RACK1 interacts with filamin-A to regulate plasma membrane levels of the cystic fibrosis transmembrane conductance regulator

Smith, Laura A.; Litman, Paul; Kohli, Ekta; Amick, Joseph; Page, Richard C.; Misra, Saurav; Liedtke, Carole M.

doi:10.1152/ajpcell.00026.2013

Cited by 6 publications

(3 citation statements)

References 67 publications

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“…In addition, liver fibrosis can induce a hypoxic environment, which promotes the production of angiogenic factors, including VEGF and angiopoietin, and these factors lead to increased angiogenesis 43–45. Excessive levels of ECM deposition and angiogenic factors lead to LSEC capillarization, which results in increased vascular resistance and portal hypertension 46. The data demonstrated that the levels of angiogenic factors, including VEGF and angiopoietin-2, were significantly increased in the serum of CCl 4 -treated mice.…”

Section: Discussionmentioning

confidence: 94%

<p>Carvedilol attenuates carbon tetrachloride-induced liver fibrosis and hepatic sinusoidal capillarization in mice</p>

Wu¹,

Li²,

Xiu³

et al. 2019

DDDT

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Aim To investigate the effect of carvedilol on liver fibrosis and hepatic sinusoidal capillarization in mice with carbon tetrachloride (CCl 4 )-induced fibrosis. Methods A liver fibrosis mouse model was induced by intraperitoneal CCl 4 injection for 8 weeks. The mice were divided into five experimental groups: the normal group, the oil group, the CCl 4 group, the CCl 4 +carvedilol (5 mg/kg/d) group, and the CCl 4 +carvedilol (10 mg/kg/d) group. The extent of liver fibrosis was evaluated by histopathological staining, and the changes in fenestrations of hepatic sinus endothelial cells were observed by scanning electron microscope (SEM). The expression of α-smooth muscle actin (α-SMA) and vascular endothelial markers was detected by immunohistochemistry and Western blot assays. The effect of carvedilol on cell apoptosis was studied via Terminal deoxynucleotidyl Transferase Mediated dUTP Nick End Labeling (TUNEL) assay, and the serum levels of matrix metalloproteinase-8 (MMP-8), vascular endothelial growth factor (VEGF), and angiopoietin-2 were detected through a Luminex assay. Results Liver fibrosis in CCl 4 -treated mice was attenuated by reduced accumulation of collagen and the reaction of inflammation with carvedilol treatment. Carvedilol reduced the activation of hepatic stellate cells (HSCs) and increased the number of apoptotic cells. The expression of α-SMA, CD31, CD34 and VWF (von Willebrand factor) was significantly decreased after carvedilol treatment. In addition, the number of fenestrae in the hepatic sinusoid showed notable differences between the groups, and the serum levels of MMP-8, VEGF and angiopoietin-2 were increased in the mice with liver fibrosis and reduced by carvedilol treatment. Conclusion The study demonstrated that carvedilol could prevent further development of liver fibrosis and hepatic sinusoidal capillarization in mice with CCl 4 -induced fibrosis.

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Section: Discussionmentioning

confidence: 94%

<p>Carvedilol attenuates carbon tetrachloride-induced liver fibrosis and hepatic sinusoidal capillarization in mice</p>

Wu¹,

Li²,

Xiu³

et al. 2019

DDDT

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“…A major interaction candidate revealed by the MS analysis was RACK1 (Table 1 ), which is known to play important roles in protein targeting and membrane retention 31 , 33 – 37 . Therefore, we hypothesized that RACK1 contributes to NBCn1 membrane targeting and/or retention.…”

Section: Resultsmentioning

confidence: 99%

Trafficking, localization and degradation of the Na+,HCO3− co-transporter NBCn1 in kidney and breast epithelial cells

