Figure 5. Phosphorylation at Thr389 by PKA inhibits MAST3 activity.
(a) WT MAST3-HA and the unphosphorylable T389A-MAST3-HA or the phosphomimetic T389D-MAST3-HA were expressed in HEK293T cells and isolated by immunoprecipitation. Upper panel shows immunoblotting of WT and mutant MAST-HA proteins. T389A-MAST3 was pre-incubated for 20 min with PKA prior to the assay with ARPP-16. All MAST3 proteins were then incubated with ARPP-16 in the presence of ATP-γ-32P for different times. The proteins were resolved by SDS-PAGE and phosphorylation of Ser46 was measured by autoradiography. The results are expressed in arbitrary densitometric units (a.u.) as mean ± SE of three independent experiments. (b) ARPP-16-HA was expressed in HEK293T cells alone or with WT MAST3-HA and the phospho-mutants T389D orT389A-MAST3-HA. Cells were treated without or with 10 μM forskolin (FSK) for 30 min. Phosphorylation at Ser46 or Ser88 ARPP-16 were measured by immunoblotting with phospho-specific antibodies. Phospho-site signals were normalized for total ARPP-16-HA expression and then to MAST3-HA proteins, each measured by immunoblotting. (c) Graph of summary data shows quantification of phosphorylation on P-S46- and P-S88-ARPP-16 sites expressed in arbitrary densitometric units (a.u.) as mean ± SE of three independent experiments. A one-way ANOVA, multiple comparison test (post-hoc test Tukey) was used for data analysis. P-S46-ARPP-16: ****p<0.001 ARPP-16/MAST3 vs ARPP-16/MAST3/FSK; ****p<0.001, ARPP-16/MAST3 vs ARPP-16/MAST3-T389D; $$$$ p<0.001, ARPP-16/MAST3-T389A vs ARPP-16/MAST3-T389A/FSK. P-S88-ARPP-16: ####p<0.001, ARPP-16/MAST3 vs ARPP-16/MAST3/FSK, ####p<0.001, ARPP-16/MAST3 vs ARPP-16/MAST3-T389A/FSK. The effect of FSK on MAST3 activity was significantly greater than that of the effect of FSK on T389A-MAST3A, two-tailed T test p=0.0146 (not shown).