scholarly journals Adjacent positioning of cellular structures enabled by a Cdc42 GTPase-activating protein–mediated zone of inhibition

2007 ◽  
Vol 179 (7) ◽  
pp. 1375-1384 ◽  
Author(s):  
Zongtian Tong ◽  
Xiang-Dong Gao ◽  
Audrey S. Howell ◽  
Indrani Bose ◽  
Daniel J. Lew ◽  
...  
Keyword(s):  
Cell Division ◽  
Nucleotide Exchange ◽  
Zone Of Inhibition ◽  
Exclusion Zone ◽  
Yeast Saccharomyces Cerevisiae ◽  
Division Site ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Guanosine Triphosphatase ◽  
Preceding Cell

Cells of the budding yeast Saccharomyces cerevisiae are born carrying localized transmembrane landmark proteins that guide the subsequent establishment of a polarity axis and hence polarized growth to form a bud in the next cell cycle. In haploid cells, the relevant landmark proteins are concentrated at the site of the preceding cell division, to which they recruit Cdc24, the guanine nucleotide exchange factor for the conserved polarity regulator Cdc42. However, instead of polarizing at the division site, the new polarity axis is directed next to but not overlapping that site. Here, we show that the Cdc42 guanosine triphosphatase–activating protein (GAP) Rga1 establishes an exclusion zone at the division site that blocks subsequent polarization within that site. In the absence of localized Rga1 GAP activity, new buds do in fact form within the old division site. Thus, Cdc42 activators and GAPs establish concentric zones of action such that polarization is directed to occur adjacent to but not within the previous cell division site.

scholarly journals Concentration of Sec12 at ER exit sites via interaction with cTAGE5 is required for collagen export

2014 ◽  
Vol 206 (6) ◽  
pp. 751-762 ◽  
Author(s):  
Kota Saito ◽  
Koh Yamashiro ◽  
Noriko Shimazu ◽  
Tomoya Tanabe ◽  
Kenji Kontani ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Secretory Proteins ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Transport Vesicles ◽  
Nucleotide Exchange Factor ◽  
Receptor Component ◽  
Er Exit Sites ◽  
Guanosine Triphosphatase ◽  
Trimeric Structure

Mechanisms for exporting variably sized cargo from the endoplasmic reticulum (ER) using the same machinery remain poorly understood. COPII-coated vesicles, which transport secretory proteins from the ER to the Golgi apparatus, are typically 60–90 nm in diameter. However, collagen, which forms a trimeric structure that is too large to be accommodated by conventional transport vesicles, is also known to be secreted via a COPII-dependent process. In this paper, we show that Sec12, a guanine-nucleotide exchange factor for Sar1 guanosine triphosphatase, is concentrated at ER exit sites and that this concentration of Sec12 is specifically required for the secretion of collagen VII but not other proteins. Furthermore, Sec12 recruitment to ER exit sites is organized by its direct interaction with cTAGE5, a previously characterized collagen cargo receptor component, which functions together with TANGO1 at ER exit sites. These findings suggest that the export of large cargo requires high levels of guanosine triphosphate–bound Sar1 generated by Sec12 localized at ER exit sites.

scholarly journals Induction of cell retraction by the combined actions of Abl–CrkII and Rho–ROCK1 signaling

2008 ◽  
Vol 183 (4) ◽  
pp. 711-723 ◽  
Author(s):  
XiaoDong Huang ◽  
Diana Wu ◽  
Hua Jin ◽  
Dwayne Stupack ◽  
Jean Y.J. Wang
Keyword(s):  
Nucleotide Exchange ◽  
Downstream Target ◽  
Abl Tyrosine Kinase ◽  
Guanine Nucleotide Exchange ◽  
Cell Matrix ◽  
Nucleotide Exchange Factor ◽  
Wide Range ◽  
Important Regulator ◽  
Guanosine Triphosphatase ◽  
Cell Retraction

