566 research outputs found
Cavin 2 and cavin 3 form distinct subcomplexes with cavin 1 and caveolin 1.
No full text<p>(A) Gradient fractions (10–40% sucrose) of non-cross-linked and detergent solubilised (1% Triton X-100) HeLa cell extracts were analysed by Western blotting. Gradients were prepared from cells expressing either GFP (as control cells), cavin 1-GFP, cavin 2-GFP, or cavin 3-GFP. Membranes were probed with antibodies against caveolin 1, cavin 1, or GFP. Pooled fractions 3–5 and 6–8 used for immuno-isolation shown in (C) are boxed. (B) Quantification of the distribution of cavin 1, 2, and-3-GFP, cavin 1, and caveolin-1 in velocity gradients as shown in (A). Relative protein amounts were determined by densitometry of Western blots. The data are expressed as an average percentage of protein in each fraction calculated from four independent experiments. (C) Cavin 1, 2, and -3-GFP, flotillin-2-GFP, caveolin-1-GFP, or GFP were immuno-isolated from gradient fractions 3–5 or 6–8 as shown in (A). Eluted proteins were analysed by Western blotting using antibodies against GFP, cavin 1, or caveolin 1. (D) Caveolar coat complexes immuno-isolated from cross-linked HeLa cells stably expressing cavin-2-GFP were incubated with increasing concentrations of DTT to partially reduce crosslinks and analyzed by Western blotting using anti-caveolin 1 or anti-cavin 1 antibodies. Monomeric and oligomeric species of caveolin 1 and cavin 1 are indicated. Note the 180 kDa cavin 1 trimer.</p
The Effects of Cavin-2 on Angiogenesis
No full textCaveolae are bulb-shaped invaginations about 50-60nm in size on the plasma membrane of mammalian cells that play a key role in cell signaling, endocytosis, plasma membrane adaptations, and lipid homeostasis. Caveolae are known to be important in angiogenesis and are found in the microvasculature of the skeletal muscle, heart and lung, making up to 60% of the plasma membrane. While the mechanisms of caveolae regulation are not completely understood, Cavin family proteins appear to be critical regulators of caveolae. Cavin proteins form subcomplexes through homo- and hetero-oligomerization which regulate caveolae dynamics. Cavin-2, also known as serum deprivation-response protein, or SDPR, is a key regulator of caveolae dynamics by binding to Cavin-1 and introducing membrane tubules. To discover genes important for the transition of endothelial cells from a quiescent to an activated state, a switch that is critical for initiation of angiogenesis, we performed RNA sequencing analysis comparing unstimulated to activated, invading endothelial cells using a three-dimensional assay that mimics angiogenesis. We found expression of Cavin-2 was downregulated 73% in invading cells compared to non-invading cells. This study tested whether Cavin-2 might suppress endothelial sprouting responses. We found that overexpression of Cavin-2 had no statistically significant effect on cell invasion using this three-dimensional model of angiogenesis, indicating that Cavin-2 does not have a suppressive effect on endothelial sprout initiation
MURC/Cavin-4 and cavin family members form tissue-specific caveolar complexes
No full textPolymerase I and transcript release factor (PTRF)/Cavin is a cytoplasmic protein whose expression is obligatory for caveola formation. Using biochemistry and fluorescence resonance energy transfer–based approaches, we now show that a family of related proteins, PTRF/Cavin-1, serum deprivation response (SDR)/Cavin-2, SDR-related gene product that binds to C kinase (SRBC)/Cavin-3, and muscle-restricted coiled-coil protein (MURC)/Cavin-4, forms a multiprotein complex that associates with caveolae. This complex can constitutively assemble in the cytosol and associate with caveolin at plasma membrane caveolae. Cavin-1, but not other cavins, can induce caveola formation in a heterologous system and is required for the recruitment of the cavin complex to caveolae. The tissue-restricted expression of cavins suggests that caveolae may perform tissue-specific functions regulated by the composition of the cavin complex. Cavin-4 is expressed predominantly in muscle, and its distribution is perturbed in human muscle disease associated with Caveolin-3 dysfunction, identifying Cavin-4 as a novel muscle disease candidate caveolar protein
Cavin-3 knockout mice show that cavin-3 is not essential for caveolae formation, for maintenance of body composition, or for glucose tolerance.
