Reciprocal regulation of ARPP-16 by PKA and MAST3 kinases provides a cAMP-regulated switch in protein phosphatase 2A inhibition

Figures

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  Figure 1.. PKA-mediated Ser88 phosphorylation attenuates the ability of MAST3 to phosphorylate S46-ARPP-16, and MAST3-mediated phosphorylation of Ser46 attenuates the ability of PKA to phosphorylate S88-ARPP-16. (a) Recombinant purified ARPP-16 or P-S88-ARPP-16 (100 nM) were incubated with ATP-γ-32P and MAST3 (overexpressed in HEK293T cells and immunoprecipitated), for various times; proteins were separated by SDS-PAGE and phosphorylation of Ser46 was measured by autoradiography. The resulting values for phosphorylation are expressed in arbitrary densitometric units (a.u.) as mean ± SE of five independent experiments. (b) Assays were carried out as in a. but with increasing concentrations (10–500 nM) of ARPP-16 or P-S88-ARPP-16 for 2 min. Kinetic parameters, determined from double-reciprocal plots, of the data are indicated in Table 1. (c) Recombinant purified ARPP-16 or P-S46-ARPP-16 were incubated with ATP-γ-32P and PKA for various times; proteins were analyzed as described in panel a. The resulting values for phosphorylation are expressed in arbitrary densitometric units (a.u.) as mean ± SE of five independent experiments. (d) Assays were carried out as in c. with increasing…

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  Figure 1—figure supplement 1.. A phospho-mimetic mutation of S88-ARPP-16 attenuates the ability of MAST3 to phosphorylate S46-ARPP-16. (a) Recombinant purified ARPP-16 or the phosphomimetic S88A-ARPP-16 (100 nM) were incubated with ATP-γ-32P and MAST3 for various times; the proteins were separated by SDS-PAGE and phosphorylation of Ser46 was measured by autoradiography. The resulting values for phosphorylation at Ser46 site are expressed in arbitrary densitometric units (a.u.) as mean ± SE of three independent experiments. (b) Assays were carried out as in a. with increasing concentrations (10–500 nM) of ARPP-16 or of the phosphomimetic S88A-ARPP-16 for 2 min. Kinetic parameters, determined from double-reciprocal plots, of the data are presented in Table 1. DOI: http://dx.doi.org/10.7554/eLife.24998.003

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  Figure 2.. cAMP signaling increases phosphorylation of Ser88 and decreases phosphorylation of Ser46 in HEK293T cells. (a) ARPP-16-HA (WT) or the phosphomutants S46D-ARPP-16 or S88D-ARPP-16 were expressed in HEK293T cells alone or in the presence of MAST3-HA kinase. Cells were incubated without or with 10 μM forskolin (FSK) for 30 min. Levels of phosphorylation for Ser46 and Ser88 were measured by immunoblotting with phospho-specific antibodies on SDS-PAGE-resolved cell lysates. Phospho-site signals were normalized for total ARPP-16-HA expression assayed by immunoblotting using anti-HA antibody (A16–HA). For P-S88-ARPP-16 the signal for total ARPP-16-HA was quantified from a separate blot of the same samples. (b) Graph of summary data shows phosphorylation at the different sites expressed in arbitrary densitometric units (a.u.) as mean ± SE of six independent experiments, and analyzed using a one-way ANOVA, multiple comparison test (post-hoc test Tukey). For P-S46-ARPP-16: ****p<0.001, ARPP-16/MAST3 vs ARPP-16 ctrl; $$$ p<0.005, ARPP-16/MAST3 vs ARPP-16/MAST3/FSK; $$$$p<0.001 ARPP-16/MAST3 vs ARPP-16-S88D/MAST3. For P-S88-ARPP-16: ####p<0.001, ARPP-16/FSK vs ARPP-16 ctrl; ####p<0.001,…

