Interaction of kindlin-2 with integrin β3 promotes outside-in signaling responses by the αVβ3 vitronectin receptor

Liao, Zhongji; Kato, Hisashi; Pandey, Manjula; Cantor, Joseph M.; Ablooglu, Ararat J.; Ginsberg, Mark H.; Shattil, Sanford J.

doi:10.1182/blood-2014-09-603035

Cited by 32 publications

(36 citation statements)

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“…β3 null mouse lung endothelial cells (mLECs) isolated from the lungs of β3 −/− mice and immortalized by retroviral delivery of a SV40 large T antigen essentially as described (Liao et al, 2015) were generously provided by Mark Ginsberg and Brian Petrich (University of California at San Diego, CA). β3 WT, D723R and L138I human integrin cDNAs, gifts from Mark Ginsberg and Timothy Springer (Harvard Medical School, Boston, MA), were cloned into pBOB, packaged into lentivirus and used to infect the immortalized β3 null mLECs as described (Coon et al, 2015).…”

Section: Methodsmentioning

confidence: 99%

Force regulated conformational change of integrin αVβ3

et al. 2017

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Integrins mediate cell adhesion to extracellular matrix and transduce signals bidirectionally across the membrane. Integrin αVβ3 has been shown to play an essential role in tumor metastasis, angiogenesis, hemostasis and phagocytosis. Integrins can take several conformations, including the bent and extended conformations of the ectodomain, which regulate integrin functions. Using a biomembrane force probe, we characterized the bending and unbending conformational changes of a single integrin αVβ3 molecule on a living cell surface in real-time. We measured the probabilities of conformational changes, rates and speeds of conformational transitions, and the dynamic equilibrium between the two conformations, which were regulated by tensile force, dependent on the ligand, and altered by point mutations. These findings provide insights into how αVβ3 acts as a molecular machine and how its physiological function and molecular structure are coupled at the single molecule level.

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Section: Methodsmentioning

confidence: 99%

Force regulated conformational change of integrin αVβ3

et al. 2017

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“…Recently, β3 integrin mutant mice and lung endothelial cells derived from these mice were employed to investigate mechanisms of kindlin-2 interaction with αVβ3 (Liao et al, 2015). Kindlin-2 was found to interact directly with β3 in a manner that requires the Cterminal three residues of the β3 cytoplasmic tail (Arg-Gly-Thr; RGT), potentially similar to the requirement for the C-terminal three residues of β1 for its interaction with kindlin-2 (Li et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

“…β3ΔRGT knock-in mice expressing β3 that lacks these three Cterminal residues exhibited reduced physiological (retinal) and pathological (tumor) angiogenesis when compared to wild-type littermates, suggesting a positive role for the αVβ3-kindlin-2 interaction in angiogenesis. Moreover, enforced interaction of kindlin-2 with β3ΔRGT using a chemical dimerizer strategy rescued endothelial cell sprout formation in an in vitro fibrin gel assay (Liao et al, 2015). However, these studies did not examine temporal or spatial details of the αVβ3-kindlin-2 interaction in endothelial cells, nor did they focus on the role of kindlin-2 interactions with other intracellular binding partners.…”

Section: Introductionmentioning

confidence: 99%

Optogenetic interrogation of integrin αVβ3 function in endothelial cells

Liao

Kasirer-Friede

Shattil

2017

Journal of Cell Science

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The integrin αVβ3 is reported to promote angiogenesis in some model systems but not in others. Here, we used optogenetics to study the effects of αVβ3 interaction with the intracellular adapter kindlin-2 (Fermt2) on endothelial cell functions potentially relevant to angiogenesis. Because interaction of kindlin-2 with αVβ3 requires the C-terminal three residues of the β3 cytoplasmic tail (Arg-Gly-Thr; RGT), optogenetic probes LOVpep and ePDZ1 were fused to β3ΔRGT-GFP and mCherry-kindlin-2, respectively, and expressed in β3 integrin-null microvascular endothelial cells. Exposure of the cells to 450 nm (blue) light caused rapid and specific interaction of kindlin-2 with αVβ3 as assessed by immunofluorescence and total internal reflection fluorescence (TIRF) microscopy, and it led to increased endothelial cell migration, podosome formation and angiogenic sprouting. Analyses of kindlin-2 mutants indicated that interaction of kindlin-2 with other kindlin-2 binding partners, including c-Src, actin, integrin-linked kinase and phosphoinositides, were also likely necessary for these endothelial cell responses. Thus, kindlin-2 promotes αVβ3-dependent angiogenic functions of endothelial cells through its simultaneous interactions with β3 integrin and several other binding partners. Optogenetic approaches should find further use in clarifying spatiotemporal aspects of vascular cell biology.