No full textThe cavins are a family of proteins associated with caveolae, cavin-1, -2 and -3 being widely expressed while cavin-4 is restricted to striated muscle. Deletion of cavin-1 results in phenotypes including metabolic changes consistent with adipocyte dysfunction, and caveolae are completely absent. Deletion of cavin-2 causes tissue-specific loss of caveolae. The consequences of cavin-3 deletion are less clear, as there are divergent data on the abundance of caveolae in cavin-3 null mice. Here we examine the consequences of cavin-3 deficiency in vivo by making cavin-3 knockout mice. We find that loss of cavin-3 has minimal or no effects on the levels of other caveolar proteins, does not appear to play a major role in formation of protein complexes important for caveolar morphogenesis, and has no significant effect on caveolae abundance. Cavin-3 null mice have the same body weight and fat mass as wild type animals at ages 8 through 30 weeks on both normal chow and high fat diets. Likewise, the two mouse strains exhibit identical glucose tolerance tests on both diets. Microarray analysis from adipose tissue shows that the changes in mRNA expression between cavin-3 null and wild type mouse are minimal. We conclude that cavin-3 is not absolutely required for making caveolae, and suggest that the mechanistic link between cavin-3 and metabolic regulation remains uncertain
Generation of Cavin-3 knockout mouse.
No full text<p><i>Cavin-3</i> knockout mice were generated as described in “Method”. A. PCR genotyping was performed by using primers against LacZ and Cavin-3. B. Adipose tissue lysates (10 µg) from wild type (W), heterozygous (H) and knockout (K) mouse were separated by SDS-PAGE and transferred to PVDF membrane for immunoblotting analysis using the antibodies indicated.</p
Interaction of suppressor of cytokine signalling 3 with cavin-1 links SOCS3 function and cavin-1 stability
No full textEffective suppression of JAK–STAT signalling by the inducible inhibitor “suppressor of cytokine signalling 3” (SOCS3) is essential for limiting signalling from cytokine receptors. Here we show that cavin-1, a component of caveolae, is a functionally significant SOCS3-interacting protein. Biochemical and confocal imaging demonstrate that SOCS3 localisation to the plasma membrane requires cavin-1. SOCS3 is also critical for cavin-1 stabilisation, such that deletion of SOCS3 reduces the expression of cavin-1 and caveolin-1 proteins, thereby reducing caveola abundance in endothelial cells. Moreover, the interaction of cavin-1 and SOCS3 is essential for SOCS3 function, as loss of cavin-1 enhances cytokine-stimulated STAT3 phosphorylation and abolishes SOCS3-dependent inhibition of IL-6 signalling by cyclic AMP. Together, these findings reveal a new functionally important mechanism linking SOCS3-mediated inhibition of cytokine signalling to localisation at the plasma membrane via interaction with and stabilisation of cavin-1
Down-regulation of the cavin family proteins in breast cancer
No full textCaveolae are abundant membrane domain on the cell surface of many mammalian cell types and are implicated in a wide range of physiological processes. The caveolae structural protein caveolin-1 is often mutated or deregulated in cancer, and cavin family protein serum deprivation response factor-related gene product that binds to C-kinase (SRBC) has been found to be epigenetically inactivated in lung, breast, and gastric cancer. Both caveolin-1 and SRBC have been proposed to function as tumor suppressors. Polymerase 1 and transcript release factor (PTRF) is the essential component for caveolae formation. The regulation of PTRF expression in cancer has not been characterized. We report here that the cavin family protein PTRF, SRBC and serum deprivation response protein were down regulated in breast cancer cell lines and breast tumor tissue. We further show that down-regulation of PTRF in breast cancer cells was associated with the promoter methylation. As caveolin-1 and cavin family proteins are required for caveolae formation and function, the reported tumor suppression function of caveolin-1 and SRBC may be due to the deregulation of caveolae and its down-stream signaling. Thus, the caveolae is a potential therapeutic target and the expression of cavin family proteins could be a useful prognostic indicator of breast cancer progression. J. Cell. Biochem. 113: 322328, 2012. (C) 2011 Wiley Periodicals, Inc.Biochemistry & Molecular BiologyCell BiologySCI(E)0ARTICLE1322-32811
Identification of Critical Genes for Growth in Olive Brine byTransposon Mutagenesis of Lactobacillus pentosus C11