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  Figure 2—figure supplement 1.. Regulation of ARPP-16 phosphorylation by the D1 receptor agonist, SKF-81297, in striatal slices. Striatal slices were treated with forskolin (10 μM) (panel a and b), SKF-81297 (panel c and d) or DMSO (as control, panel a, b, c and d) for 15 min. Phosphorylation of ARPP-16 at Ser46 and Ser88 (and GluA1 at Ser845, DARPP-32 at Thr34, assayed as controls) were determined by immunoblotting using phosphorylation state-specific antibodies. Quantitation of phosphorylation was calculated for GluA1-Ser845, DARPP-32-Thr34, ARPP-16-Ser46 and ARPP-16-Ser88 and normalized to total GluA1, DARPP-32, or ARPP-16, respectively. Total proteins were also normalized to GAPDH (panel b. and d.). Graphs represent means ± SEM for 6–8 experiments compared with control slices; **p=0.0011, ***p=0.0006, ****p<0.0001; unpaired T test. DOI: http://dx.doi.org/10.7554/eLife.24998.006

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  Figure 3.. Mathematical modeling shows bistability derived from the reciprocal interactions of ARPP-16 phosphorylation sites. (a) Nullclines of P-S46-ARPP-16 and P-S88-ARPP-16. The steady states of P-S46-ARPP-16 (blue) and P-S88-ARPP-16 (red) are plotted as functions of the concentrations of each other. The intersections of the red and blue curve define the steady states of the system at different cAMP concentrations. Filled circles represent stable steady states, whereas the empty circle indicates an unstable state. (b) Antagonism between P-S46-ARPP-16 and P-S88-ARPP-16 creates a switch-like cAMP response. The figure shows bifurcation diagrams of P-S46-ARPP-16 (blue) and P-S88-ARPP-16 (red) plotted as functions of cAMP concentration. Solid lines show stable steady state solutions whereas dashed lines indicate unstable state values. DOI: http://dx.doi.org/10.7554/eLife.24998.007

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  Figure 3—figure supplement 1.. Bifurcation diagrams of P-S46-ARPP-16 under mutual inhibition (blue, layer 1), PKA inhibition of MAST3 (green, this effect is in addition to previous effect, layers 1 + 2), P-S88-ARPP-16 dominant negative effect on P-S46-ARPP-16 inhibiting PP2A (pink, this effect is in addition to previous effects, layers 1 + 2 + 3), and mutual inhibition with P-S88-ARPP-16 dominant negative effect (black, layers 1 + 3). DOI: http://dx.doi.org/10.7554/eLife.24998.008

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  Figure 3—figure supplement 2.. Bifurcation diagrams of inhibited PP2A under three different layers of regulation described in Figure 3—figure supplement 1. DOI: http://dx.doi.org/10.7554/eLife.24998.009

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  Figure 3—figure supplement 3.. Bifurcation diagrams of P-S46-ARPP-16 under different total MAST3 concentrations. Thin black dashed lines show an example range of cAMP concentrations required for bistability. LP: limit points. DOI: http://dx.doi.org/10.7554/eLife.24998.010

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  Figure 3—figure supplement 4.. Bifurcation diagram of P-S46-ARPP-16, in terms of total MAST3 concentration changes at a fixed cAMP concentration (1.5 µM). DOI: http://dx.doi.org/10.7554/eLife.24998.011

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  Figure 3—figure supplement 5.. Double-parameter bifurcation diagram (cAMP and MAST3tot) showing how total concentration of MAST3 affects the two cAMP thresholds (LP: limit points shown in Figure 3—figure supplement 3); and how cAMP concentration affects the two MASTtot thresholds. DOI: http://dx.doi.org/10.7554/eLife.24998.012

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  Figure 3—figure supplement 6.. Bifurcation diagrams of P-S46-ARPP-16, with increasing P-S46-ARPP-16 inhibitory effect on Ser88 phosphorylation (k46). Thin black dash lines show an example range of cAMP concentrations required for bistability. LP: limit points. DOI: http://dx.doi.org/10.7554/eLife.24998.013

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  Figure 3—figure supplement 7.. Double-parameter bifurcation diagram (cAMP and k46) showing how inhibitory effect of P-S46-ARPP-16 affects the two cAMP thresholds. (LP: limit points corresponding to Figure 3—figure supplement 6). DOI: http://dx.doi.org/10.7554/eLife.24998.014