50 publications

Multisite Phosphorylation of the Guanine Nucleotide Exchange Factor Cdc24 during Yeast Cell Polarization

Multisite Phosphorylation of the Guanine Nucleotide Exchange Factor Cdc24 during Yeast Cell Polarization

Cla4 kinase triggers destruction of the Rac1-GEF Cdc24 during polarized growth inUstilago maydis

N-terminal Dbl domain of the RhoGEF, Kalirin

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