Olesen

Vogensen

Axholm

et al. 2018

Sci Rep

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The Na+;HCO3− co-transporter NBCn1 (SLC4A7) is a major regulator of intracellular pH yet its trafficking and turnover are essentially unstudied. Here, we used MDCK-II and MCF-7 cells to investigate these processes in epithelial cells. GFP-NBCn1 membrane localization was abolished by truncation of the full NBCn1 C-terminal tail (C-tail) yet did not require the C-terminal PDZ-binding motif (ETSL). Glutathione-S-Transferase-pulldown of the C-tail followed by mass spectrometry analysis revealed putative interactions with multiple sorting-, degradation- and retention factors, including the scaffolding protein RACK1. Pulldown of FLAG-tagged deletion constructs mapped the RACK1 interaction to the proximal NBCn1 C-tail. Proximity Ligation Assay and co-immunoprecipitation confirmed that native NBCn1 interacts with RACK1 in a cellular context. Consistent with a functional role of this complex, RACK1 knockdown reduced NBCn1 membrane localization without affecting total NBCn1 expression. Notably, only non-confluent cells exhibited detectable NBCn1-RACK1 plasma membrane co-localization, suggesting that RACK1 regulates the trafficking of NBCn1 to the membrane. Whereas total NBCn1 degradation was slow, with a half-life of more than 24 h, one-third of surface NBCn1 was constitutively endocytosed from the basolateral membrane within 60 min. This suggests that a fraction of NBCn1 exhibits recycling between the basolateral membrane and intracellular compartment(s). Our findings have important implications for understanding NBCn1 regulation as well as its dysregulation in disease.

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“…This method was also used in the study identifying the specific site phosphorylated by PKCδ, which phosphorylates Nrf2 at serine 40 3 . Since peptides competitively bind to a protein, the principle may also be applied to an in vitro GST pull-down assay to identify a binding motif 10,11 . Moreover, this peptidomimetic method may also be applicable to the identification of other post-translational modifications, such as acetylation at lysine residues.…”

Section: Discussionmentioning

confidence: 99%

Oligopeptide Competition Assay for Phosphorylation Site Determination

Joo

Koo

Shin

et al. 2017

JoVE

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Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects biological functions and characteristics of the cell. Currently, the most common method for discovering phosphorylation sites is by liquid chromatography/mass spectrometry (LC/MS) analysis, a rapid and sensitive method. However, relatively labile phosphate moieties are often released from phosphopeptides during the fragmentation step, which often yields false-negative signals. In such cases, a traditional in vitro kinase assay using site-directed mutants would be more accurate, but this method is laborious and time-consuming. Therefore, an alternative method using peptide competition may be advantageous. The consensus recognition motif of 5' adenosine monophosphate-activated protein kinase (AMPK) has been established and was validated using a positional scanning peptide library assay. Thus, AMPK phosphorylation sites for a novel substrate could be predicted and confirmed by the peptide competition assays. In this report, we describe the detailed steps and procedures for the in vitro oligopeptide-competing kinase assay by illustrating AMPK-mediated nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation. To authenticate the phosphorylation site, we carried out a sequential in vitro kinase assay using a site-specific mutant. Overall, the peptide competition assay provides a method to screen multiple potential phosphorylation sites and to identify sites for validation by the phosphorylation site mutants.

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RACK1 interacts with filamin-A to regulate plasma membrane levels of the cystic fibrosis transmembrane conductance regulator

Abstract: Smith L, Litman P, Kohli E, Amick J, Page RC, Misra S, Liedtke CM. RACK1 interacts with filamin-A to regulate plasma membrane levels of the cystic fibrosis transmembrane conductance regulator.

Cited by 6 publications

References 67 publications

<p>Carvedilol attenuates carbon tetrachloride-induced liver fibrosis and hepatic sinusoidal capillarization in mice</p>

<p>Carvedilol attenuates carbon tetrachloride-induced liver fibrosis and hepatic sinusoidal capillarization in mice</p>

Trafficking, localization and degradation of the Na+,HCO3− co-transporter NBCn1 in kidney and breast epithelial cells

Oligopeptide Competition Assay for Phosphorylation Site Determination

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