No full textOlive brine represents a stressful environment due to the high NaCl concentration, presence of phenolic compounds known as antimicrobials, and low availability of nutrients. Thus, only a few strains of lactic acid bacteria (LAB) are adapted to grow in and ferment table olives. To identify the mechanisms by which these few strains are able to grow in olive brine, Lactobacillus pentosus C11, a particularly resistant strain isolated from naturally fermented table olives, was mutagenized by random transposition using the Pjunc-TpaseIS1223 system (H. Licandro-Seraut, S. Brinster, M. van de Guchte, H. Scornec, E. Maguin, P. Sansonetti, J. F. Cavin, and P. Serror, Appl. Environ. Microbiol. 78:5417–5423, 2012). A library of 6,000 mutants was generated and screened for adaptation and subsequent growth in a medium, named BSM (brine screening medium), which presents the stressful conditions encountered in olive brine. Five transposition mutants impaired in growth on BSM were identified. Transposition occurred in two open reading frames and in three transcription terminators affecting stability of transcripts. Thus, several essential genes for adaptation and growth of L. pentosus C11 in olive brine were identified
Cavin‐2 promotes fibroblast‐to‐myofibroblast trans‐differentiation and aggravates cardiac fibrosis
No full textAbstract Aims Transforming growth factor β (TGF‐β) signalling is one of the critical pathways in fibroblast activation, and several drugs targeting the TGF‐β/Smad signalling pathway in heart failure with cardiac fibrosis are being tested in clinical trials. Some caveolins and cavins, which are components of caveolae on the plasma membrane, are known for their association with the regulation of TGF‐β signalling. Cavin‐2 is particularly abundant in fibroblasts; however, the detailed association between Cavin‐2 and cardiac fibrosis is still unclear. We tried to clarify the involvement and role of Cavin‐2 in fibroblasts and cardiac fibrosis. Methods and results To clarify the role of Cavin‐2 in cardiac fibrosis, we performed transverse aortic constriction (TAC) operations on four types of mice: wild‐type (WT), Cavin‐2 null (Cavin‐2 KO), Cavin‐2flox/flox, and activated fibroblast‐specific Cavin‐2 conditional knockout (Postn‐Cre/Cavin‐2flox/flox, Cavin‐2 cKO) mice. We collected mouse embryonic fibroblasts (MEFs) from WT and Cavin‐2 KO mice and investigated the effect of Cavin‐2 in fibroblast trans‐differentiation into myofibroblasts and associated TGF‐β signalling. Four weeks after TAC, cardiac fibrotic areas in both the Cavin‐2 KO and the Cavin‐2 cKO mice were significantly decreased compared with each control group (WT 8.04 ± 1.58% vs. Cavin‐2 KO 0.40 ± 0.03%, P < 0.01; Cavin‐2flox/flox, 7.19 ± 0.50% vs. Cavin‐2 cKO 0.88 ± 0.44%, P < 0.01). Fibrosis‐associated mRNA expression (Col1a1, Ctgf, and Col3) was significantly attenuated in the Cavin‐2 KO mice after TAC. α1 type I collagen deposition and non‐vascular αSMA‐positive cells (WT 43.5 ± 2.4% vs. Cavin‐2 KO 25.4 ± 3.2%, P < 0.01) were reduced in the heart of the Cavin‐2 cKO mice after TAC operation. The levels of αSMA protein (0.36‐fold, P < 0.05) and fibrosis‐associated mRNA expression (Col1a1, 0.69‐fold, P < 0.01; Ctgf, 0.27‐fold, P < 0.01; Col3, 0.60‐fold, P < 0.01) were decreased in the Cavin‐2 KO MEFs compared with the WT MEFs. On the other hand, αSMA protein levels were higher in the Cavin‐2 overexpressed MEFs compared with the control MEFs (2.40‐fold, P < 0.01). TGF‐β1‐induced Smad2 phosphorylation was attenuated in the Cavin‐2 KO MEFs compared with WT MEFs (0.60‐fold, P < 0.01). Heat shock protein 90 protein levels were significantly reduced in the Cavin‐2 KO MEFs compared with the WT MEFs (0.69‐fold, P < 0.01). Conclusions Cavin‐2 loss suppressed fibroblast trans‐differentiation into myofibroblasts through the TGF‐β/Smad signalling. The loss of Cavin‐2 in cardiac fibroblasts suppresses cardiac fibrosis and may maintain cardiac function
Data from: Cavin-3 dictates the balance between ERK and Akt signaling
No full textCavin-3 is a tumor suppressor protein of unknown function. Using a combination of in vivo knockout and in vitro gain/loss of function approaches, we show that cavin-3 dictates the balance between ERK and Akt signaling. Loss of cavin-3 increases Akt signaling at the expense of ERK, while gain of cavin-3 increases ERK signaling at the expense Akt. Cavin-3 facilitates signal transduction to ERK by anchoring caveolae, a lipid-raft specialization that contains an ERK activation module, to the membrane skeleton of the plasma membrane. Loss of cavin-3 reduces the number of caveolae, thereby separating this ERK activation module from signaling receptors. Loss of cavin-3 promotes Akt signaling through suppression of EGR1 and PTEN. The in vitro consequences of the loss of cavin-3 include induction of Warburg metabolism (aerobic glycolysis), accelerated cell proliferation and resistance to apoptosis. The in vivo consequences of cavin-3 loss are increased lactate production and cachexia
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