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Receptor-Specific Mechanisms Regulate Phosphorylation of AKT at Ser473: Role of RICTOR in β1 Integrin-Mediated Cell Survival
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2012 (English)In: PLoS ONE, ISSN 1932-6203, E-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.

Place, publisher, year, edition, pages
2012. Vol. 7, no 2, e32081- p.
National Category
Basic Medicine
Identifiers
URN: urn:nbn:se:uu:diva-170264DOI: 10.1371/journal.pone.0032081ISI: 000302875500073PubMedID: 22384145OAI: oai:DiVA.org:uu-170264DiVA: diva2:508814
Available from: 2012-03-09 Created: 2012-03-09 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Integrin Signaling in Cell Adhesion and Mechanotransduction: Regulation of PI3K, AKT, and ROS
Open this publication in new window or tab >>Integrin Signaling in Cell Adhesion and Mechanotransduction: Regulation of PI3K, AKT, and ROS
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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 750
Keyword
integrin, ROS, PI3K, AKT, mechanosignaling, actin polymerization, spreading
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-170267 (URN)978-91-554-8301-2 (ISBN)
Public defence
2012-04-27, C10:301, BMC, Husargatan 3, Uppsala, 10:00 (English)
Opponent
Supervisors
Available from: 2012-04-04 Created: 2012-03-09 Last updated: 2012-04-19Bibliographically approved
2. Adhesion Dependent Signals: Cell Survival, Receptor Crosstalk and Mechanostimulation
Open this publication in new window or tab >>Adhesion Dependent Signals: Cell Survival, Receptor Crosstalk and Mechanostimulation
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The integrin family of cell surface receptors is evolutionary conserved and found in all multicellular animals. In humans 8-alpha and 18-beta integrins are non-covalently associated into 24 dimers. Integrins mediate cell-extracellular matrix and cell-cell interactions and participate in cell signalling. This ideally places integrins to regulate vital processes such as cell adhesion, migration, differentiation and cytoskeleton dynamics. Integrins also play a fundamental role in regulating cell survival and anoikis. In this thesis molecular mechanisms employed by integrins to induce signal transduction, independently or through crosstalk with other receptors, were characterised.

Rictor-mTOR (mTORC2) was required for Akt Ser473 phosphorylation in response to β1 integrin-mediated adhesion as well as EGF-, PDGF- or LPA-stimulation of MCF7 cells. ILK and PAK were dispensable for Akt Ser473 phosphorylation upon β1 integrin-engagement or EGF-stimulation. PAK was needed when this phosphorylation was induced by PDGF or LPA. β1 integrin-promoted cell survival during serum starvation conditions was mTORC2 dependent, indicating the importance of Akt Ser473 phosphorylation.

mTORC2 was also required for Akt Ser473 phosphorylation induced upon heparanase treatment of cells. Heparanase preferred PI3K catalytic subunit p110α for the upstream lipid phosphorylation required for Akt activation. Interaction between this subunit and Ras was needed for optimal Akt phosphorylation upon heparanase exposure. Cell adhesion strongly promoted heparanase signalling, which was more efficient in β1 integrin-expressing fibroblasts compared to cells lacking this subunit. The cooperative signalling between integrins and heparanase involved FAK and PYK2 since simultaneous silencing of these kinases suppressed heparanase-triggered Akt activation. Furthermore, the resistance of cells to apoptosis induced by H2O2 or serum starvation was promoted by heparanase. 

Integrin stimulation by adhesion or cyclic stretching showed divergent downstream signalling responses. Cell attachment on integrin-specific ligands lead to robust phosphorylation of several intracellular integrin-effectors, e.g. p130CAS, FAK, Akt and ERK 1/2. However, mechanical cell stretching only triggered prominent phosphorylation of ERK 1/2. Signalling induced at early stages of integrin-mediated cell adhesion occurred independently of intracellular contraction. Reactive oxygen species (ROS) generated during adhesion and cell stretching influenced integrin signalling. Inhibition of mitochondrial ROS production blocked adhesion-induced Akt phosphorylation. In contrast, stretch-induced ERK 1/2 phosphorylation was elevated when extracellular ROS was scavenged. These results indicate that the two types of integrin stimuli generate signals by different mechanisms.   

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 48 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 868
Keyword
Integrins, Signal transduction, Protein kinase B, Akt, PI3K, Heparanase, ROS, Mechanosignalling
National Category
Cell and Molecular Biology
Research subject
Molecular Cellbiology; Biology with specialization in Molecular Cell Biology; Cell Research
Identifiers
urn:nbn:se:uu:diva-195712 (URN)978-91-554-8604-4 (ISBN)
Public defence
2013-04-12, BMC C4:305, Institutionen för medicinsk biokemi och mikrobiolog, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2013-03-20 Created: 2013-02-26 Last updated: 2014-01-27Bibliographically approved

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