Multiple protein kinases determine the phosphorylated state of the small heat shock protein, HSP27, in SH-SY5Y neuroblastoma cells

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.

► Muscarinic receptors are coupled to phosphorylation of HSP27 at Ser-78 and Ser-82 in SH-SY5Y cells.
► Phorbol ester-stimulated HSP27 phosphorylation at Ser-82 occurs through the PKC/PKD pathway.
► The activities of Akt and p38 MAPK are inversely related to HSP27 phosphorylation.
► Lamellipodial formation in SH-SY5Y cells does not involve phospho-(Ser-82)-HSP27.