Pxl1p, a Paxillin-like Protein inSaccharomyces cerevisiae, May Coordinate Cdc42p and Rho1p Functions during Polarized Growth

Rho-family GTPases Cdc42p and Rho1p play critical roles in the budding process of the yeast Saccharomyces cerevisiae. However, it is not clear how the functions of these GTPases are coordinated temporally and spatially during this process. Based on its ability to suppress cdc42-Ts mutants when overexpressed, a novel gene PXL1 was identified. Pxl1p resembles mammalian paxillin, which is involved in integrating various signaling events at focal adhesion. Both proteins share amino acid sequence homology and structural organization. When expressed in yeast, chicken paxillin localizes to the sites of polarized growth as Pxl1p does. In addition, the LIM domains in both proteins are the primary determinant for targeting the proteins to the cortical sites in their native cells. These data strongly suggest that Pxl1p is the “ancient paxillin” in yeast. Deletion of PXL1 does not produce any obvious phenotype. However, Pxl1p directly binds to Rho1p-GDP in vitro, and inhibits the growth of rho1-2 and rho1-3 mutants in a dosage-dependent manner. The opposite effects of overexpressed Pxl1p on cdc42 and rho1 mutants suggest that the functions of Cdc42p and Rho1p may be coordinately regulated during budding and that Pxl1p may be involved in this coordination.

Download Full-text

Direct interaction of the tau 1 transactivation domain of the human glucocorticoid receptor with the basal transcriptional machinery

Molecular and Cellular Biology ◽

10.1128/mcb.13.1.399-407.1993 ◽

1993 ◽

Vol 13 (1) ◽

pp. 399-407

Author(s):

I J McEwan ◽

A P Wright ◽

K Dahlman-Wright ◽

J Carlstedt-Duke ◽

J A Gustafsson

Keyword(s):

Glucocorticoid Receptor ◽

Protein Interactions ◽

Core Promoter ◽

Specific Interaction ◽

Direct Interaction ◽

Dependent Manner ◽

Transactivation Domain ◽

Yeast Saccharomyces Cerevisiae ◽

Concentration Dependent Manner

We have used a yeast (Saccharomyces cerevisiae) cell free transcription system to study protein-protein interactions involving the tau 1 transactivation domain of the human glucocorticoid receptor that are important for transcriptional transactivation by the receptor. Purified tau 1 specifically inhibited transcription from a basal promoter derived from the CYC1 gene and from the adenovirus 2 major late core promoter in a concentration-dependent manner. This inhibition or squelching was correlated with the transactivation activity of tau 1. Recombinant yeast TATA-binding protein (yTFIID), although active in vitro, did not specifically reverse the inhibitory effect of tau 1. In addition, no specific interaction between tau 1 and yTFIID could be shown in vitro by affinity chromatography. Taken together, these results indicate that the tau 1 transactivation domain of the human glucocorticoid receptor interacts directly with the general transcriptional apparatus through some target protein(s) that is distinct from the TATA-binding factor. Furthermore, this assay can be used to identify interacting factors, since after phosphocellulose chromatography of a whole-cell yeast extract, a fraction that contained an activity which selectively counteracted the squelching effect of tau 1 was found.

Download Full-text

Spire contains actin binding domains and is related to ascidian posterior end mark-5

Development ◽

10.1242/dev.126.23.5267 ◽

1999 ◽

Vol 126 (23) ◽

pp. 5267-5274 ◽

Cited By ~ 5

Author(s):

A. Wellington ◽

S. Emmons ◽

B. James ◽

J. Calley ◽

M. Grover ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Posterior Pole ◽

Actin Binding ◽

Binding Domains ◽

Trap System ◽

Rho Family Gtpases ◽

Rho Family ◽

Interaction Trap ◽

Anterior Posterior

Spire is a maternal effect locus that affects both the dorsal-ventral and anterior-posterior axes of the Drosophila egg and embryo. It is required for localization of determinants within the developing oocyte to the posterior pole and to the dorsal anterior corner. During mid-oogenesis, spire mutants display premature microtubule-dependent cytoplasmic streaming, a phenotype that can be mimicked by pharmacological disruption of the actin cytoskeleton with cytochalasin D. Spire has been cloned by transposon tagging and is related to posterior end mark-5, a gene from sea squirts that encodes a posteriorly localized mRNA. Spire mRNA is not, however, localized to the posterior pole. SPIRE also contains two domains with similarity to the actin monomer-binding WH2 domain, and we demonstrate that SPIRE binds to actin in the interaction trap system and in vitro. In addition, SPIRE interacts with the rho family GTPases RHOA, RAC1 and CDC42 in the interaction trap system. Thus, our evidence supports the model that SPIRE links rho family signaling to the actin cytoskeleton.

