scholarly journals Conserved NDR/LATS kinase controls RAS GTPase activity to regulate cell growth and chronological lifespan

2019 ◽  
Vol 30 (20) ◽  
pp. 2598-2616 ◽  
Author(s):  
Chuan Chen ◽  
Marbelys Rodriguez Pino ◽  
Patrick Roman Haller ◽  
Fulvia Verde
Keyword(s):  
Cell Growth ◽  
Gtpase Activity ◽  
Nucleotide Exchange ◽  
Chronological Lifespan ◽  
Protein Levels ◽  
Ras Gtpase ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Regulate Cell Growth ◽  
Cell Growth And Differentiation

Adaptation to the nutritional environment is critical for all cells. RAS GTPase is a highly conserved GTP-binding protein with crucial functions for cell growth and differentiation in response to environmental conditions. Here, we describe a novel mechanism connecting RAS GTPase to nutrient availability in fission yeast. We report that the conserved NDR/LATS kinase Orb6 responds to nutritional cues and regulates Ras1 GTPase activity. Orb6 increases the protein levels of an Ras1 GTPase activator, the guanine nucleotide exchange factor Efc25, by phosphorylating Sts5, a protein bound to efc25 mRNA. By manipulating the extent of Orb6-mediated Sts5 assembly into RNP granules, we can modulate Efc25 protein levels, Ras1 GTPase activity, and, as a result, cell growth and cell survival. Thus, we conclude that the Orb6–Sts5–Ras1 regulatory axis plays a crucial role in promoting cell adaptation, balancing the opposing demands of promoting cell growth and extending chronological lifespan.

Trio amino-terminal guanine nucleotide exchange factor domain expression promotes actin cytoskeleton reorganization, cell migration and anchorage-independent cell growth

1999 ◽  
Vol 112 (12) ◽  
pp. 1825-1834 ◽  
Author(s):  
K. Seipel ◽  
Q.G. Medley ◽  
N.L. Kedersha ◽  
X.A. Zhang ◽  
S.P. O'Brien ◽  
...  
Keyword(s):  
Cell Migration ◽  
Cell Growth ◽  
Actin Cytoskeleton ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Amino Terminal ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

Rho family GTPases regulate diverse cellular processes, including extracellular signal-mediated actin cytoskeleton reorganization and cell growth. The functions of GTPases are positively regulated by guanine nucleotide exchange factors, which promote the exchange of GDP for GTP. Trio is a complex protein possessing two guanine nucleotide exchange factor domains, each with adjacent pleckstrin homology and SH3 domains, a protein serine/threonine kinase domain with an adjacent immunoglobulin-like domain and multiple spectrin-like domains. To assess the functional role of the two Trio guanine nucleotide exchange factor domains, NIH 3T3 cell lines stably expressing the individual guanine nucleotide exchange factor domains were established and characterized. Expression of the amino-terminal guanine nucleotide exchange factor domain results in prominent membrane ruffling, whereas cells expressing the carboxy-terminal guanine nucleotide exchange factor domain have lamellae that terminate in miniruffles. Moreover, cells expressing the amino-terminal guanine nucleotide exchange factor domain display more rapid cell spreading, haptotactic cell migration and anchorage-independent growth, suggesting that Trio regulates both cell motility and cell growth. Expression of full-length Trio in COS cells also alters actin cytoskeleton organization, as well as the distribution of focal contact sites. These findings support a role for Trio as a multifunctional protein that integrates and amplifies signals involved in coordinating actin remodeling, which is necessary for cell migration and growth.

scholarly journals GTP Hydrolysis Is Not Important for Ypt1 GTPase Function in Vesicular Transport

1998 ◽  
Vol 18 (2) ◽  
pp. 827-838 ◽  
Author(s):  
Celeste J. Richardson ◽  
Sara Jones ◽  
Robert J. Litt ◽  
Nava Segev
Keyword(s):  
Membrane Fusion ◽  
Vesicular Transport ◽  
Gtpase Activity ◽  
Nucleotide Exchange ◽  
Gtp Hydrolysis ◽  
Dominant Phenotype ◽  
Guanine Nucleotide Exchange ◽  
Membrane Morphology ◽  
Nucleotide Exchange Factor ◽  
Mutant Cells

ABSTRACT GTPases of the Ypt/Rab family play a key role in the regulation of vesicular transport. Their ability to cycle between the GTP- and the GDP-bound forms is thought to be crucial for their function. Conversion from the GTP- to the GDP-bound form is achieved by a weak endogenous GTPase activity, which can be stimulated by a GTPase-activating protein (GAP). Current models suggest that GTP hydrolysis and GAP activity are essential for vesicle fusion with the acceptor compartment or for timing membrane fusion. To test this idea, we inactivated the GTPase activity of Ypt1p by using the Q67L mutation, which targets a conserved residue that helps catalyze GTP hydrolysis in Ras. We demonstrate that the mutant Ypt1-Q67L protein is severely impaired in its ability to hydrolyze GTP both in the absence and in the presence of GAP and consequently is restricted mostly to the GTP-bound form. Surprisingly, a strain with ypt1-Q67L as the only YPT1 gene in the cell has no observable growth phenotypes at temperatures ranging from 14 to 37°C. In addition, these mutant cells exhibit normal rates of secretion and normal membrane morphology as determined by electron microscopy. Furthermore, the ypt1-Q67L allele does not exhibit dominant phenotypes in cell growth and secretion when overexpressed. Together, these results lead us to suggest that, contrary to current models for Ypt/Rab function, GTP hydrolysis is not essential either for Ypt1p-mediated vesicular transport or as a timer to turn off Ypt1p-mediated membrane fusion but only for recycling of Ypt1p between compartments. Finally, the ypt1-Q67L allele, like the wild type, is inhibited by dominant nucleotide-freeYPT1 mutations. Such mutations are thought to exert their dominant phenotype by sequestration of the guanine nucleotide exchange factor (GNEF). These results suggest that the function of Ypt1p in vesicular transport requires not only the GTP-bound form of the protein but also the interaction of Ypt1p with its GNEF.

scholarly journals PtdIns(3,4,5)P3-dependent Rac Exchanger 1 (PREX1) Rac-Guanine Nucleotide Exchange Factor (GEF) Activity Promotes Breast Cancer Cell Proliferation and Tumor Growth via Activation of Extracellular Signal-regulated Kinase 1/2 (ERK1/2) Signaling

2016 ◽  
Vol 291 (33) ◽  
pp. 17258-17270 ◽  
Author(s):  
Heng-Jia Liu ◽  
Lisa M. Ooms ◽  
Nuthasuda Srijakotre ◽  
Joey Man ◽  
Jessica Vieusseux ◽  
...  
Keyword(s):  
Cell Growth ◽  
Tumor Growth ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Breast Cancers ◽  
Nucleotide Exchange ◽  
Wild Type ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

PtdIns(3,4,5)P3-dependent Rac exchanger 1 (PREX1) is a Rac-guanine nucleotide exchange factor (GEF) overexpressed in a significant proportion of human breast cancers that integrates signals from upstream ErbB2/3 and CXCR4 membrane surface receptors. However, the PREX1 domains that facilitate its oncogenic activity and downstream signaling are not completely understood. We identify that ERK1/2 MAPK acts downstream of PREX1 and contributes to PREX1-mediated anchorage-independent cell growth. PREX1 overexpression increased but its shRNA knockdown decreased ERK1/2 phosphorylation in response to EGF/IGF-1 stimulation, resulting in induction of the cell cycle regulators cyclin D1 and p21WAF1/CIP1. PREX1-mediated ERK1/2 phosphorylation, anchorage-independent cell growth, and cell migration were suppressed by inhibition of MEK1/2/ERK1/2 signaling. PREX1 overexpression reduced staurosporine-induced apoptosis whereas its shRNA knockdown promoted apoptosis in response to staurosporine or the anti-estrogen drug tamoxifen. Expression of wild-type but not GEF-inactive PREX1 increased anchorage-independent cell growth. In addition, mouse xenograft studies revealed that expression of wild-type but not GEF-dead PREX1 resulted in the formation of larger tumors that displayed increased phosphorylation of ERK1/2 but not AKT. The impaired anchorage-independent cell growth, apoptosis, and ERK1/2 signaling observed in stablePREX1knockdown cells was restored by expression of wild-type but not GEF-dead-PREX1. Therefore, PREX1-Rac-GEF activity is critical for PREX1-dependent anchorage-independent cell growth and xenograft tumor growth and may represent a possible therapeutic target for breast cancers that exhibit PREX1 overexpression.

scholarly journals C3G dynamically associates with nuclear speckles and regulates mRNA splicing

2018 ◽  
Vol 29 (9) ◽  
pp. 1111-1124 ◽  
Author(s):  
Dhruv Kumar Shakyawar ◽  
Bhattiprolu Muralikrishna ◽  
Vegesna Radha
Keyword(s):  
Rna Splicing ◽  
Glycogen Synthase ◽  
C2c12 Cells ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Nuclear Speckles ◽  
Protein Levels ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Artificial Gene

C3G (Crk SH3 domain binding guanine nucleotide releasing factor) (Rap guanine nucleotide exchange factor 1), essential for mammalian embryonic development, is ubiquitously expressed and undergoes regulated nucleocytoplasmic exchange. Here we show that C3G localizes to SC35-positive nuclear speckles and regulates splicing activity. Reversible association of C3G with speckles was seen on inhibition of transcription and splicing. C3G shows partial colocalization with SC35 and is recruited to a chromatin and RNase-sensitive fraction of speckles. Its presence in speckles is dependent on intact cellular actin cytoskeleton and is lost on expression of the kinase Clk1. Rap1, a substrate of C3G, is also present in nuclear speckles, and inactivation of Rap signaling by expression of GFP-Rap1GAP alters speckle morphology and number. Enhanced association of C3G with speckles is seen on glycogen synthase kinase 3 beta inhibition or differentiation of C2C12 cells to myotubes. CRISPR/Cas9-mediated knockdown of C3G resulted in altered splicing activity of an artificial gene as well as endogenous CD44. C3G knockout clones of C2C12 as well as MDA-MB-231 cells showed reduced protein levels of several splicing factors compared with control cells. Our results identify C3G and Rap1 as novel components of nuclear speckles and a role for C3G in regulating cellular RNA splicing activity.

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