title: Modulation of phosducin-like protein 3 (PhLP3) levels promotes cytoskeletal remodelling in a MAPK and RhoA-dependent manner.
abstract: BACKGROUND
  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 expression from mammalian cell expression systems.
  
  
  RESULTS
  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 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.
  
  
  CONCLUSIONS
  Our 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.
authors.affiliation: Centre for Molecular Processing and School of Biosciences, University of Kent, Canterbury, Kent, United Kingdom.
authors.forename: Nandini V L
authors.initials: NV
authors.lastname: Hayes
authors.forename: Lyne
authors.initials: L
authors.lastname: Jossé
authors.forename: C Mark
authors.initials: CM
authors.lastname: Smales
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Carden
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002448:Cell Adhesion; D002460:Cell Line; D049109:Cell Proliferation; D048430:Cell Shape; D006224:Cricetinae; D003412:Cricetulus; D048749:Cytokinesis; D003599:Cytoskeleton / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation; D020868:Gene Silencing; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008938:Mitosis; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000378:metabolism; D022502:Stress Fibers / Q000378:metabolism; D009626:Terminology as Topic; D014404:Tubulin / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D018076:DNA, Complementary; D009419:Nerve Tissue Proteins; C498322:PDCL3 protein, human; D034741:RNA, Small Interfering; D014404:Tubulin; D020928:Mitogen-Activated Protein Kinases; D020742:rhoA GTP-Binding Protein