Fascin lysine 471 acetylation cooperates with serine 39 phosphorylation to inhibit actin-bundling activity and tumor metastasis in esophageal squamous cell carcinoma.
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ABSTRACT:
SUBMITTER: Zeng FM
PROVIDER: S-EPMC9257986 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundFascin is crucial for cancer cell filopodium formation and tumor metastasis, and is functionally regulated by post-translational modifications. However, whether and how Fascin is regulated by acetylation remains unclear. This study explored the regulation of Fascin acetylation and its corresponding roles in filopodium formation and tumor metastasis.MethodsImmunoprecipitation and glutathione-S-transferase pull-down assays were performed to examine the interaction between Fascin and acetyltransferase P300/CBP-associated factor (PCAF), and immunofluorescence was used to investigate their colocalization. An in vitro acetylation assay was performed to identify Fascin acetylation sites by using mass spectrometry. A specific antibody against acetylated Fascin was generated and used to detect the PCAF-mediated Fascin acetylation in esophageal squamous cell carcinoma (ESCC) cells using Western blotting by overexpressing and knocking down PCAF expression. An in vitro cell migration assay was performed, and a xenograft model was established to study in vivo tumor metastasis. Live-cell imaging and fluorescence recovery after photobleaching were used to evaluate the function and dynamics of acetylated Fascin in filopodium formation. The clinical significance of acetylated Fascin and PCAF in ESCC was evaluated using immunohistochemistry.ResultsFascin directly interacted and colocalized with PCAF in the cytoplasm and was acetylated at lysine 471 (K471) by PCAF. Using the specific anti-AcK471-Fascin antibody, Fascin was found to be acetylated in ESCC cells, and the acetylation level was consequently increased after PCAF overexpression and decreased after PCAF knockdown. Functionally, Fascin-K471 acetylation markedly suppressed in vitro ESCC cell migration and in vivo tumor metastasis, whereas Fascin-K471 deacetylation exhibited a potent oncogenic function. Moreover, Fascin-K471 acetylation reduced filopodial length and density, and lifespan of ESCC cells, while its deacetylation produced the opposite effect. In the filipodium shaft, K471-acetylated Fascin displayed rapid dynamic exchange, suggesting that it remained in its monomeric form owing to its weakened actin-bundling activity. Clinically, high levels of AcK471-Fascin in ESCC tissues were strongly associated with prolonged overall survival and disease-free survival of ESCC patients.ConclusionsFascin interacts directly with PCAF and is acetylated at lysine 471 in ESCC cells. Fascin-K471 acetylation suppressed ESCC cell migration and tumor metastasis by reducing filopodium formation through the impairment of its actin-bundling activity.
| S-EPMC8696220 | biostudies-literature
Project description:Fascin is the main actin filament bundling protein in filopodia. Because of the important role filopodia play in cell migration, fascin is emerging as a major target for cancer drug discovery. However, an understanding of the mechanism of bundle formation by fascin is critically lacking. Fascin consists of four ?-trefoil domains. Here, we show that fascin contains two major actin-binding sites, coinciding with regions of high sequence conservation in ?-trefoil domains 1 and 3. The site in ?-trefoil-1 is located near the binding site of the fascin inhibitor macroketone and comprises residue Ser-39, whose phosphorylation by protein kinase C down-regulates actin bundling and formation of filopodia. The site in ?-trefoil-3 is related by pseudo-2-fold symmetry to that in ?-trefoil-1. The two sites are ?5 nm apart, resulting in a distance between actin filaments in the bundle of ?8.1 nm. Residue mutations in both sites disrupt bundle formation in vitro as assessed by co-sedimentation with actin and electron microscopy and severely impair formation of filopodia in cells as determined by rescue experiments in fascin-depleted cells. Mutations of other areas of the fascin surface also affect actin bundling and formation of filopodia albeit to a lesser extent, suggesting that, in addition to the two major actin-binding sites, fascin makes secondary contacts with other filaments in the bundle. In a high resolution crystal structure of fascin, molecules of glycerol and polyethylene glycol are bound in pockets located within the two major actin-binding sites. These molecules could guide the rational design of new anticancer fascin inhibitors.
