ARPP-16 Is a Striatal-Enriched Inhibitor of Protein Phosphatase 2A Regulated by Microtubule-Associated Serine/Threonine Kinase 3 (Mast 3 Kinase)

Abstract

ARPP-16 (cAMP-regulated phospho-protein of molecular weight 16 kDa) is one of several small acid-soluble proteins highly expressed in medium spiny neurons of striatum that are phosphorylated in response to dopamine acting via D1 receptor/protein kinase A (PKA) signaling. We show here that ARPP-16 is also phosphorylated in vitro and in vivo by microtubule-associated serine/threonine kinase 3 (MAST3 kinase), an enzyme of previously unknown function that is enriched in striatum. We find that ARPP-16 interacts directly with the scaffolding A subunit of the serine/threonine protein phosphatase, PP2A, and that phosphorylation of ARPP-16 at Ser46 by MAST3 kinase converts the protein into a selective inhibitor of B55α- and B56δ-containing heterotrimeric forms of PP2A. Ser46 of ARPP-16 is phosphorylated to a high basal stoichiometry in striatum, suggestive of basal inhibition of PP2A in striatal neurons. In support of this hypothesis, conditional knock-out of ARPP-16 in CaMKIIα::cre/floxed ARPP-16/19 mice results in dephosphorylation of a subset of PP2A substrates including phospho-Thr75-DARPP-32, phospho-T308-Akt, and phospho-T202/Y204-ERK. Conditional knock-out of ARPP-16/19 is associated with increased motivation measured on a progressive ratio schedule of food reinforcement, yet an attenuated locomotor response to acute cocaine. Our previous studies have shown that ARPP-16 is phosphorylated at Ser88 by PKA. Activation of PKA in striatal slices leads to phosphorylation of Ser88, and this is accompanied by marked dephosphorylation of Ser46. Together, these studies suggest that phospho-Ser46-ARPP-16 acts to basally control PP2A in striatal medium spiny neurons but that dopamine acting via PKA inactivates ARPP-16 leading to selective potentiation of PP2A signaling.SIGNIFICANCE STATEMENT We describe a novel mechanism of signal transduction enriched in medium spiny neurons of striatum that likely mediates effects of the neurotransmitter dopamine acting on these cells. We find that the protein ARPP-16, which is highly expressed in striatal medium spiny neurons, acts as a selective inhibitor of certain forms of the serine/threonine protein phosphatase, PP2A, when phosphorylated by the kinase, MAST3. Under basal conditions, ARPP-16 is phosphorylated by MAST3 to a very high stoichiometry. However, the actions of MAST3 are antagonized by dopamine and cAMP-regulated signaling leading to disinhibition of ARPP-16 and increased PP2A action.

Keywords: cocaine; dopamine; medium spiny neuron; motivation; protein kinase A.

Figure 1.