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“…Kindlin-2 has been shown to cooperate with talin to mediate integrin activation; multiple lines of evidence suggest that kindlin-2 plays pivotal roles in controlling both the outside-in and inside-out signaling of integrin (14,24,25). To study whether kindlin-2 is important for RNA polymerase I transcriptional activity, we analyzed RNA transcription in MEFs in the absence of kindlin-2.…”

Section: Figmentioning

confidence: 99%

Phosphatidylinositol 3-Kinase/Akt Mediates Integrin Signaling To Control RNA Polymerase I Transcriptional Activity

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et al. 2016

Molecular and Cellular Biology

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c RNA polymerase I-mediated rRNA production is a key determinant of cell growth. Despite extensive studies, the signaling pathways that control RNA polymerase I-mediated rRNA production are not well understood. Here we provide original evidence showing that RNA polymerase I transcriptional activity is tightly controlled by integrin signaling. Furthermore, we show that a signaling axis consisting of focal adhesion kinase (FAK), Src, phosphatidylinositol 3-kinase (PI3K), Akt, and mTOR mediates the effect of integrin signaling on rRNA transcription. Additionally, we show that in kindlin-2 knockout mouse embryonic fibroblasts, overactivation of Ras, Akt, and Src can successfully rescue the defective RNA polymerase I activity induced by the loss of kindlin-2. Finally, through experiments with inhibitors of FAK, Src, and PI3K and rescue experiments in MEFs, we found that the FAK/Src/PI3K/Akt signaling pathway to control rRNA transcription is linear. Collectively, these studies reveal, for the first time, a pivotal role of integrin signaling in regulation of RNA polymerase I transcriptional activity and shed light on the downstream signaling axis that participates in regulation of this key aspect of cell growth. RNA polymerase I (Pol I) plays a central role in regulating cellular growth and proliferation (1). Eukaryotic cells contain hundreds of ribosomal DNA (rDNA) copies that occupy several different chromosomal locations (2). The production of rRNA can be divided into several steps, i.e., rRNA transcription, modification, and processing, all of which occur in the nucleolus (3, 4). The rate-limiting step is rRNA transcription (1, 5). On sensing of outside stimuli, a preinitiation complex comprised of the transcriptional factors upstream binding factor (UBF), SL1, TBP, Rrn3, and TTF assembles in the promoter region of rDNA. This complex then recruits RNA polymerase I to rDNA loci, and rRNA transcription starts (6-8). In mammalian cells, a single precursor rRNA transcript, 47S rRNA (14.3 kb), is transcribed from rDNA by the RNA polymerase I complex. This large polycistronic transcript encompasses 18S, 5.8S, and 28S rRNAs and includes several spacer regions, which are later processed into distinct rRNA species before assembly into preribosomal subunits (9).The transcriptional activity of Pol I is a fundamental determinant of cell proliferation capacity (3). In rapidly proliferating cells, rRNA production takes more than 50% of all nuclear transcriptional activity. In yeast cells, this percentage can reach more than 80% (10). As such, the tremendous energy consumption demands tight control.At the tissue level, cells attach to the extracellular matrix (ECM) through cell surface receptors termed integrins (11). Integrins are heterodimeric transmembrane receptors comprised of ␣ subunits and ␤ subunits that bind to extracellular ligands, such as laminin, collagen, vitronectin, and fibronectin. Different combinations of the 18 ␣ subunits and 8 ␤ subunits confer specificity on the integrin-ECM interactions (12). After binding ...

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Interaction of kindlin-2 with integrin β3 promotes outside-in signaling responses by the αVβ3 vitronectin receptor

Abstract: Key Points Interaction of the integrin β3 cytoplasmic tail with kindlin-2 selectively promotes outside-in signaling through αVβ3. Disruption of the kindlin-2/αVβ3 interaction impairs outside-in signaling and endothelial cell functions, both in vitro and in vivo.

Cited by 32 publications

References 65 publications

Force regulated conformational change of integrin αVβ3

Force regulated conformational change of integrin αVβ3

Optogenetic interrogation of integrin αVβ3 function in endothelial cells

Phosphatidylinositol 3-Kinase/Akt Mediates Integrin Signaling To Control RNA Polymerase I Transcriptional Activity

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