Download Full-text

Dependence of Dbl and Dbs Transformation on MEK and NF-κB Activation

Molecular and Cellular Biology ◽

10.1128/mcb.19.11.7759 ◽

1999 ◽

Vol 19 (11) ◽

pp. 7759-7770 ◽

Cited By ~ 85

Author(s):

Ian P. Whitehead ◽

Que T. Lambert ◽

Judith A. Glaven ◽

Karon Abe ◽

Kent L. Rossman ◽

...

Keyword(s):

Cyclin D1 ◽

Structural Integrity ◽

Small Gtpases ◽

Dependent Manner ◽

Nucleotide Exchange ◽

Ph Domain ◽

Transforming Activity ◽

Rho Family ◽

Altered Gene

ABSTRACT Dbs was identified initially as a transforming protein and is a member of the Dbl family of proteins (>20 mammalian members). Here we show that Dbs, like its rat homolog Ost and the closely related Dbl, exhibited guanine nucleotide exchange activity for the Rho family members RhoA and Cdc42, but not Rac1, in vitro. Dbs transforming activity was blocked by specific inhibitors of RhoA and Cdc42 function, demonstrating the importance of these small GTPases in Dbs-mediated growth deregulation. Although Dbs transformation was dependent upon the structural integrity of its pleckstrin homology (PH) domain, replacement of the PH domain with a membrane localization signal restored transforming activity. Thus, the PH domain of Dbs (but not Dbl) may be important in modulating association with the plasma membrane, where its GTPase substrates reside. Both Dbs and Dbl activate multiple signaling pathways that include activation of the Elk-1, Jun, and NF-κB transcription factors and stimulation of transcription from the cyclin D1 promoter. We found that Elk-1 and NF-κB, but not Jun, activation was necessary for Dbl and Dbs transformation. Finally, we have observed that Dbl and Dbs regulated transcription from the cyclin D1 promoter in a NF-κB-dependent manner. Previous studies have dissociated actin cytoskeletal activity from the transforming potential of RhoA and Cdc42. These observations, when taken together with those of the present study, suggest that altered gene expression, and not actin reorganization, is the critical mediator of Dbl and Rho family protein transformation.

Download Full-text

Identification of Yeast IQGAP (Iqg1p) as an Anaphase-Promoting-Complex Substrate and Its Role in Actomyosin-Ring-Independent Cytokinesis

Molecular Biology of the Cell ◽

10.1091/mbc.e07-05-0509 ◽

2007 ◽

Vol 18 (12) ◽

pp. 5139-5153 ◽

Cited By ~ 42

Author(s):

Nolan Ko ◽

Ryuichi Nishihama ◽

Gregory H. Tully ◽

Denis Ostapenko ◽

Mark J. Solomon ◽

...

Keyword(s):

Myosin Ii ◽

In Vitro Assays ◽

Dependent Manner ◽

Anaphase Promoting Complex ◽

Recognition Sequence ◽

Yeast Saccharomyces Cerevisiae ◽

Genes Encoding ◽

Mitotic Cyclin ◽

Strain Background

In the yeast Saccharomyces cerevisiae, a ring of myosin II forms in a septin-dependent manner at the budding site in late G1. This ring remains at the bud neck until the onset of cytokinesis, when actin is recruited to it. The actomyosin ring then contracts, septum formation occurs concurrently, and cytokinesis is soon completed. Deletion of MYO1 (the only myosin II gene) is lethal on rich medium in the W303 strain background and causes slow-growth and delayed-cell-separation phenotypes in the S288C strain background. These phenotypes can be suppressed by deletions of genes encoding nonessential components of the anaphase-promoting complex (APC/C). This suppression does not seem to result simply from a delay in mitotic exit, because overexpression of a nondegradable mitotic cyclin does not suppress the same phenotypes. Overexpression of either IQG1 or CYK3 also suppresses the myo1Δ phenotypes, and Iqg1p (an IQGAP protein) is increased in abundance and abnormally persistent after cytokinesis in APC/C mutants. In vitro assays showed that Iqg1p is ubiquitinated directly by APC/CCdh1via a novel recognition sequence. A nondegradable Iqg1p (lacking this recognition sequence) can suppress the myo1Δ phenotypes even when expressed at relatively low levels. Together, the data suggest that compromise of APC/C function allows the accumulation of Iqg1p, which then promotes actomyosin-ring-independent cytokinesis at least in part by activation of Cyk3p.