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Project description:Actin filament turnover plays a central role in shaping actin networks, yet the feedback mechanism between network architecture and filament assembly dynamics remains unclear. The activity of ADF/cofilin, the main protein family responsible for filament disassembly, has been mainly studied at the single filament level. This study unveils that fascin, by crosslinking filaments into bundles, strongly slows down filament disassembly by cofilin. We show that this is due to a markedly slower initiation of the first cofilin clusters, which occurs up to 100-fold slower on large bundles compared with single filaments. In contrast, severing at cofilin cluster boundaries is unaffected by fascin bundling. After the formation of an initial cofilin cluster on a filament within a bundle, we observed the local removal of fascin. Notably, the formation of cofilin clusters on adjacent filaments is highly enhanced, locally. We propose that this interfilament cooperativity arises from the local propagation of the cofilin-induced change in helicity from one filament to the other filaments of the bundle. Overall, taking into account all the above reactions, we reveal that fascin crosslinking slows down the disassembly of actin filaments by cofilin. These findings highlight the important role played by crosslinkers in tuning actin network turnover by modulating the activity of other regulatory proteins.
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Project description:Cells use a large repertoire of proteins to remodel the actin cytoskeleton. Depending on the proteins involved, F-actin is organized in specialized protrusions such as lamellipodia or filopodia, which serve diverse functions in cell migration and sensing. Although factors responsible for directed filament assembly in filopodia have been extensively characterized, the mechanisms of filament disassembly in these structures are mostly unknown. We investigated how the actin-depolymerizing factor cofilin-1 affects the dynamics of fascincrosslinked actin filaments in vitro and in live cells. By multicolor total internal reflection fluorescence microscopy and fluorimetric assays, we found that cofilin-mediated severing is enhanced in fascin-crosslinked bundles compared with isolated filaments, and that fascin and cofilin act synergistically in filament severing. Immunolabeling experiments demonstrated for the first time that besides its known localization in lamellipodia and membrane ruffles, endogenous cofilin can also accumulate in the tips and shafts of filopodia. Live-cell imaging of fluorescently tagged proteins revealed that cofilin is specifically targeted to filopodia upon stalling of protrusion and during their retraction. Subsequent electron tomography established filopodial actin filament and/or bundle fragmentation to precisely correlate with cofilin accumulation. These results identify a new mechanism of filopodium disassembly involving both fascin and cofilin.
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Project description:We have previously shown that angiotensin-converting enzyme 2 (ACE2), an enzyme counterbalancing the deleterious effects of angiotensin type 1 receptor activation by production of vasodilatory peptides Angiotensin (Ang)-(1-9) and Ang-(1-7), is internalized and degraded in lysosomes following chronic Ang-II treatment. However, the molecular mechanisms involved in this effect remain unknown. In an attempt to identify the accessory proteins involved in this effect, we conducted a proteomic analysis in ACE2-transfected HEK293T cells. A single protein, fascin-1, was found to differentially interact with ACE2 after Ang-II treatment for 4 h. The interactions between fascin-1 and ACE2 were confirmed by confocal microscopy and co-immunoprecipitation. Overexpression of fascin-1 attenuates the effects of Ang-II on ACE2 activity. In contrast, downregulation of fascin-1 severely decreased ACE2 enzymatic activity. Interestingly, in brain homogenates from hypertensive mice, we observed a significant reduction of fascin-1, suggesting that the levels of this protein may change in cardiovascular diseases. In conclusion, we identified fascin-1 as an ACE2-accessory protein, interacting with the enzyme in an Ang-II dependent manner and contributing to the regulation of enzyme activity.
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Project description:High expression of the actin bundling protein Fascin increases the malignancy of tumor cells. Here we show that fascin expression is up-regulated in more malignant sub-cell lines of MDA-MB-231 cells as compared to parental cells. Since also parental MDA-MB-231 cells exhibit high fascin levels, increased fascin expression was termed as "hyperexpression". To examine the effect of fascin hyperexpression, fascin was hyperexpressed in parental MDA-MB-231 cells and metastasis was analyzed in NOD scid gamma (NSG) mice. In addition, the effect of fascin mutants with inactive or constitutively active actin bundling activity was examined. Unexpectedly, we found that hyperexpression of both, wildtype (wt) and mutant fascin strongly increased metastasis in vivo, showing that the effect of fascin hyperexpression did not depend on its actin bundling activity. Cellular assays revealed that hyperexpression of wt and mutant fascin increased adhesion of MDA-MB-231 cells while transmigration and proliferation were not affected. Since it has been shown that fascin controls adhesion by directly interacting with microtubules (MTs), we analyzed if fascin hyperexpression affects MT dynamics. We found that at high concentrations fascin significantly increased MT dynamics in cells and in cell-free approaches. In summary our data show that strong expression of fascin in breast cancer cells increases metastasis independent of its actin bundling activity. Thus, it seems that the mechanism of fascin-stimulated metastasis depends on its concentration.
