Unique in Vivo Associations with SmgGDS and RhoGDI and Different Guanine Nucleotide Exchange Activities Exhibited by RhoA, Dominant Negative RhoAAsn-19, and Activated RhoAVal-14

Strassheim, Derek; Porter, Rebecca A.; Phelps, Scott H.; Williams, Carol L.

doi:10.1074/jbc.275.10.6699

Cited by 28 publications

(35 citation statements)

References 31 publications

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“…Nuclear proteins are solubilized by this method, as indicated by our ability to detect nuclear proteins such as RCC1 in Western blots of the lysate supernatants (data not shown). The supernatants from the centrifuged lysates were immunoprecipitated using the 9E10 myc antibody (Santa Cruz Biotechnology), the HA antibody (Covance), or the p120ctn antibody (BD Transduction Laboratories), as previously described (15). The immunoprecipitates were subjected to SDS-PAGE followed by autoradiography as previously described (15).…”

Section: Methodsmentioning

confidence: 99%

“…The cells were cultured for an additional 16 h (37°C, 5% CO 2 ) in the presence or absence of compactin (Sigma, St. Louis, MO) and then suspended in ice-cold lysis buffer (50 mM Tris-HCl, 120 mM NaCl, 2.5 mM EDTA, 1 mM dithiothreitol, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 10 M leupeptin, pH 7.4) containing phosphatase inhibitors (15). The cells were incubated in the lysis buffer for 20 min on ice with periodic vigorous vortexing of the cell suspension, followed by centrifugation (13,000 ϫ g, 10 min, 4°C).…”

Section: Methodsmentioning

confidence: 99%

“…To co-localize GFP-tagged proteins with HA-tagged proteins or with endogenous p120ctn, the cells were fixed with 3% formaldehyde in phosphate-buffered saline (PBS) (15 min, 4°C) and incubated with 50 mM ammonium chloride in PBS to quench formaldehyde fluorescence (10 min, 25°C). The fixed cells were permeabilized with acetone (10 s, 4°C) or with 0.2% Triton X-100 (TX-100) in PBS (10 min, 25°C), as described previously (15,22). After incubating with PBS containing 1% bovine serum albumin (30 min, 25°C), the cells were incubated with mouse antibody to HA (Covance) or to p120ctn (BD Transduction Laboratories, San Diego, CA) (1 h, 25°C), followed by incubation with TRITC-anti-mouse IgG (1 h, 25°C).…”

Section: Methodsmentioning

confidence: 99%

“…SmgGDS interacts with nuclear proteins (23,24) and cytoplasmic proteins (11)(12)(13)(14)(15)18), we looked for sequences in SmgGDS that might promote nucleocytoplasmic shuttling. Although classic NLS sequences are not apparent in SmgGDS, putative NES sequences, consisting of LX (2)(3) LXXLXL in which the leucines are sometimes replaced by isoleucines (reviewed in Ref.…”

Section: Smggds Has a Nuclear Export Signal Sequence-becausementioning

confidence: 99%

“…SmgGDS promotes guanine nucleotide exchange by small GTPases containing a C-terminal polybasic region (PBR), which is a series of adjacent lysines or arginines (11)(12)(13)(14)(15)18). We noticed a striking sequence similarity between the PBR of small GTPases that interact with Smg-GDS and the NLS sequence of proteins that associate with the ARM protein karyopherin ␣.…”

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confidence: 99%

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Novel Mechanism of the Co-regulation of Nuclear Transport of SmgGDS and Rac1