Download Full-text

The integrin cytoplasmic domain-associated protein ICAP-1 binds and regulates Rho family GTPases during cell spreading

The Journal of Cell Biology ◽

10.1083/jcb.200108030 ◽

2002 ◽

Vol 156 (2) ◽

pp. 377-388 ◽

Cited By ~ 36

Author(s):

Simona Degani ◽

Fiorella Balzac ◽

Mara Brancaccio ◽

Simona Guazzone ◽

Saverio Francesco Retta ◽

...

Keyword(s):

Cell Spreading ◽

Cytoplasmic Domain ◽

Nih3t3 Cells ◽

Cell Matrix ◽

Rho Family Gtpases ◽

Rho Family ◽

First Time ◽

Cell Matrix Adhesion

Using two-hybrid screening, we isolated the integrin cytoplasmic domain-associated protein (ICAP-1), an interactor for the COOH terminal region of the β1A integrin cytoplasmic domain. To investigate the role of ICAP-1 in integrin-mediated adhesive function, we expressed the full-length molecule in NIH3T3 cells. ICAP-1 expression strongly prevents NIH3T3 cell spreading on extracellular matrix. This inhibition is transient and can be counteracted by coexpression of a constitutively activated mutant of Cdc42, suggesting that ICAP-1 acts upstream of this GTPase. In addition, we found that ICAP-1 binds both to Cdc42 and Rac1 in vitro, and its expression markedly inhibits activation of these GTPases during integrin-mediated cell adhesion to fibronectin as detected by PAK binding assay. In the attempt to define the molecular mechanism of this inhibition, we show that ICAP-1 reduces both the intrinsic and the exchange factor–induced dissociation of GDP from Cdc42; moreover, purified ICAP-1 displaces this GTPase from cellular membranes. Together, these data show for the first time that ICAP-1 regulates Rho family GTPases during integrin-mediated cell matrix adhesion, acting as guanine dissociation inhibitor.

Download Full-text

Targeting and functional mechanisms of the cytokinesis‑related F‑BAR protein Hof1 during the cell cycle

Molecular Biology of the Cell ◽

10.1091/mbc.e12-11-0804 ◽

2013 ◽

Vol 24 (9) ◽

pp. 1305-1320 ◽

Cited By ~ 34

Author(s):

Younghoon Oh ◽

Jennifer Schreiter ◽

Ryuichi Nishihama ◽

Carsten Wloka ◽

Erfei Bi

Keyword(s):

Cell Cycle ◽

Budding Yeast ◽

Coiled Coil ◽

Dependent Manner ◽

Polarized Growth ◽

Yeast Saccharomyces Cerevisiae ◽

Septum Formation ◽

Primary Septum ◽

Bud Neck ◽

Ring Constriction

F-BAR proteins are membrane‑associated proteins believed to link the plasma membrane to the actin cytoskeleton in cellular processes such as cytokinesis and endocytosis. In the budding yeast Saccharomyces cerevisiae, the F‑BAR protein Hof1 localizes to the division site in a complex pattern during the cell cycle and plays an important role in cytokinesis. However, the mechanisms underlying its localization and function are poorly understood. Here we show that Hof1 contains three distinct targeting domains that contribute to cytokinesis differentially. The N‑terminal half of Hof1 localizes to the bud neck and the sites of polarized growth during the cell cycle. The neck localization is mediated mainly by an interaction between the second coiled‑coil region in the N‑terminus and the septin Cdc10, whereas the localization to the sites of polarized growth is mediated entirely by the F‑BAR domain. In contrast, the C‑terminal half of Hof1 interacts with Myo1, the sole myosin‑II heavy chain in budding yeast, and localizes to the bud neck in a Myo1‑dependent manner from the onset to the completion of cytokinesis. We also show that the SH3 domain in the C‑terminus plays an important role in maintaining the symmetry of Myo1 ring constriction during cytokinesis and that Hof1 interacts with Chs2, a chitin synthase that is required for primary septum formation. Together these data define a mechanism that accounts for the localization of Hof1 during the cell cycle and suggest that Hof1 may function in cytokinesis by coupling actomyosin ring constriction to primary septum formation through interactions with Myo1 and Chs2.