| S-EPMC5746366 | biostudies-literature
Project description:Fascin is an actin-bundling protein involved in filopodia assembly and cancer invasion and metastasis of multiple epithelial cancer types. Fascin forms stable actin bundles with slow dissociation kinetics in vitro and is regulated by phosphorylation of serine 39 by protein kinase C (PKC). Cancer cells use invasive finger-like protrusions termed invadopodia to invade into and degrade extracellular matrix. Invadopodia have highly dynamic actin that is assembled by both Arp2/3 complex and formins; they also contain components of membrane trafficking machinery such as dynamin and cortactin and have been compared with focal adhesions and podosomes. We show that fascin is an integral component of invadopodia and that it is important for the stability of actin in invadopodia. The phosphorylation state of fascin at S39, a PKC site, contributes to its regulation at invadopodia. We further implicate fascin in invasive migration into collagen I-Matrigel gels and particularly in cell types that use an elongated mesenchymal type of motility in 3D. We provide a potential molecular mechanism for how fascin increases the invasiveness of cancer cells, and we compare invadopodia with invasive filopod-like structures in 3D.
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Project description:Fascin actin-bundling protein 1 (FSCN1) is a highly conserved actin-bundling protein that cross links F-actin microfilaments into tight, parallel bundles. Elevated FSCN1 levels have been reported in many types of human cancers and have been correlated with aggressive clinical progression, poor prognosis, and survival outcomes. The overexpression of FSCN1 in cancer cells has been associated with tumor growth, migration, invasion, and metastasis. Currently, FSCN1 is recognized as a candidate biomarker for multiple cancer types and as a potential therapeutic target. The aim of this study was to provide a brief overview of the FSCN1 gene and protein structure and elucidate on its actin-bundling activity and physiological functions. The main focus was on the role of FSCN1 and its upregulatory mechanisms and significance in cancer cells. Up-to-date studies on FSCN1 as a novel biomarker and therapeutic target for human cancers are reviewed. It is shown that FSCN1 is an unusual biomarker and a potential therapeutic target for cancer.
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Project description:Protein acetylation is a rapid mechanism for control of protein function. Acetyl-CoA synthetase (AMP-forming, Acs) is the paradigm for the control of metabolic enzymes by lysine acetylation. In many bacteria, type I or II protein acetyltransferases acetylate Acs, however, in actinomycetes type III protein acetyltransferases control the activity of Acs. We measured changes in the activity of the Streptomyces lividans Acs (SlAcs) enzyme upon acetylation by PatB using in vitro and in vivo analyses. In addition to the acetylation of residue K610, residue S608 within the acetylation motif of SlAcs was also acetylated (PKTRSGK610 ). S608 acetylation rendered SlAcs inactive and non-acetylatable by PatB. It is unclear whether acetylation of S608 is enzymatic, but it was clear that this modification occurred in vivo in Streptomyces. In S. lividans, an NAD+ -dependent sirtuin deacetylase from Streptomyces, SrtA (a homologue of the human SIRT4 protein) was needed to maintain SlAcs function in vivo. We have characterized a sirtuin-dependent reversible lysine acetylation system in Streptomyces lividans that targets and controls the Acs enzyme of this bacterium. These studies raise questions about acetyltransferase specificity, and describe the first Acs enzyme in any organism whose activity is modulated by O-Ser and N? -Lys acetylation.
| S-EPMC5796852 | biostudies-literature
Project description:A cDNA for fascin, an actin-bundling protein in echinoderms, has been cloned, sequenced, and expressed. The predicted mass of the protein is approximately 55 kDa, similar to that observed for fascin purified from sea urchin eggs. Bacterially expressed fascin reacts with antibodies prepared against sea urchin egg fascin. Fascin has a strong sequence similarity to the singed gene (sn) product in Drosophila and has similarities with a 55-kDa human actin-bundling protein. No extensive similarities were found with other known actin-binding/bundling proteins, indicating that this is a separate gene family.
| S-EPMC47512 | biostudies-other