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Integrin Signaling in Cell Adhesion and Mechanotransduction: Regulation of PI3K, AKT, and ROS
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Integrins are a family of conserved cell surface receptors found throughout the animal kingdom. They comprise 24 dimers in mammals, and regulate a number of processes including cell survival, differentiation, and migration. These complex cellular responses involve processes such as cell attachment, spreading, and various signaling pathways, which in turn depend on the composition of the extracellular environment, on its mechanical properties, and involved integrin types. This thesis focuses on identifying molecules that signal downstream of integrins and how integrin-induced signals may differ dependent on the type of mechanical stimulus that is given.

In Paper I, we show that cell spreading and the activation of AKT is regulated by the catalytic PI3K isoform p110α. An intact β1 integrin cytoplasmic tail and actin polymerization was needed for spreading, whereas the presence of FAK or SRC, or the interaction between p110α and RAS was dispensable.

Paper II reports that the RICTOR-mTOR complex (TORC2) acts as the kinase downstream of β1 integrins in order to phosphorylate AKT on Ser473, which was functionally linked to cell survival. β1 integrins activated both AKT1 and AKT2, but seemed to prefer AKT2. The investigation of several receptor types with regard to their requirement of TORC2, PAK, and ILK for AKT Ser473 phosphorylation revealed that different kinds of receptors engage specific enzyme combinations depending on cell type and context.

In the third paper, we demonstrate that adhesion- and mechanical stretch-induced integrin signaling lead to divergent protein phosphorylation patterns, and that most signals from cell adhesion were not dependent on intracellular contractility. This indicates that integrin ligand binding and mechanical stretch induce signaling via distinct mechanisms. Reactive oxygen species (ROS) derived from different cellular sources modulated these responses. Stretching primarily induced phosphorylation of ERK1/2, and this signal was markedly increased by a derivative of the antioxidant ascorbate and extracellularly administered catalase. The robust AKT phosphorylation in response to adhesion was almost completely abolished with an inhibitor targeting mitochondrial ROS, whereas phosphorylation levels were only marginally affected in stretch assays. Similar results were obtained with siRNA knock-down of a critical subunit of ROS-producing NADPH oxidases.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. , 46 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 750
Keyword [en]
integrin, ROS, PI3K, AKT, mechanosignaling, actin polymerization, spreading
National Category
Cell and Molecular Biology
URN: urn:nbn:se:uu:diva-170267ISBN: 978-91-554-8301-2OAI: diva2:509315
Public defence
2012-04-27, C10:301, BMC, Husargatan 3, Uppsala, 10:00 (English)
Available from: 2012-04-04 Created: 2012-03-09 Last updated: 2012-04-19Bibliographically approved
List of papers
1. Receptor-Specific Mechanisms Regulate Phosphorylation of AKT at Ser473: Role of RICTOR in β1 Integrin-Mediated Cell Survival
Open this publication in new window or tab >>Receptor-Specific Mechanisms Regulate Phosphorylation of AKT at Ser473: Role of RICTOR in β1 Integrin-Mediated Cell Survival
2012 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 7, no 2, e32081- p.Article in journal (Refereed) Published
Abstract [en]

A tight control over AKT/PKB activation is essential for cells, and they realise this in part by regulating the phosphorylation of Ser473 in the "hydrophobic motif" of the AKT carboxy-terminal region. The RICTOR-mTOR complex (TORC2) is a major kinase for AKT Ser473 phosphorylation after stimulation by several growth factors, in a reaction proposed to require p21-activated kinase (PAK) as a scaffold. However, other kinases may catalyse this reaction in stimuli-specific manners. Here we characterised the requirement of RICTOR, ILK, and PAK for AKT Ser473 phosphorylation downstream of selected family members of integrins, G protein-coupled receptors, and tyrosine-kinase receptors and analysed the importance of this phosphorylation site for adhesion-mediated survival. siRNA-mediated knockdown in HeLa and MCF7 cells showed that RICTOR-mTOR was required for phosphorylation of AKT Ser473, and for efficient phosphorylation of the downstream AKT targets FOXO1 Thr24 and BAD Ser136, in response to β1 integrin-stimulation. ILK and PAK1/2 were dispensable for these reactions. RICTOR knockdown increased the number of apoptotic MCF7 cells on β1 integrin ligands up to 2-fold after 24 h in serum-free conditions. β1 integrin-stimulation induced phosphorylation of both AKT1 and AKT2 but markedly preferred AKT2. RICTOR-mTOR was required also for LPA-induced AKT Ser473 phosphorylation in MCF7 cells, but, interestingly, not in HeLa cells. PAK was needed for the AKT Ser473 phosphorylation in response to LPA and PDGF, but not to EGF. These results demonstrate that different receptors utilise different enzyme complexes to phosphorylate AKT at Ser473, and that AKT Ser473 phosphorylation significantly contributes to β1 integrin-mediated anchorage-dependent survival of cells.

