Receptor-Specific Mechanisms Regulate Phosphorylation of AKT at Ser473: Role of RICTOR in β1 Integrin-Mediated Cell Survival

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

Integrin Signaling in Cell Adhesion and Mechanotransduction : Regulation of PI3K, AKT, and ROS

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

Integrin Signaling in Cell Adhesion and Mechanotransduction : Regulation of PI3K, AKT, and ROS

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

Integrin Signaling in Cell Adhesion and Mechanotransduction : Regulation of PI3K, AKT, and ROS

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

Common and Diverging Integrin Signals Downstreamof Adhesion and Mechanical Stimuli and TheirInterplay with Reactive Oxygen Species

The integrin family of adhesion receptors regulates basic functions of cells, and the signalsthey induce are altered in tumor cells. In this review we discuss how different integrindependentsignals are generated during cell adhesion and by physical forces acting oncells.We also describe how reactive oxygen species are integral parts of integrin signalingand highlight a few important questions in the field. Answers to those may improve ourunderstanding of integrins and their role in the development of cancer

Evaluation of cellular stress responses in magnetomotive ultrasound

Early and accurate diagnoses are important for successful cancer treatment. Lymph node involvement is often critical, and magnetomotive ultrasound (MMUS) has been proposed for its detection and characterization. MMUS relies on a magnetic contrast agent, for example, iron oxide nanoparticles, delivered to the tissue of interest, magnetically set in motion and detected using ultrasound. The magneto-mechanical interaction has not previously been evaluated on a cellular level. Here we demonstrate uptake and dose-dependent retention of magnetic nanoparticles in two human adenocarcinoma cell lines, with <10% cytotoxicity which did not increase following magnetic excitation. Further, the oxidative stress levels were not affected by magnetic particles or force. Thus, we found no evidence of adverse effects from the magneto-mechanical interactions under these conditions

GARD : Genomic allergen rapid detection

Genomic allergen rapid detection (GARD) is an in vitro assay for the prediction of skin sensitizing capacity of chemicals. The GARD assay monitors changes in the expression of 196 genes, termed the GARD prediction signature. In short, MUTZ-3 cells, representing a well-characterized in vitro model of human dendritic cells (DCs), are exposed to the test substances for 24 h. After RNA extraction and analysis of expression levels, compounds are predicted as either sensitizers or non-sensitizers by a support vector machine model. Due to high information content, the test can deliver novel insights into pathway utilization and potency. Based on blinded test evaluations, GARD has been shown to have an accuracy of 89%

Effects of RICTOR, ILK or PAK knockdown on LPA-induced AKT Ser473 phosphorylation in HeLa and MCF7 cells.

<p>(A) HeLa cells were transfected with RICTOR-directed siRNA or non-target siRNA and then stimulated with LPA (10 µM) for 20 minutes. Cell lysates were subjected to SDS-PAGE (4–15% gradient gel) and LPA-induced AKT Ser473 phosphorylation was determined by western blotting. A representative western blot is shown and the graph below provides quantification of AKT pSer473 (mean ± s.e.m.; <i>n</i> = 3). (B) HeLa cells transfected with ILK-directed siRNA and stimulated by LPA were analysed as described above. The graph below shows quantification of AKT pSer473 (mean ± s.e.m.; <i>n</i> = 2). (C) MCF7 cells transfected as indicated were stimulated with LPA (5 µM) for 5 min and analysed as described above. A representative western blot is presented and the graph below shows quantified levels of AKT pSer473 (mean ± s.e.m.; <i>n</i> = 3, * represents <i>p</i><0.005). (D) MCF7 cells were transfected simultaneously with PAK1 and PAK2 siRNA or with non-target siRNA and then stimulated with LPA (5 µM) for 5 min. A representative western blot is shown. The graph below is a quantification of AKT pSer473 levels (mean ± s.e.m.; <i>n</i> = 3, ** represents <i>p</i><0.05). (E) HeLa cells were treated with increasing concentrations of the PAK inhibitor IPA3 or with DMSO and then stimulated with LPA (10 µM) for 20 min. The graph below is the quantification of AKT pSer473 levels after LPA-stimulation in cells treated with IPA3 (30 µM) normalised to the pSer473 level of this protein in LPA-stimulated cells without the inhibitor (mean ± s.e.m.; <i>n</i> = 3, * represents <i>p</i><0.005).</p

RICTOR knockdown reduces β1 integrin-induced AKT Ser473 phosphorylation but ILK or PAK knockdown has no effect.

<p>(A) HeLa cells were transfected with siRNA directed against RICTOR and then allowed to adhere to plates coated with invasin (β1 integrin-ligand) or Pluronic (non-adhesive control) for 60 min. Cell lysates were subjected to SDS-PAGE (4–15% gradient gel) followed by western blotting using the different antibodies as indicated. A representative western blot is shown and quantifications of AKT pSer473, FOXO1 pThr24, and BAD pSer136 on Pluronic and invasin are given below (mean ± s.e.m.; <i>n</i> = 3; * represents <i>p</i><0.005 and ** represents <i>p</i><0.05). (B) MCF7 cells were transfected and treated in the same manner as in (A). (C) HeLa cells transfected with ILK-directed siRNA or non-target siRNA were allowed to adhere to plates coated with Pluronic, collagen type I or invasin. Cells were lysed and analysed as explained above. A representative western blot is shown and the graph to the right shows quantification of AKT pSer473 levels on Pluronic and invasin (mean ± s.e.m.; <i>n</i> = 3). (D) HeLa cells were transfected simultaneously with PAK1- and PAK2-directed siRNAs or non-target siRNA. Adhesion assays were performed on plates coated with Pluronic, invasin or collagen type I. Cell lysates were subjected to SDS-PAGE (10% gel) followed by western blotting using the different antibodies as indicated. A representative western blot is shown and the graph below presents quantification of AKT pSer473 levels on Pluronic and invasin (mean ± s.e.m.; <i>n</i> = 3). (E) Adhesion assay with MCF7 cells transfected with PAK1- and PAK2-directed siRNAs or non-target siRNA, performed as described in (A).</p