Dataset Information

PI3K/Akt signalling-mediated protein surface expression sensed by 14-3-3 interacting motif.

ABSTRACT: The regulation of protein expression on the cell surface membrane is an important component of the cellular response to extracellular signalling. The translation of extracellular signalling into specific protein localization often involves the post-translational modification of cargo proteins. Using a genetic screen of random peptides, we have previously identified a group of C-terminal sequences, represented by RGRSWTY-COOH (termed'SWTY'), which are capable of overriding an endoplasmic reticulum localization signal and directing membrane proteins to the cell surface via specific binding to 14-3-3 proteins. The identity of the kinase signalling pathways that drive phosphorylation and 14-3-3 binding of the SWTY sequence is not known. In this study, we report that the activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway by the over-expression of active kinases, stimulation with fetal bovine serum or growth factors can: (a) phosphorylate the SWTY sequence; (b) recruit 14-3-3 proteins to SWTY; and (c) promote surface expression of the chimeric potassium channel fused with the SWTY sequence. The expression of the dominant negative Akt inhibited the enhancement of surface expression by fetal bovine serum. In addition, the activation of PI3K significantly enhanced the 14-3-3 association and cell surface expression of GPR15, a G protein-coupled receptor which carries an endogenous SWTY-like, C-terminal, 14-3-3 binding sequence and is known to serve as a HIV co-receptor. Given the wealth and specificity of both kinase activity and 14-3-3 binding sequences, our results suggest that the C-terminal SWTYlike motif may serve as a sensor that can selectively induce the cell surface expression of membrane proteins in response to different extracellular signals.

SUBMITTER: Chung JJ

PROVIDER: S-EPMC4301307 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

PI3K/Akt signalling-mediated protein surface expression sensed by 14-3-3 interacting motif.

Chung Jean-Ju JJ Okamoto Yukari Y Coblitz Brian B Li Min M Qiu Yun Y Shikano Sojin S

The FEBS journal 20090819 19

The regulation of protein expression on the cell surface membrane is an important component of the cellular response to extracellular signalling. The translation of extracellular signalling into specific protein localization often involves the post-translational modification of cargo proteins. Using a genetic screen of random peptides, we have previously identified a group of C-terminal sequences, represented by RGRSWTY-COOH (termed'SWTY'), which are capable of overriding an endoplasmic reticulu ...[more]

PMID: 19691494

Similar Datasets

Project description:Background and aimsActivation of the cAMP/Epac signalling stabilises endothelial barrier function. Moreover, its activation is accompanied by an activation of PI3K/Akt and MEK/ERK signalling in diverse cell types but their impact on endothelial barrier function is largely unknown. Here the role of PI3K/Akt and MEK/ERK signalling in cAMP/Epac-mediated endothelial barrier stabilisation was analysed.MethodsEndothelial barrier function was analysed in cultured human umbilical vein endothelial cells (HUVECs) by measuring flux of albumin. A modified cAMP analogue 8-pCPT-2'-O-Me-cAMP (Epac agonist) was used to specifically activate cAMP/Epac signalling.ResultsEpac agonist reduces the basal and attenuates thrombin-induced endothelial hyperpermeability accompanied by an activation of PI3K/Akt and MEK/ERK signalling. The qPCR data demonstrate HUVECs express PI3Kα, PI3Kβ, and PI3Kγ but not PI3Kδ isoforms. The western blot data demonstrate Epac agonist activates PI3Kα and PI3Kβ isoforms. Inhibition of MEK/ERK but not PI3K/Akt pathway potentiates the endothelial barrier protective effects of cAMP/Epac signalling. Inhibition of MEK/ERK signalling in the presence of Epac agonist induces a reorganisation of actin cytoskeleton to the cell periphery, enhanced VE-cadherin localisation at cell-cell junctions, and dephosphorylation of myosin light chains (MLC) but not inhibition of RhoA/Rock signalling. Moreover, Epac agonist promotes endothelial cell (EC) survival via reduction in activities of pro-apoptotic caspases in a PI3K/Akt and MEK/ERK signalling-dependent manner.ConclusionOur data demonstrate that the Epac agonist simultaneously activates diverse signalling pathways in ECs, which may have differential effects on endothelial barrier function. It activates PI3K/Akt and MEK/ERK signalling which mainly govern its pro-survival effects on ECs. Inhibition of MEK/ERK but not PI3K/Akt signalling enhances barrier stabilising and barrier protective effects of cAMP/Epac activation.Chemical compounds used in this study8-pCPT-2'-O-Me-cAMP (PubChem CID: 9913268); Akt inhibitor VIII (PubChem CID: 10196499); AS-252424 (PubChem CID: 11630874); IC-87114 (PubChem CID: 9908783); PD 98059 (PubChem CID: 4713); PIK-75 (PubChem CID: 10275789); TGX-221 (PubChem CID: 9907093); Thrombin (PubChem CID: 90470996); U0126 (PubChem CID: 3006531); Wortmannin (PubChem CID: 312145).

Project description:BackgroundGrowth factors, such as EGF, activate the PI3K/Akt/mTORC1 signalling pathway, which regulates a distinct program of protein synthesis leading to cell growth. This pathway relies on mTORC1 sensing sufficient levels of intracellular amino acids, such as leucine, which are required for mTORC1 activation. However, it is currently unknown whether there is a direct link between these external growth signals and intracellular amino acid levels. In primary prostate cancer cells, intracellular leucine levels are regulated by L-type amino acid transporter 3 (LAT3/SLC43A1), and we therefore investigated whether LAT3 is regulated by growth factor signalling.MethodsTo investigate how PI3K/Akt signalling regulates leucine transport, prostate cancer cells were treated with different PI3K/Akt inhibitors, or stable knock down of LAT3 by shRNA, followed by analysis of leucine uptake, western blotting, immunofluorescent staining and proximity ligation assay.ResultsInhibition of PI3K/Akt signalling significantly reduced leucine transport in LNCaP and PC-3 human prostate cancer cell lines, while growth factor addition significantly increased leucine uptake. These effects appeared to be mediated by LAT3 transport, as LAT3 knockdown blocked leucine uptake, and was not rescued by growth factor activation or further inhibited by signalling pathway inhibition. We further demonstrated that EGF significantly increased LAT3 protein levels when Akt was phosphorylated, and that Akt and LAT3 co-localised on the plasma membrane in EGF-activated LNCaP cells. These effects were likely due to stabilisation of LAT3 protein levels on the plasma membrane, with EGF treatment preventing ubiquitin-mediated LAT3 degradation.ConclusionGrowth factor-activated PI3K/Akt signalling pathway regulates leucine transport through LAT3 in prostate cancer cell lines. These data support a direct link between growth factor and amino acid uptake, providing a mechanism by which the cells rapidly coordinate amino acid uptake for cell growth.

Dataset Information

PI3K/Akt signalling-mediated protein surface expression sensed by 14-3-3 interacting motif.

Publications

PI3K/Akt signalling-mediated protein surface expression sensed by 14-3-3 interacting motif.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets