Figure 3
Activation of the p38 MAPK and PKA pathways can provoke phosphorylation of HSP27. (A) HEK293 cells were transfected with an expression vector for Flag-tagged HSP27 and 24 h after transfection left untreated (-) or exposed to forskolin (FSK; 10 μM for 30 min) or sodium arsenite (SA; 250 μM for 30 min). The phosphorylation levels of phosphoSer-78 HSP27 were monitored. Equal loading was verified by examining the total levels of HSP27 and actin in the samples. To ensure that the stimuli were active, phosphorylation of the PKA substrate CREB at Ser-133 and of the p38MAPK was tested. The double bands observed in the blots with (phospho)HSP27 antibodies represent Flag-tagged HSP27 (upper band) and endogenous HSP27 (lower band). (B) HEK293 cells transfected with the Flag-HSP27 expression plasmid and cells were either left untreated, exposed for 30 min with the PKA inhibitor H89 (10 μM) or the p38MAPK inhibitor SB203580 (10 μM) before forskolin or arsenite was added, or were treated with forskolin or arsenite as described in (A). Lane 1: untreated Flag-Hsp27 transfected HEK293 cells; lane 2: stimulated with forskolin; lane 3: stimulated with sodium arsenite; lane 4: exposed to H89; lane 5: pretreated with H89 and then stimulated with forskolin; lane 6: pretreated with H89 and then stimulated with arsenite; lane 7: exposed to SB203580; lane 8: pretreated with SB203580 and then stimulated with forskolin; lane 9: pretreated with SB203590 and then stimulated with arsenite. PhosphoSer78 HSP27 and phosphoSer82 HSP27 were examined. Membranes were stripped and the lysates were assayed for phosphorylation of CREB and p38MAPK. Lower panel shows the expression levels of endogenous and Flag-tagged HSP27. (C) Cells were cotransfected with expression vectors for Flag-tagged HSP27 and the catalytic subunit of PKA (Cα) or p38MAPK and a constitutive active mutant of its upstream activator MKK6 (MKK). PhosphoSer78 HSP27 protein levels were examined by western blotting. M is the protein molecular mass (in kD) marker. RDU (relative densitometry units) indicates the increase in HSP27 phosphorylation and was calculated as follows. The densitometry values obtained for the signals of phosphorylated HSP27 were determined and corrected for the values obtained for actin (in A) or for total HSP27 (in B). This ratio obtained for untreated cells was arbitrarily set as 1.0 and the ratios obtained for stimulated cells were related to this. Values were calculated separately for flag-tagged HSP27 and endogenous HSP27.