Lanning

Ruiz-Velasco

Williams

2003

Journal of Biological Chemistry

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131

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The armadillo protein SmgGDS promotes guanine nucleotide exchange by small GTPases containing a C-terminal polybasic region (PBR), such as Rac1 and RhoA. Because the PBR resembles a nuclear localization signal (NLS) sequence, we investigated the nuclear transport of SmgGDS with Rac1 or RhoA. We show that the Rac1 PBR has significant NLS activity when it is fused to green fluorescent protein (GFP) or in the context of full-length Rac1. In contrast, the RhoA PBR has very poor NLS activity when it is fused to GFP or in the context of full-length RhoA. The nuclear accumulation of both Rac1 and SmgGDS is enhanced by Rac1 activation and diminished by mutation of the Rac1 PBR. Conversely, SmgGDS nuclear accumulation is diminished by interactions with RhoA. An SmgGDS nuclear export signal sequence that we identified promotes SmgGDS nuclear export. These results suggest that SmgGDS⅐ Rac1 complexes accumulate in the nucleus because the Rac1 PBR has NLS activity and because Rac1 supplies the appropriate GTP-dependent signal. In contrast, SmgGDS⅐RhoA complexes accumulate in the cytoplasm because the RhoA PBR does not have NLS activity. This model may be applicable to other armadillo proteins in addition to SmgGDS, because we demonstrate that activated Rac1 and RhoA also provide stimulatory and inhibitory signals, respectively, for the nuclear accumulation of p120 catenin. These results indicate that small GTPases with a PBR can regulate the nuclear transport of armadillo proteins.Armadillo (ARM) 1 family proteins that contain multiple copies of the ϳ42-amino acid (aa) ARM motif include SmgGDS, p120 catenin (p120ctn), ␤-catenin, plakoglobin, APC, karyopherin ␣ (also known as importin ␣), and several other proteins (reviewed in Refs. 1-3). Nucleocytoplasmic shuttling by many ARM proteins allows them to regulate events in different cellular compartments, including gene transcription and cell adhesion (reviewed in Refs. 2-7). ARM proteins enter the nucleus by different mechanisms (reviewed in Refs. 2-7). Karyopherin ␣ enters the nucleus when it associates with proteins containing a nuclear localization signal (NLS) sequence consisting of a series of adjacent lysines or arginines (reviewed in Ref. 3). The NLS sequence is believed to anchor within the long surface groove formed by the multiple ARM repeats of karyopherin ␣, promoting the nuclear import of both the NLS-containing protein and karyopherin ␣ (3, 5). APC possesses two NLS sequences and may enter the nucleus by associating with karyopherin ␣ or related proteins (4, 7). The mechanisms by which other ARM proteins enter the nucleus are less clear, because some of these proteins neither possess classic NLS sequences, nor have they been reported to associate with NLS-containing proteins.Several ARM proteins interact with the Rho family of small GTPases (8 -15) or with guanine nucleotide exchange factors (GEFs) for these GTPases (16,17). SmgGDS promotes guanine nucleotide exchange by small GTPases containing a C-terminal polybasic region (PBR), which is a series of adjacent l...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Smggds Has a Nuclear Export Signal Sequence-becausementioning

confidence: 99%

mentioning

confidence: 99%

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Novel Mechanism of the Co-regulation of Nuclear Transport of SmgGDS and Rac1

Lanning

Ruiz-Velasco

Williams

2003

Journal of Biological Chemistry

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131

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Identification and characterization of the unique guanine nucleotide exchange factor, SmgGDS, in vascular smooth muscle cells

Thill

Campbell

Williams

2008

J of Cellular Biochemistry

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The guanine nucleotide exchange factor (GEF), SmgGDS, promotes nucleotide exchange by several GTPases in both the Ras and Rho families, especially by RhoA. Because RhoA plays an important role in regulating the contraction of vascular smooth muscle cells (VSMC), we examined the expression and function of SmgGDS in VSMC. SmgGDS is expressed in primary rat aortic smooth muscle (ASM) cells, primary bovine coronary artery smooth muscle (BCASM) cells, and the immortalized A7r5 line of rat ASM cells. Down regulation of SmgGDS expression by siRNA transfection resulted in a decrease of RhoA-GTP levels, enhanced cell spreading, and loss of the characteristic elongated morphology of VSMC. A similar morphology was also observed following treatment with the Rho-kinase inhibitor, Y27632. In contrast, cells with reduced RhoA expression exhibit an elongated shape. Subsequent immunofluorescent staining revealed a disruption of the myosin filament organization in the cells with reduced SmgGDS expression. Further studies analyzed the effect of SmgGDS siRNA transfection on the contraction of A7r5 cells and BCASM cells, which is also a Rho-regulated pathway. Transfection of SmgGDS siRNA or RhoA siRNA resulted in an impaired ability of the A7r5 and BCASM cells to undergo contraction in a collagen gel matrix. However, phosphorylation of the myosin-binding subunit of myosin phosphatase (MYPT1) or the light chain of myosin II (MLC) was not altered by downregulating expression of either SmgGDS or RhoA GTPase. Taken together these results identify SmgGDS as a novel regulator of myosin organization and contraction in VSMC.

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Ras Family Proteins

Gunzburg

2006

RAS Family GTPases

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Unique in Vivo Associations with SmgGDS and RhoGDI and Different Guanine Nucleotide Exchange Activities Exhibited by RhoA, Dominant Negative RhoAAsn-19, and Activated RhoAVal-14

Cited by 28 publications

References 31 publications

Novel Mechanism of the Co-regulation of Nuclear Transport of SmgGDS and Rac1

Novel Mechanism of the Co-regulation of Nuclear Transport of SmgGDS and Rac1

Identification and characterization of the unique guanine nucleotide exchange factor, SmgGDS, in vascular smooth muscle cells

Ras Family Proteins

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