Dynamic modulation of cell adhesion is integral to a wide range of biological processes. The small guanosine triphosphatase (GTPase) Rap1 is an important regulator of cell–cell and cell–matrix adhesions. We show here that induced expression of activated Abl tyrosine kinase reduces Rap1-GTP levels through phosphorylation of Tyr221 of CrkII, which disrupts interaction of CrkII with C3G, a guanine nucleotide exchange factor for Rap1. Abl-dependent down-regulation of Rap1-GTP causes cell rounding and detachment only when the Rho–ROCK1 pathway is also activated, for example, by lysophosphatidic acid (LPA). During ephrin-A1–induced retraction of PC3 prostate cancer cells, we show that endogenous Abl is activated and disrupts the CrkII–C3G complex to reduce Rap1-GTP. Interestingly, ephrin-A1–induced PC3 cell retraction also requires LPA, which stimulates Rho to a much higher level than that is activated by ephrin-A1. Our results establish Rap1 as another downstream target of the Abl–CrkII signaling module and show that Abl–CrkII collaborates with Rho–ROCK1 to stimulate cell retraction.

scholarly journals The Rac activator Tiam1 is required for α3β1-mediated laminin-5 deposition, cell spreading, and cell migration

2005 ◽  
Vol 171 (5) ◽  
pp. 871-881 ◽  
Author(s):  
Irene H.L. Hamelers ◽  
Cristina Olivo ◽  
Alexander E.E. Mertens ◽  
D. Michiel Pegtel ◽  
Rob A. van der Kammen ◽  
...  
Keyword(s):  
Mouse Skin ◽  
Nucleotide Exchange ◽  
Laminin 5 ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
T Lymphoma ◽  
Guanosine Triphosphatase ◽  
And Migration ◽  
Migration Of Cells

The Rho-like guanosine triphosphatase Rac1 regulates various signaling pathways, including integrin-mediated adhesion and migration of cells. However, the mechanisms by which integrins signal toward Rac are poorly understood. We show that the Rac-specific guanine nucleotide exchange factor Tiam1 (T-lymphoma invasion and metastasis 1) is required for the integrin-mediated laminin (LN)-5 deposition, spreading, and migration of keratinocytes. In contrast to wild-type keratinocytes, Tiam1-deficient (Tiam1−/−) keratinocytes are unable to adhere to and spread on a glass substrate because they are unable to deposit their own LN5 substrate. Both Tiam1 and V12Rac1 can rescue the defects of Tiam1−/− keratinocytes, indicating that these deficiencies are caused by impaired Tiam1-mediated Rac activation. Tiam1−/− cells are unable to activate Rac upon α3β1-mediated adhesion to an exogenous LN5 substrate. Moreover, Tiam1 deficiency impairs keratinocyte migration in vitro and reepithelialization of excision wounds in mouse skin. Our studies indicate that Tiam1 is a key molecule in α3β1-mediated activation of Rac, which is essential for proper production and secretion of LN5, a requirement for the spreading and migration of keratinocytes.

scholarly journals Identification of Novel, Evolutionarily Conserved Cdc42p-interacting Proteins and of Redundant Pathways Linking Cdc24p and Cdc42p to Actin Polarization in Yeast

2000 ◽  
Vol 11 (2) ◽  
pp. 773-793 ◽  
Author(s):  
Erfei Bi ◽  
John B. Chiavetta ◽  
Herman Chen ◽  
Guang-Chao Chen ◽  
Clarence S. M. Chan ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Double Mutant ◽  
Synthetic Lethality ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Yeast Saccharomyces Cerevisiae ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

In the yeast Saccharomyces cerevisiae, Cdc24p functions at least in part as a guanine-nucleotide-exchange factor for the Rho-family GTPase Cdc42p. A genetic screen designed to identify possible additional targets of Cdc24p instead identified two previously known genes, MSB1 and CLA4, and one novel gene, designated MSB3, all of which appear to function in the Cdc24p–Cdc42p pathway. Nonetheless, genetic evidence suggests that Cdc24p may have a function that is distinct from its Cdc42p guanine-nucleotide-exchange factor activity; in particular, overexpression of CDC42 in combination withMSB1 or a truncated CLA4 in cells depleted for Cdc24p allowed polarization of the actin cytoskeleton and polarized cell growth, but not successful cell proliferation.MSB3 has a close homologue (designatedMSB4) and two more distant homologues (MDR1 and YPL249C) in S. cerevisiae and also has homologues inSchizosaccharomyces pombe, Drosophila(pollux), and humans (the oncogenetre17). Deletion of either MSB3 orMSB4 alone did not produce any obvious phenotype, and the msb3 msb4 double mutant was viable. However, the double mutant grew slowly and had a partial disorganization of the actin cytoskeleton, but not of the septins, in a fraction of cells that were larger and rounder than normal. Like Cdc42p, both Msb3p and Msb4p localized to the presumptive bud site, the bud tip, and the mother-bud neck, and this localization was Cdc42p dependent. Taken together, the data suggest that Msb3p and Msb4p may function redundantly downstream of Cdc42p, specifically in a pathway leading to actin organization. From previous work, the BNI1, GIC1, andGIC2 gene products also appear to be involved in linking Cdc42p to the actin cytoskeleton. Synthetic lethality and multicopy suppression analyses among these genes, MSB, andMSB4, suggest that the linkage is accomplished by two parallel pathways, one involving Msb3p, Msb4p, and Bni1p, and the other involving Gic1p and Gic2p. The former pathway appears to be more important in diploids and at low temperatures, whereas the latter pathway appears to be more important in haploids and at high temperatures.

