title: Phosphorylation status of heat shock protein 27 influences neurite growth in adult dorsal root ganglion sensory neurons in vitro.
abstract: The small heat shock protein Hsp27 influences neurite growth, potentially via phosphorylation-dependent interactions of Hsp27 with actin. To investigate the contribution of Hsp27 phosphorylation to neurite growth in adult DRG neurons, we employed hamster Hsp27 cDNA constructs (in pIRES-EGFP) with mutations in the phosphorylation sites, either mimicking constitutively phosphorylated Hsp27 (with substitution of serines 15 and/or 90 by glutamate) or preventing phosphorylation at the site (serines 15/90 replaced by alanine). Five mutant constructs were employed in this study in addition to wild-type hamster Hsp27; siRNA directed against the rat Hsp27 was used to depress endogenous Hsp27. Neurite growth was assessed in EGFP-expressing cells following immunocytochemistry and tracing of neurite growth. Hsp27 staining and phalloidin labelling were used to examine Hsp27 and actin colocalization in neurons and growth cones. Overall, our results demonstrate that the role that Hsp27 plays in neurite growth can be affected by phosphorylation, oligomerization, or a combination of both. Hsp27 constructs that are able to dimerize and/or form large oligomers [WT, Hsp27-AA, Hsp27-AE, Hsp27-Δ(5-23)] rescued siRNA-depressed neurite growth, whereas Hsp27 mutants that do not form dimers or oligomers (Hsp27-EE and Hsp27-EA) were unable to rescue the effect of the siRNA. The phalloidin labelling qualitatively showed a higher level of localization of actin with the Hsp27-AA compared with the other constructs. Although phosphorylation appears to be important in growth, the ability of Hsp27 to exist in both phospho- and nonphospho- states is likely key to its role in regulating cytoskeletal elements involved in neurite growth.
authors.affiliation: Division of BioMedical Sciences, Memorial University of Newfoundland, St. John's, Newfoundland, Canada.
authors.forename: Kristy L
authors.initials: KL
authors.lastname: Williams
authors.forename: Karen M
authors.initials: KM
authors.lastname: Mearow
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D006224:Cricetinae; D005727:Ganglia, Spinal / Q000378:metabolism*; D055551:HSP27 Heat-Shock Proteins / Q000378:metabolism*; D016501:Neurites / Q000378:metabolism*; D010766:Phosphorylation; D020671:Posterior Horn Cells / Q000378:metabolism*; D034741:RNA, Small Interfering; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection
chemical_list: D000199:Actins; D055551:HSP27 Heat-Shock Proteins; D034741:RNA, Small Interfering