Download Full-text

Proximity-dependent proteomics reveals extensive interactions of Protocadherin-19 with regulators of Rho GTPases and the microtubule cytoskeleton

10.1101/2020.09.11.293589 ◽

2020 ◽

Author(s):

Michelle R. Emond ◽

Sayantanee Biswas ◽

Matthew L. Morrow ◽

James D. Jontes

Keyword(s):

Nervous System ◽

Behavioral Problems ◽

Rho Gtpases ◽

Protein Complexes ◽

Epileptic Seizures ◽

Centrosomal Protein ◽

Surface Receptors ◽

Rho Family Gtpases ◽

Rho Family

AbstractProtocadherin-19 belongs to the cadherin family of cell surface receptors and has been shown to play essential roles in the development of the vertebrate nervous system. Mutations in human Protocadherin-19 (PCDH19) lead to PCDH19 Female-limited epilepsy (PCDH19 FLE) in humans, characterized by the early onset of epileptic seizures in children and a range of cognitive and behavioral problems in adults. Despite being considered the second most prevalent gene in epilepsy, very little is known about the intercellular pathways in which it participates. In order to characterize the protein complexes within which Pcdh19 functions, we generated Pcdh19-BioID fusion proteins and utilized proximity-dependent biotinylation to identify neighboring proteins. Proteomic identification and analysis revealed that the Pcdh19 interactome is enriched in proteins that regulate Rho family GTPases, microtubule binding proteins and proteins that regulate cell divisions. We cloned the centrosomal protein Nedd1 and the RacGEF Dock7 and verified their interactions with Pcdh19 in vitro. Our findings provide the first comprehensive insights into the interactome of Pcdh19, and provide a platform for future investigations into the cellular and molecular biology of this protein critical to the proper development of the nervous system.

Download Full-text

Control of neutrophil pseudopods by fluid shear: role of Rho family GTPases

AJP Cell Physiology ◽

10.1152/ajpcell.00358.2004 ◽

2005 ◽

Vol 288 (4) ◽

pp. C863-C871 ◽

Cited By ~ 28

Author(s):

Ayako Makino ◽

Michael Glogauer ◽

Gary M. Bokoch ◽

Shu Chien ◽

Geert W. Schmid-Schönbein

Keyword(s):

Signal Transduction ◽

Shear Stress ◽

Fluid Shear Stress ◽

Small Gtpases ◽

Dependent Manner ◽

Fluid Shear ◽

Rho Family

Blood vessels and blood cells are under continuous fluid shear. Studies on vascular endothelium and smooth muscle cells have shown the importance of this mechanical stress in cell signal transduction, gene expression, vascular remodeling, and cell survival. However, in circulating leukocytes, shear-induced signal transduction has not been investigated. Here we examine in vivo and in vitro the control of pseudopods in leukocytes under the influence of fluid shear stress and the role of the Rho family small GTPases. We used a combination of HL-60 cells differentiated into neutrophils (1.4% dimethyl sulfoxide for 5 days) and fresh leukocytes from Rac knockout mice. The cells responded to shear stress (5 dyn/cm2) with retraction of pseudopods and reduction of their projected cell area. The Rac1 and Rac2 activities were decreased by fluid shear in a time- and magnitude-dependent manner, whereas the Cdc42 activity remained unchanged (up to 5 dyn/cm2). The Rho activity was transiently increased and recovered to static levels after 10 min of shear exposure (5 dyn/cm2). Inhibition of either Rac1 or Rac2 slightly but significantly diminished the fluid shear response. Transfection with Rac1-positive mutant enhanced the pseudopod formation during shear. Leukocytes from Rac1-null and Rac2-null mice had an ability to form pseudopods in response to platelet-activating factor but did not respond to fluid shear in vitro. Leukocytes in wild-type mice retracted pseudopods after physiological shear exposure, whereas cells in Rac1-null mice showed no retraction during equal shear. On leukocytes from Rac2-null mice, however, fluid shear exerted a biphasic effect. Leukocytes with extended pseudopods slightly decreased in length, whereas initially round cells increased in length after shear application. The disruption of Rac activity made leukocytes nonresponsive to fluid shear, induced cell adhesion and microvascular stasis, and decreased microvascular density. These results suggest that deactivation of Rac activity by fluid shear plays an important role in stable circulation of leukocytes.

Download Full-text

Mechanism and role of localized activation of Rho-family GTPases in growth factor-stimulated fibroblasts and neuronal cells

Biochemical Society Transactions ◽

10.1042/bst0330631 ◽

2005 ◽

Vol 33 (4) ◽

pp. 631-634 ◽

Cited By ~ 48

Author(s):

K. Kurokawa ◽

T. Nakamura ◽

K. Aoki ◽

M. Matsuda

Keyword(s):

Plasma Membrane ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Cell Function ◽

Leading Edge ◽

Dependent Manner ◽

Rhoa Activity ◽

Rho Family Gtpases ◽

Epidermal Growth ◽

Rho Family

Rho-family GTPases regulate various aspects of cell function by controlling cytoskeletal changes; however, their spatial regulation within the cells remains largely unknown. To understand this regulation, we have studied the spatiotemporal activity of Rho-family GTPases in migrating cells and growth factor-stimulated cells by using probes based on the principle of fluorescence resonance energy transfer. In migrating fibroblasts and epithelial cells, the level of RhoA activity is high both at the contractile tail and at the leading edge, whereas Rac1 and Cdc42 activities are high only at the leading edge. In cells stimulated with epidermal growth factor or nerve growth factor, activities of Rac1 and Cdc42 were transiently elevated in a broad area of the plasma membrane, followed by a localized activation at nascent lamellipodia. In contrast, on epidermal growth factor stimulation, RhoA activity decreased diffusely at the plasma membrane. Notably, RhoA activity persisted at the tip of growth factor-induced membrane ruffles and, in agreement with this finding, RhoA is required for membrane ruffling. These observations suggest that the activities of Rho-family GTPases are elaborately regulated in a time- and space-dependent manner to control cytoskeletal changes and that the basic mechanism of controlling cell shape via Rho-family GTPases is common to various cell types.

Download Full-text

The LIM domain-containing Dbm1 GTPase-activating protein is required for normal cellular morphogenesis in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.16.4.1376 ◽

1996 ◽

Vol 16 (4) ◽

pp. 1376-1390 ◽

Cited By ~ 28

Author(s):

G C Chen ◽

L Zheng ◽

C S Chan

Keyword(s):

Saccharomyces Cerevisiae ◽

Site Selection ◽

Yeast Cells ◽

Gtpase Activating Protein ◽

Lim Domain ◽

Yeast Saccharomyces Cerevisiae ◽

Lim Domains ◽

Bud Site

Normal cell growth in the yeast Saccharomyces cerevisiae involves the selection of genetically determined bud sites where most growth is localized. Previous studies have shown that BEM2, which encodes a GTPase-activating protein (GAP) that is specific for the Rho-type GTPase Rho1p in vitro, is required for proper bud site selection and bud emergence. We show here that DBM1, which encodes another putative Rho-type GAP with two tandemly arranged cysteine-rich LIM domains, also is needed for proper bud site selection, as haploid cells lacking Dbm1p bud predominantly in a bipolar, rather than the normal axial, manner. Furthermore, yeast cells lacking both Bem2p and Dbm1p are inviable. The nonaxial budding defect of dbm1 mutants can be rescued partially by overproduction of Bem3p and is exacerbated by its absence. Since Bem3p has previously been shown to function as a GAP for Cdc42p, and also less efficiently for Rho1p, our results suggest that Dbm1p, like Bem2p and Bem3p, may function in vivo as a GAP for Cdc42p and/or Rho1p. Both LIM domains of Dbm1p are essential for its normal function. Point mutations that alter single conserved cysteine residues within either LIM domain result in mutant forms of Dbm1p that can no longer function in bud site selection but instead are capable of rescuing the inviability of bem2 mutants at 35 degrees C.

Download Full-text