scholarly journals Rab5 GTPases are required for optimal TORC2 function

2018 ◽  
Vol 218 (3) ◽  
pp. 961-976 ◽  
Author(s):  
Melissa N. Locke ◽  
Jeremy Thorner
Keyword(s):  
Protein Kinase ◽  
Nucleotide Exchange ◽  
Gtpase Activating Protein ◽  
Yeast Saccharomyces Cerevisiae ◽  
Guanine Nucleotide Exchange ◽  
Downstream Effector ◽  
Nucleotide Exchange Factor ◽  
Pronounced Reduction

Target of rapamycin complex-2 (TORC2), a conserved protein kinase complex, is an indispensable regulator of plasma membrane homeostasis. In budding yeast (Saccharomyces cerevisiae), the essential downstream effector of TORC2 is protein kinase Ypk1 and its paralog Ypk2. Muk1, a Rab5-specific guanine nucleotide exchange factor (GEF), was identified in our prior global screen for candidate Ypk1 targets. We confirm here that Muk1 is a substrate of Ypk1 and demonstrate that Ypk1-mediated phosphorylation stimulates Muk1 function in vivo. Strikingly, yeast lacking its two Rab5 GEFs (Muk1 and Vps9) or its three Rab5 paralogs (Vps21/Ypt51, Ypt52, and Ypt53) or overexpressing Msb3, a Rab5-directed GTPase-activating protein, all exhibit pronounced reduction in TORC2-mediated phosphorylation and activation of Ypk1. Vps21 coimmunoprecipitates with TORC2, and immuno-enriched TORC2 is less active in vitro in the absence of Rab5 GTPases. Thus, TORC2-dependent and Ypk1-mediated activation of Muk1 provides a control circuit for positive (self-reinforcing) up-regulation to sustain TORC2-Ypk1 signaling.

scholarly journals Tuba, a Cdc42 GEF, is required for polarized spindle orientation during epithelial cyst formation

2010 ◽  
Vol 189 (4) ◽  
pp. 661-669 ◽  
Author(s):  
Yi Qin ◽  
Walter H. Meisen ◽  
Yi Hao ◽  
Ian G. Macara
Keyword(s):  
Cyst Formation ◽  
Cell Polarization ◽  
Spindle Orientation ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Lateral Plane ◽  
Similar Phenotype ◽  
Guanosine Triphosphatase

The Cdc42 guanosine triphosphatase is essential for cell polarization in several organisms and in vitro for the organization of polarized epithelial cysts. A long-standing question concerns the identity of the guanine nucleotide exchange factor (GEF) that controls this process. Using Madin–Darby canine kidney cells grown in Matrigel, we screened 70 GEFs by RNA interference. Of these, six positives were identified that caused a multilumen phenotype, including Tuba, a Cdc42-specific GEF localized below the apical cortex. Loss of Tuba abolishes Cdc42 enrichment at the apical cortex. Normal lumen formation is rescued by human Tuba or active Cdc42 but not by a GEF-negative Tuba mutant. Silencing Cdc42 causes a similar phenotype, including multilumen formation and reduced atypical protein kinase C (aPKC) activity. Lumen disorganization after depletion of Tuba or Cdc42 or inhibition of aPKC is caused by defective spindle orientation. Together, our findings implicate Tuba as a key activator of the Cdc42 GTPase during epithelial ductal morphogenesis, which in turn activates apical aPKC to ensure that spindles orient parallel to the lateral plane.

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