Dataset Information

Modulation of phosducin-like protein 3 (PhLP3) levels promotes cytoskeletal remodelling in a MAPK and RhoA-dependent manner.

ABSTRACT: Phosducin-like protein 3 (PhLP3) forms a ternary complex with the ATP-dependent molecular chaperone CCT and its folding client tubulin. In vitro studies suggest PhLP3 plays an inhibitory role in ?-tubulin folding while conversely in vivo genetic studies suggest PhLP3 is required for the correct folding of ?-tubulin. We have a particular interest in the cytoskeleton, its chaperones and their role in determining cellular phenotypes associated with high level recombinant protein expression from mammalian cell expression systems.As studies into PhLP3 function have been largely carried out in non mammalian systems, we examined the effect of human PhLP3 over-expression and siRNA silencing using a single murine siRNA on both tubulin and actin systems in mammalian Chinese hamster ovary (CHO) cell lines. We show that over-expression of PhLP3 promotes an imbalance of ? and ? tubulin subunits, microtubule disassembly and cell death. In contrast, ?-actin levels are not obviously perturbed. On-the-other-hand, RNA silencing of PhLP3 increases RhoA-dependent actin filament formation and focal adhesion formation and promotes a dramatic elongated fibroblast-like change in morphology. This was accompanied by an increase in phosphorylated MAPK which has been associated with promoting focal adhesion assembly and maturation. Transient overexpression of PhLP3 in knockdown experiments rescues cells from the morphological change observed during PhLP3 silencing but mitosis is perturbed, probably reflecting a tipping back of the balance of PhLP3 levels towards the overexpression state.Our results support the hypothesis that PhLP3 is important for the maintenance of ?-tubulin levels in mammalian cells but also that its modulation can promote actin-based cytoskeletal remodelling by a mechanism linked with MAPK phosphorylation and RhoA-dependent changes. PhLP3 levels in mammalian cells are thus finely poised and represents a novel target for engineering industrially relevant cell lines to evolve lines more suited to suspension or adherent cell growth.

SUBMITTER: Hayes NV

PROVIDER: S-EPMC3235111 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Modulation of phosducin-like protein 3 (PhLP3) levels promotes cytoskeletal remodelling in a MAPK and RhoA-dependent manner.

Hayes Nandini V L NV Jossé Lyne L Smales C Mark CM Carden Martin J MJ

PloS one 20111209 12

<h4>Background</h4>Phosducin-like protein 3 (PhLP3) forms a ternary complex with the ATP-dependent molecular chaperone CCT and its folding client tubulin. In vitro studies suggest PhLP3 plays an inhibitory role in β-tubulin folding while conversely in vivo genetic studies suggest PhLP3 is required for the correct folding of β-tubulin. We have a particular interest in the cytoskeleton, its chaperones and their role in determining cellular phenotypes associated with high level recombinant protein ...[more]

PMID: 22174782

Similar Datasets

Project description:Phosducin (Phd) and Phd-like proteins (PhLPs) selectively bind guanine nucleotide protein (G protein) betagamma subunits (Gbetagamma), while Phd-like orphan proteins (PhLOPs) lack the major functional domain for the binding of Gbetagamma. A retina- and pineal gland-specific transcription factor, cone-rod homeobox (CRX), was identified by a yeast two-hybrid screen using PhLOP1 as the bait. Direct protein-protein interactions between Phd or PhLOP1 and CRX were demonstrated using a beta-galactosidase quantitative assay in the yeast two-hybrid system and were confirmed by an in vitro binding assay and a glutathione S-transferase (GST) pull-down assay. To determine if the interaction with Phd or PhLOP1 affected CRX transactivation, a 120-bp interphotoreceptor retinoid binding protein (IRBP) promoter-luciferase reporter construct containing a CRX consensus element (GATTAA) was cotransfected into either COS-7 or retinoblastoma Weri-Rb-1 cells with expression constructs for CRX and either Phd or PhLOP1. Phd and PhLOP1 inhibited the transcriptional activation activity of CRX by 50% during transient cotransfection in COS-7 cells and by 70% in Weri-Rb-1 cells and COS-7 cells stably transfected with CRX. Phd inhibited CRX transactivation in a dose-dependent manner. Whereas Phd is a cytoplasmic phosphoprotein, coexpression of Phd with CRX results in Phd being localized both in the cytoplasm and nucleus. By contrast, PhLOP1 is found in the nucleus even without CRX coexpression. To address the physiological relevance of these potential protein interacting partners, we identified immunoreactive proteins for Phd and CRX in retinal cytosolic and nuclear fractions. Immunohistochemical analysis of bovine retinas reveals colocalization of Phd isoforms with CRX predominantly in the inner segment of cone cells, with additional costaining in the outer nuclear layer and the synaptic region. Our findings demonstrate that both Phd and PhLOP1 interact directly with CRX and that each diminishes the transactivation activity of CRX on the IRBP promoter. A domain that interacts with CRX is found in the carboxyl terminus of the Phd isoforms. Phd antibody-immunoreactive peptides are seen in light-adapted mouse retinal cytosolic and nuclear extracts. Neither Phd nor PhLOP1 affected CRX binding to its consensus DNA element in electrophoretic mobility shift assays. A model that illustrates separate functional roles for interactions between Phd and either SUG1 or CRX is proposed. The model suggests further a mechanism by which Phd isoforms could inhibit CRX transcriptional activation.

Dataset Information

Modulation of phosducin-like protein 3 (PhLP3) levels promotes cytoskeletal remodelling in a MAPK and RhoA-dependent manner.

Publications

Modulation of phosducin-like protein 3 (PhLP3) levels promotes cytoskeletal remodelling in a MAPK and RhoA-dependent manner.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets