title: Multiple protein kinases determine the phosphorylated state of the small heat shock protein, HSP27, in SH-SY5Y neuroblastoma cells.
abstract: In SH-SY5Y human neuroblastoma cells, the cholinergic agonist, carbachol, stimulates phosphorylation of the small heat shock protein 27 (HSP27). Carbachol increases phosphorylation of both Ser-82 and Ser-78 while the phorbol ester, phorbol-12, 13-dibutyrate (PDB) affects only Ser-82. Muscarinic receptor activation by carbachol was confirmed by sensitivity of Ser-82 phosphorylation to hyoscyamine with no effect of nicotine or bradykinin. This response to carbachol is partially reduced by inhibition of protein kinase C (PKC) with GF 109203X and p38 mitogen-activated protein kinase (MAPK) with SB 203580. In contrast, phosphorylation produced by PDB is completely reversed by GF 109203X or CID 755673, an inhibitor of PKD. Inhibition of phosphatidylinositol 3-kinase or Akt with LY 294002 or Akti-1/2 stimulates HSP27 phosphorylation while rapamycin, which inhibits mTORC1, does not. The stimulatory effect of Akti-1/2 is reversed by SB 203580 and correlates with increased p38 MAPK phosphorylation. SH-SY5Y cells differentiated with a low concentration of PDB and basic fibroblast growth factor to a more neuronal phenotype retain carbachol-, PDB- and Akti-1/2-responsive HSP27 phosphorylation. Immunofluorescence microscopy confirms increased HSP27 phosphorylation in response to carbachol or PDB. At cell margins, PDB causes f-actin to reorganize forming lamellipodial structures from which phospho-HSP27 is segregated. The resultant phenotypic change in cell morphology is dependent upon PKC, but not PKD, activity. The major conclusion from this study is that the phosphorylated state of HSP27 in SH-SY5Y cells results from integrated signaling involving PKC, p38 MAPK and Akt.
authors.affiliation: Department of Pharmacology, College of Pharmacy, 3000 Arlington Avenue, The University of Toledo, Toledo, OH 43614, USA. ldokas@umich.edu
authors.forename: Linda A
authors.initials: LA
authors.lastname: Dokas
authors.forename: Amy M
authors.initials: AM
authors.lastname: Malone
authors.forename: Frederick E
authors.initials: FE
authors.lastname: Williams
authors.forename: Surya M
authors.initials: SM
authors.lastname: Nauli
authors.forename: William S
authors.initials: WS
authors.lastname: Messer
mesh_terms: D045744:Cell Line, Tumor; D055551:HSP27 Heat-Shock Proteins / Q000378:metabolism*; D006360:Heat-Shock Proteins; D006801:Humans; D018832:Molecular Chaperones; D009447:Neuroblastoma / Q000201:enzymology*; D010766:Phosphorylation / Q000502:physiology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000378:metabolism / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D055551:HSP27 Heat-Shock Proteins; C057725:HSPB1 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D047428:Protein Kinase Inhibitors; D011976:Receptors, Muscarinic; D011494:Protein Kinases; D011493:Protein Kinase C; D048051:p38 Mitogen-Activated Protein Kinases