Localization and structure of the ankyrin-binding site on beta2-spectrin.
ABSTRACT Spectrins are tetrameric actin-cross-linking proteins that form an elastic network, termed the membrane skeleton, on the cytoplasmic surface of cellular membranes. At the plasma membrane, the membrane skeleton provides essential support, preventing loss of membrane material to environmental shear stresses. The skeleton also controls the location, abundance, and activity of membrane proteins that are critical to cell and tissue function. The ability of the skeleton to modulate membrane stability and function requires adaptor proteins that bind the skeleton to membranes. The principal adaptors are the ankyrin proteins, which bind to the beta-subunit of spectrin and to the cytoplasmic domains of numerous integral membrane proteins. Here, we present the crystal structure of the ankyrin-binding domain of human beta2-spectrin at 1.95 A resolution together with mutagenesis data identifying the binding surface for ankyrins on beta2-spectrin.
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ABSTRACT: Ankyrin-G and βII-spectrin colocalize at sites of cell-cell contact in columnar epithelial cells and promote lateral membrane assembly. This study identifies two critical inputs from lipids that together provide a rationale for how ankyrin-G and βII-spectrin selectively localize to Madin-Darby canine kidney (MDCK) cell lateral membranes. We identify aspartate-histidine-histidine-cysteine 5/8 (DHHC5/8) as ankyrin-G palmitoyltransferases required for ankyrin-G lateral membrane localization and for assembly of lateral membranes. We also find that βII-spectrin functions as a coincidence detector that requires recognition of both ankyrin-G and phosphoinositide lipids for its lateral membrane localization. DHHC5/8 and βII-spectrin colocalize with ankyrin-G in micrometer-scale subdomains within the lateral membrane that are likely sites for palmitoylation of ankyrin-G. Loss of either DHHC5/8 or ankyrin-G-βII-spectrin interaction or βII-spectrin-phosphoinositide recognition through its pleckstrin homology domain all result in failure to build the lateral membrane. In summary, we identify a functional network connecting palmitoyltransferases DHHC5/8 with ankyrin-G, ankyrin-G with βII-spectrin, and βII-spectrin with phosphoinositides that is required for the columnar morphology of MDCK epithelial cells.The Journal of Cell Biology 07/2014; 206(2):273-88. DOI:10.1083/jcb.201401016 · 9.69 Impact Factor
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ABSTRACT: Transforming growth factor-β (TGF-β) signaling promotes cell motility by inducing epithelial-to-mesenchymal transitions (EMTs) in normal physiology and development, as well as in pathological conditions, such as cancer. We performed a time-resolved analysis of the proteomic and phosphoproteomic changes of cultured human keratinocytes undergoing EMT and cell cycle arrest in response to stimulation with TGF-β. We quantified significant changes in 2079 proteins and 2892 phosphorylation sites regulated by TGF-β. We identified several proteins known to be involved in TGF-β-induced cellular processes, such as the cytostatic response, extracellular matrix remodeling, and epithelial dedifferentiation. In addition, we identified proteins involved in other cellular functions, such as vesicle trafficking, that were not previously associated with TGF-β signaling. Although many TGF-β responses are mediated by phosphorylation of the transcriptional regulators of the SMAD family by the TGF-β receptor complex, we observed rapid kinetics of changes in protein phosphorylation, indicating that many responses were mediated through SMAD-independent TGF-β signaling. Combined analysis of changes in protein abundance and phosphorylation and knowledge of protein interactions and transcriptional regulation provided a comprehensive representation of the dynamic signaling events underlying TGF-β-induced changes in cell behavior. Our data suggest that in epithelial cells stimulated with TGF-β, early signaling is a mixture of both pro- and antiproliferative signals, whereas later signaling primarily inhibits proliferation.Science Signaling 01/2014; 7(335):rs5. DOI:10.1126/scisignal.2004856 · 7.65 Impact Factor
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ABSTRACT: The spectrin superfamily is composed of proteins involved in cytolinker functions. Their main structural feature is a large central subdomain with numerous repeats folded in triple helical coiled-coils. Their similarity of sequence was considered to be low without detailed quantification of the intra- and intermolecular levels. Among the superfamily, we considered as essential to propose an overview of the surface properties of all the repeats of the five proteins of the spectrin family, namely α- and β-spectrins, α-actinin, dystrophin and utrophin. Therefore, the aim of this work was to obtain a quantitative comparison of all the repeats at both the primary sequence and the three-dimensional levels. For that purpose, we applied homology modelling methods to obtain structural models for successive and overlapping tandem repeats of the human erythrocyte α- and β-spectrins and utrophin, as previously undertaken for dystrophin, and we used the known structure of α-actinin. The matrix calculation of the pairwise similarities of all the repeat sequences and the electrostatic and hydrophobic surface properties throughout the protein family support the view that spectrins and α-actinin on one hand and utrophin and dystrophin on the other hand share some structural similarities, but a detailed molecular characterisation highlights substantial differences. The repeats within the family are far from identical, which is consistent with their multiple interactions with different cellular partners, including proteins and membrane lipids.Journal of Structural Biology 03/2014; 186(3). DOI:10.1016/j.jsb.2014.03.011 · 3.37 Impact Factor