National Category
Basic Medicine
urn:nbn:se:uu:diva-170264 (URN)10.1371/journal.pone.0032081 (DOI)000302875500073 ()22384145 (PubMedID)
Available from: 2012-03-09 Created: 2012-03-09 Last updated: 2013-03-22Bibliographically approved
2. PI3-kinase p110 alpha mediates beta 1 integrin-induced Akt activation and membrane protrusion during cell attachment and initial spreading
Open this publication in new window or tab >>PI3-kinase p110 alpha mediates beta 1 integrin-induced Akt activation and membrane protrusion during cell attachment and initial spreading
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2010 (English)In: Cellular Signalling, ISSN 0898-6568, E-ISSN 1873-3913, Vol. 22, no 12, 1838-1848 p.Article in journal (Refereed) Published
Abstract [en]

Integrin-mediated cell adhesion activates several signaling effectors, including phosphatidylinositol 3-kinase (PI3K), a central mediator of cell motility and survival. To elucidate the molecular mechanisms of this important pathway the specific members of the PI3K family activated by different integrins have to be identified. Here, we studied the role of PI3K catalytic isoforms in beta 1 integrin-induced lamellipodium protrusion and activation of Akt in fibroblasts. Real-time total internal reflection fluorescence imaging of the membrane substrate interface demonstrated that beta 1 integrin-mediated attachment induced rapid membrane spreading reaching essentially maximal contact area within 5-10 min. This process required actin polymerization and involved activation of PI3K. Isoform-selective pharmacological inhibition identified p110 alpha as the PI3K catalytic isoform mediating both beta 1 integrin-induced cell spreading and Akt phosphorylation. A K756L mutation in the membrane-proximal part of the beta 1 integrin subunit, known to cause impaired Akt phosphorylation after integrin stimulation, induced slower cell spreading. The initial beta 1 integrin-regulated cell spreading as well as Akt phosphorylation were sensitive to the tyrosine kinase inhibitor PP2, but were not dependent on Src family kinases, FAK or EGF/PDGF receptor transactivation. Notably, cells expressing a Ras binding-deficient p110 alpha mutant were severely defective in integrin-induced Akt phosphorylation, but exhibited identical membrane spreading kinetics as wild-type p110 alpha cells. We conclude that p110 alpha mediates beta 1 integrin-regulated activation of Akt and actin polymerization important for survival and lamellipodia dynamics. This could contribute to the tumorigenic properties of cells expressing constitutively active p110 alpha.

beta 1 integrin, PI3K, p110 alpha, Cell spreading, Akt
National Category
Medical and Health Sciences
urn:nbn:se:uu:diva-134169 (URN)10.1016/j.cellsig.2010.07.011 (DOI)000282857200006 ()

Correction in: Cellular signaling, vol. 24, issue 4, p: 971-972, DOI: 10.1016/j.cellsig.2011.12.010

Available from: 2010-11-22 Created: 2010-11-22 Last updated: 2012-04-19Bibliographically approved
3. The role of mechanical force and ROS in integrin-dependent signals
Open this publication in new window or tab >>The role of mechanical force and ROS in integrin-dependent signals
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 5, e64897Article in journal (Refereed) Published
Abstract [en]

Cells are exposed to several types of integrin stimuli, which generate responses generally referred to as "integrin signals", but the specific responses to different integrin stimuli are poorly defined. In this study, signals induced byintegrin ligation during cell attachment, mechanical force from intracellular contraction, or cell stretching by external force were compared. The elevated phosphorylation levels of several proteins during the early phase of cell attachment and spreading of fibroblast cell lines were not affected by inhibition ofROCK and myosin II activity, i.e. the reactions occurred independently ofintracellular contractile force acting on the adhesion sites. The contraction-independent phosphorylation sites included ERK1/2 T202/Y204, AKT S473, p130CAS Y410, and cofilin S3. In contrast to cell attachment, cyclic stretching ofthe adherent cells induced a robust phosphorylation only of ERK1/2 and thephosphorylation levels of the other investigated proteins were not or only moderately affected by stretching. No major differences between signaling via alpha 5 beta 1 or alpha v beta 3 integrins were detected. The importance ofmitochondrial ROS for the integrin-induced signaling pathways was investigated using rotenone, a specific inhibitor of complex I in the respiratory chain. While rotenone only moderately reduced ATP levels and hardly affected the signalsinduced by cyclic cell stretching, it abolished the activation of AKT and reduced theactin polymerization rate in response to attachment in both cell lines. In contrast, scavenging of extracellular ROS with catalase or the vitamin C analog Asc-2P did not significantly influence the attachment-derived signaling, but caused a selective and pronounced enhancement of ERK1/2 phosphorylation in response to stretching. In conclusion, the results showed that "integrin signals" are composedof separate sets of reactions triggered by different types of integrin stimulation. Mitochondrial ROS and extracellular ROS had specific and distinct effects on theintegrin signals induced by cell attachment and mechanical stretching.

integrin, ROS, mechanosignaling
National Category
Cell and Molecular Biology
Research subject
Biology with specialization in Molecular Cell Biology
urn:nbn:se:uu:diva-170266 (URN)10.1371/journal.pone.0064897 (DOI)000321394700069 ()23738008 (PubMedID)
Swedish Cancer SocietySwedish Research Council
Available from: 2012-03-12 Created: 2012-03-09 Last updated: 2016-08-09Bibliographically approved

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