| 1. |
- Costa, Tânia D F, et al.
(författare)
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PAK4 suppresses RELB to prevent senescence-like growth arrest in breast cancer
- 2019
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 10:1, s. 1-18
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Tidskriftsartikel (refereegranskat)abstract
- Overcoming cellular growth restriction, including the evasion of cellular senescence, is a hallmark of cancer. We report that PAK4 is overexpressed in all human breast cancer subtypes and associated with poor patient outcome. In mice, MMTV-PAK4 overexpression promotes spontaneous mammary cancer, while PAK4 gene depletion delays MMTV-PyMT driven tumors. Importantly, PAK4 prevents senescence-like growth arrest in breast cancer cells in vitro, in vivo and ex vivo, but is not needed in non-immortalized cells, while PAK4 overexpression in untransformed human mammary epithelial cells abrogates H-RAS-V12-induced senescence. Mechanistically, a PAK4 - RELB - C/EBPβ axis controls the senescence-like growth arrest and a PAK4 phosphorylation residue (RELB-Ser151) is critical for RELB-DNA interaction, transcriptional activity and expression of the senescence regulator C/EBPβ. These findings establish PAK4 as a promoter of breast cancer that can overcome oncogene-induced senescence and reveal a selective vulnerability of cancer to PAK4 inhibition.
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| 2. |
- Elliott, Kerryn, et al.
(författare)
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A novel function of the monomeric CCT epsilon subunit connects the serum response factor pathway to chaperone-mediated actin folding
- 2015
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 26:15, s. 2801-2809
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Tidskriftsartikel (refereegranskat)abstract
- Correct protein folding is fundamental for maintaining protein homeostasis and avoiding the formation of potentially cytotoxic protein aggregates. Although some proteins appear to fold unaided, actin requires assistance from the oligomeric molecular chaperone CCT. Here we report an additional connection between CCT and actin by identifying one of the CCT subunits, CCT epsilon, as a component of the myocardin-related cotranscription factor-A (MRTF-A)/serum response factor (SRF) pathway. The SRF pathway registers changes in G-actin levels, leading to the transcriptional up-regulation of a large number of genes after actin polymerization. These genes encode numerous actin-binding proteins as well as actin. We show that depletion of the CCT epsilon subunit by siRNA enhances SRF signaling in cultured mammalian cells by an actin assembly-independent mechanism. Overexpression of CCTe in its monomeric form revealed that CCT epsilon binds via its substrate-binding domain to the C-terminal region of MRTF-A and that CCT epsilon is able to alter the nuclear accumulation of MRTF-A after stimulation by serum addition. Given that the levels of monomeric CCT epsilon conversely reflect the levels of CCT oligomer, our results suggest that CCT epsilon provides a connection between the actin-folding capacity of the cell and actin expression.
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| 3. |
- Nejedla, Michaela, et al.
(författare)
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A Fluorophore Fusion Construct of Human Profilin I with Non-Compromised Poly(L-Proline) Binding Capacity Suitable for Imaging
- 2017
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 429:7, s. 964-976
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Tidskriftsartikel (refereegranskat)abstract
- Profilin is vital for actin organisation in eukaryotic cells. It controls actin filament formation by binding monomeric actin and numerous proteins involved in polarised actin assembly. Important for the latter is the interaction surface formed by the N- and C-terminal helices, which pack close to each other on one side of the molecule at a distance from the actin site and mediate binding to poly-proline sequences present in many of the targeted proteins. Via these interactions, profilin contributes to the spatiotemporal control of actin filament growth. Studies of profilin dynamics in living cells by imaging techniques have been hampered by problems to generate fusion constructs with fluorophore proteins without negatively impacting on its poly-proline binding. With the object to circumvent this problem, we have generated an internal fusion of profilin with the green fluorescent variant citrine, here referred to as citrine profilin. The characterisation of citrine profilin (CIT-Pfn) demonstrates that it has full capacity to interact with poly-proline and also binds phosphatidylinositol lipids and actin, albeit with 10 times reduced affinity for the latter. Imaging of living cells expressing CIT-Pfn showed a distribution of the fusion protein similar to endogenous profilin. Furthermore, CIT-Pfn rescued the phenotypes observed after the Crispr/Cas9 knockout of the profilin 1 gene, including the lost migratory capacity characterising the knockout cells. Based on this, we conclude that the CIT-Pfn construct will be useful as a tool for displaying profilin localisation in living cells and obtaining information on its dynamic organisation under different conditions and activations of the actin microfilament and microtubule systems.
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| 4. |
- Spiess, Matthias, et al.
(författare)
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Active and inactive beta 1 integrins segregate into distinct nanoclusters in focal adhesions
- 2018
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Ingår i: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 217:6, s. 1929-1940
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Tidskriftsartikel (refereegranskat)abstract
- Integrins are the core constituents of cell-matrix adhesion complexes such as focal adhesions (FAs) and play key roles in physiology and disease. Integrins fluctuate between active and inactive conformations, yet whether the activity state influences the spatial organization of integrins within FAs has remained unclear. In this study, we address this question and also ask whether integrin activity may be regulated either independently for each integrin molecule or through locally coordinated mechanisms. We used two distinct superresolution microscopy techniques, stochastic optical reconstruction microscopy (STORM) and stimulated emission depletion microscopy (STED), to visualize active versus inactive beta 1 integrins. We first reveal a spatial hierarchy of integrin organization with integrin molecules arranged in nanoclusters, which align to form linear substructures that in turn build FAs. Remarkably, within FAs, active and inactive beta 1 integrins segregate into distinct nanoclusters, with active integrin nanoclusters being more organized. This unexpected segregation indicates synchronization of integrin activities within nanoclusters, implying the existence of a coordinate mechanism of integrin activity regulation.
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| 5. |
- Spiess, Matthias, et al.
(författare)
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Over-Expression Analysis of All Eight Subunits of the Molecular Chaperone CCT in Mammalian Cells Reveals a Novel Function for CCTdelta
- 2015
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 427:17, s. 2757-2764
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Tidskriftsartikel (refereegranskat)abstract
- Chaperonin containing tailless complex polypeptide 1 (CCT) forms a classical chaperonin barrel structure where two rings of subunits surround a central cavity. Each ring consists of eight distinct subunits, creating a complex binding interface that makes CCT unique among the chaperonins. In addition to acting as a multimeric chaperonin, there is increasing evidence indicating that the CCT subunits, when monomeric, possess additional functions. Here we assess the role of the CCT subunits individually, using a GFP (green fluorescent protein) tagging approach to express each of the subunits in their monomeric form in cultured mammalian cells. Over-expression of CCTdelta, but not the other seven CCT subunits, results in the appearance of numerous protrusions at the cell surface. Two point mutations, one in the apical domain and one in the ATP binding pocket of CCTdelta, that abolish protrusion formation have been identified, consistent with the apical domain containing a novel interaction site that is influenced by the ATPase activity in the equatorial domain. Structured illumination microscopy, together with sub-cellular fractionation, reveals that only the wild-type CCTdelta is associated with the plasma membrane, thus connecting spatial organization with surface protrusion formation. Expression of the equivalent subunit in yeast, GFP-Cct4, rescues growth of the temperature-sensitive strain cct4-1 at the non-permissive temperature, indicative of conserved subunit-specific activities for CCTdelta. (C) 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-ncV4.0/).
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