Isoproterenol- and forskolin-induced phosphoprotein signaling in the presence of the PKA inhibitor H89.

<p><b>A</b>, MEFs were pretreated with H89 (10 μM) for 1 hour and then were stimulated with isoproterenol (Iso) (10 μM) for various times (0, 5, 10, 20, 30, or 60 minutes). The heat map shows phosphorylation (yellow) and dephosphorylation (blue) events after isoproterenol stimulation. The color scale shows fold change as compared with unstimulated MEFs. Data are means of three independent stimulation experiments. * denotes pathways that are significantly altered by pretreatment with H89 as assessed using the SAM algorithm. A two-class unpaired time course analysis was performed comparing signed-areas under the curve for each pathway. <b>B</b>, MEFs were pretreated with H89 as in (A) but were stimulated with forskolin (50 μM). Data are means of three independent stimulation experiments. * denotes pathways that are significantly altered by pretreatment with H89 as assessed using the SAM algorithm. <b>C</b>, The phosphorylation kinetics of ERK1/2(T202/Y204), PP1α(T320), GSK3β(S9) in response to isoproterenol (10 μM) stimulation in the presence (dashed line) or absence (solid line) of H89. The phosphorylation of ERK1/2(T202/Y204) is blocked by H89, dephosphorylation of PP1α(T320) is unchanged by H89, and dephosphorylation of GSK3β(S9) is enhanced by H89. Graphs show means ± SEM for three independent experiments. Values in the graph represent fold change as compared with unstimulated MEFs. D, Rapid phosphorylation kinetics of GSK3β(S9) by isoproterenol (10 μM) in the presence (dashed line) or absence (solid line) of H89. The graphs show means ± SEM of three independent experiments. * indicates a significant difference (P<0.05) from baseline phosphorylation level as determined using a Student’s T-Test.