Abstract
BackgroundPhosducin-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 expression from mammalian cell expression systems.Methodology/Principal FindingsAs 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 α and β tubulin subunits, microtubule disassembly and cell death. In contrast, β-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.ConclusionsOur results support the hypothesis that PhLP3 is important for the maintenance of β-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.
Highlights
The phosducin-like family of proteins were first identified, through phosducin itself, as proteins proposed to sequester the b and c subunit dimer of G protein (Gbc) thereby inhibiting its interaction with the a subunit (Ga) and regulating signalling involving trimeric G-protein coupled receptors in multicellular organisms [1]
From studies to date it appears that phosducin-like protein 1 (PhLP1)-3 may all be important as co-chaperones during chaperonin containing TCP1 (CCT)-assisted protein folding whilst only Pdc and PhLP1 have a role in G protein signalling, phosducin itself being a relatively recent evolutionary product that has lost interaction with CCT
Authentic human Phosducin-like protein 3 (PhLP3) is expressed in the adherent CHOK1 cell line
Summary
The phosducin-like family of proteins were first identified, through phosducin itself, as proteins proposed to sequester the b and c subunit dimer of G protein (Gbc) thereby inhibiting its interaction with the a subunit (Ga) and regulating signalling involving trimeric G-protein coupled receptors in multicellular organisms [1]. Stirling and co-workers [4] suggested that PhLP3 ( referred to as APACD or TXNDC9 in mammals, see [4] and Table 1) may be involved during the early stages of actin and btubulin folding (independent of the prefoldin complex). These in vitro studies demonstrated that PhLP3 had a negative effect on. 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
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