ARPP-16 interacts with the A subunit of PP2A. a, Rat brain S2 fractions were incubated with immobilized His-ARPP-16, and then eluted proteins were separated using DIGE. The striatal sample was labeled with Cy3, whereas the control sample (nonspecific binding of striatal samples to beads with no ARPP-16) was labeled with Cy2. Samples were mixed and analyzed by DIGE. White arrow indicates a spot corresponding to PP2A-A. Arrowhead indicates a spot corresponding to tubulin. b, Independent striatal S2 samples were incubated with control beads (Control) or beads with immobilized His-ARPP-16 (His-A16), and bound proteins were analyzed by SDS-PAGE and immunoblotting with antibodies to PP2A-A (red) and PP2A-C (green). c, Increasing amounts of recombinant, SEC-purified PP2A-A (10, 20, 40, or 80 ng) were incubated with immobilized His-ARPP-16 (50 μg) and bound protein analyzed by SDS-PAGE and immunoblotting with antibody to PP2A-A. The PP2A-A input (10 ng) is shown in lane 1, and the eluant from beads with no His-ARPP-16 that were incubated with PP2A-A (10 ng) is shown in lane 2. d, Striatal lysate fraction S2 was incubated with immobilized His-ARRP-16 (100 μg) or beads alone (empty beads, negative control). Eluted proteins were separated by SDS-PAGE and immunoblotted with antibody against PP2A-A, PP2A-C, PP1, PP2B, or synaptophysin. The lysate input (1%) is showed in lane 1; 20% of the eluates were analyzed in lanes 2 and 3. e, Full-length GST-PP2A-A, GST-tagged truncation mutants of PP2A-A that included HEAT repeats HEAT1–7, HEAT1–9, HEAT3-END, or a GST tag control (50 μg), were immobilized onto glutathione Sepharose 4 Fast Flow beads and incubated with increasing amounts of recombinant, SEC-purified His-ARPP-16 (A16, 10, 100, or 1000 nm) or SEC-purified His-ARPP-19 (A19, 10, 100, or 1000 nm). Recombinant RCS (100 or 1000 nm) was incubated with beads to determine nonspecific binding. Eluted samples were analyzed by SDS-PAGE and immunoblotted for ARPP-16, ARPP-19, or RCS.

Figure 2.

ARPP-16 is phosphorylated at Ser46 in intact cells and in vitro by MAST3 kinase. a, Purified ARPP-16 was phosphorylated in vitro with MAST3 kinase, and unphosphorylated (left lane) or phosphorylated (right lane) samples analyzed by immunoblotting with total ARPP-16 (bottom) or a phospho-Ser46 antibody (top). b, HA-ARPP-16 was expressed in HEK293 cells without or with HA-MAST3 kinase, and the phosphorylation at Ser46 of ARPP-16 was analyzed by immunoblotting using the phospho-Ser46-specific antibody. HA-MAST3 and HA-ARPP-16 expression was analyzed by immunoblotting with an HA antibody. c, Recombinant purified ARPP-16 (1 μm) was incubated with immunoprecipitated MAST3 for various times (as indicated), and phosphorylation of Ser46 was measured by immunoblotting. Ser46 phosphorylation was normalized to total ARPP-16 levels, and to the zero time value in each experiment. Results shown represent the average from three experiments. Error bars indicate SEM. d, Purified HA-ARPP16 (1 μm) was phosphorylated in vitro with MAST3 kinase in the presence of thio-γ-ATP. P-γ-Ser-46-ARPP-16 was then incubated without or with PP2A, and phosphorylation of Ser46 was measured by immunoblotting.

Figure 3.

ARPP-16 inhibits PP2A activity in vitro. a, Purified PP2A-AC dimer (0.01 U/μl, Millipore) was incubated with increasing concentration (0, 0.05, 0.1, and 1 μm) of recombinant, purified dephospho-ARPP-16 (top) or P-γ-Ser46-ARPP-16 (bottom) for 10 min at 37°C and phosphatase activity measured using 500 μm phosphopeptide as substrate. Phosphate release was detected using a malachite green assay with absorbance measured at 650 nm. b, Recombinant Flag-Bα, Flag-B56δ, or Flag-PR72 subunits were individually overexpressed in HEK293 cells, and PP2A heterotrimers were isolated by immunoprecipitation with anti-Flag antibody. PP2A heterotrimeric forms were incubated with 200 nm dephospho-ARPP-16 or P-γ-Ser46-ARPP-16 for 10 min at 37°C with [³²P]-T75-DARPP-32 as substrate. ³²P release was measured following TCA precipitation and scintillation counting. Results are all expressed as percentage changes with respect to PP2A alone (white bars). *p < 0.05 (Newman–Keuls test). **p < 0.01 (Newman–Keuls test). ***p < 0.001 (Newman–Keuls test). Error bars indicate SEM.

Figure 4.

Regulation of phosphorylation of ARPP-16 at Ser46 and ARPP-19/ENSA at Ser62 in striatal slices. a, Rat striatal slices (n = 6 per condition) were treated without (Control, ctrl) or with 10 μm forskolin (fsk) for 10 min. Samples (30 μg total lysate) were analyzed by immunoblotting for phospho-Ser46 and total ARPP-16 or phospho-Ser62 and total ENSA (top). Phospho-site signals were each normalized to total protein, and data for forskolin treatment were normalized to controls (bottom bar graph). **p < 0.01 (Student's t test). ***p < 0.001 (Student's t test). Error bars indicate SEM. b, Striatal slices (n = 3 or 4 per condition) were treated without (ctrl) or with 100 μm NMDA for 5 min (5m), or 15 min (15m), and differences in phosphorylation of ARPP-16 at Ser46 and ENSA at Ser62 were analyzed as in a. *p < 0.05 (multivariate ANOVA with Bonferroni post hoc analysis). Error bars indicate SEM.

Figure 5.

ARPP-16/19 regulates PP2A in a substrate-specific manner in striatum. a, Samples from different brain regions (as indicated) from WT and conditional ARPP-16/19 knock-out (cKO) mice were analyzed by immunoblotting with an antibody that recognizes ARPP-16, ARPP-19, and ENSA. Striatal slices from ARPP-16/19 cKOs (n = 4) or WT littermate controls (n = 3 or 4) were isolated and basal phosphorylation of the PP2A targets, (b) DARPP-32 at Thr75, or (c) Akt at Thr308 were analyzed by SDS-PAGE and immunoblotting. b, Phospho-Thr75 (pD32–T75) and total DARPP-32 (totD32) blots. c, Phospho-Thr308 (pAkt-t308) and total Akt (totAkt) blots. Bottom bar graphs represent cumulative data. *p < 0.05 (Student's t test). Error bars indicate SEM. d, Striatal slices from ARPP-16/19 cKOs (n = 4) or WT littermate controls (n = 4) were incubated with 10 μm forskolin for 0, 10, 30, or 60 min. Samples were analyzed by SDS-PAGE, and immunoblotting was done for phospho-Thr202/204 and total ERK1/2 (top); quantitation of phosphorylation as done for Thr202/204 normalized to ERK1. Bottom graph represents cumulative data. **p < 0.01 (multivariate ANOVA). b–d, Phospho-site signals were each normalized to total protein, and then data for cKO mouse samples were normalized to controls.

Figure 6.

ARPP-16/19 does not regulate Ser845 phosphorylation of GluA1 or Thr34 phosphorylation of DARPP-32. Striatal slices from ARPP-16/19 cKOs (n = 4) or WT littermate controls (n = 3 or 4) were isolated and basal phosphorylation of the PP2A targets, (a) elongation factor-2 (EF2) at Thr56 or (b) tau at Ser396, were analyzed by immunoblotting. a, Phospho-Thr56 (pEF2) and total EF2 (TotEF2) blots. b, Phospho-Ser396 (pTau) and total tau (TotTau) blots. Bar graphs represent cumulative data (multivariate ANOVA p > 0.05). Data for cKO mouse samples were normalized to controls. Striatal slices from ARPP-16/19 cKOs (n = 4) or WT littermate controls (n = 4) were incubated with 10 μm forskolin for 0, 10, 30, or 60 min. Samples were analyzed by immunoblotting for (c) phospho-Ser845 and total GluA1 and (d) phospho-Thr34 and total DARPP-32 (bottom); quantitation of phosphorylation was calculated for Ser845 or Thr34 normalized to total GluA1 or total DARPP-32, respectively. Graphs represents cumulative data (multivariate ANOVA, p > 0.05). Data for cKO mouse samples were normalized to controls.

Figure 7.

ARPP-16/19 cKO mice show increased motivation in progressive ratio responding and an altered locomotor response to cocaine. a, ARPP-16/19 cKOs (open circles, n = 11) and WT littermate controls (closed circles, n = 9) were trained on a VR2 schedule of reinforcement to respond by nose-poking into an assigned nose aperture to receive a food reinforcer until stable acquisition of reward was measured. No significant difference in acquisition of this task was detected between the cKOs and WT controls (repeated-measures ANOVA). Data are mean ± SEM. b, Once the WT (black bars) and ARPP-16/19 cKO (white bars) mice were stably trained, the number of responses required to receive a food reinforcer increased using an escalating schedule of reinforcement (1, 5, 9… x + 4 responses/reinforcer). The breakpoint ratio was defined as the response/reinforcer ratio achieved by an animal when the animal gave up nose-poking (defined as 5 min of no activity in the active nose aperture). This paradigm was completed on 2 consecutive days. *p < 0.05 (repeated-measures ANOVA with Bonferroni post hoc analysis). Data are mean ± SEM. c, d, ARPP-16/19 cKO and WT littermate controls were exposed to saline (n = 10 WT; n = 11 cKO), 5 mg/kg (n = 10 WT; n = 9 cKO), 12.5 mg/kg (n = 7 WT; n = 6 cKO), or 20 mg/kg (n = 8 WT, n = 10 cKO) cocaine (i.p.); then locomotor activity was measured using an Accuscan tracking system. c, Horizontal movement during the 60 min postinjection session were binned into 5 min increments. ARPP-16/19 cKO (open circles and squares) shows an attenuated response to 20 mg/kg cocaine compared with WT littermate controls (closed circles and squares) and no significant effect of cocaine compared with cKO animals exposed to saline. *p < 0.05 (repeated-measures ANOVA with Bonferroni post hoc analysis). **p < 0.01 (repeated-measures ANOVA with Bonferroni post hoc analysis). ***p < 0.001 (repeated-measures ANOVA with Bonferroni post hoc analysis). Data are mean ± SEM. d, Total horizontal movement over the 60 min postinjection session for saline (n = 9 for both WT and cKO), 5 mg/kg (n = 10 WT; n = 9 cKO), 12.5 mg/kg (n = 7 WT; n = 6 cKO), and 20 mg/kg (n = 8 WT, n = 10 cKO) showed significant hyperlocomotor activity in WT animals following 12.5 mg/kg and 20 mg/kg cocaine exposure. **p < 0.01 (two-way ANOVA with Bonferroni post hoc analysis). ****p < 0.0001 (two-way ANOVA with Bonferroni post hoc analysis). Data are mean ± SEM. There was no significant difference in total distance moved by cKO mice in response to cocaine exposure compared with saline exposure, and ARPP-16/19 cKO mice showed significantly less cocaine-induced locomotor activity over 60 min compared with WT controls at 20 mg/kg cocaine. *p < 0.05 (two-way ANOVA with Bonferroni post hoc analysis). Data are mean ± SEM. e, WT and ARPP-16/19 cKO mice were exposed to saline or cocaine as described in c, d, and events of vertical movement were measured and summarized for 1 h after injection of cocaine or saline. WT control animals showed significantly increased vertical movement following 20 mg/kg cocaine injection compared with WT animals injected with saline. ***p < 0.001 (two-way ANOVA with Bonferroni post hoc analysis). Data are mean ± SEM. ARPP-16/19 cKO mice showed no difference in vertical movement at any dose. At 20 mg/kg cocaine, ARPP-16/19 cKO mice showed significantly reduced vertical movement compared with WT control animals. **p < 0.01 (two-way ANOVA with Bonferroni post hoc analysis). Data are mean ± SEM. f, No significant differences were observed in stereotypic behavior in either ARPP-16/19 cKO mice or WT control animals at any dose.

Figure 8.

Schematic illustration of ARPP-16 regulation in striatal neurons. ARPP-16 is phosphorylated by MAST3 at Ser46 to a high basal stoichiometry in striatum, which leads to selective inhibition of PP2A activity. Dephosphorylation of Ser46 in ARPP-16 is increased by activation of PKA or by stimulation of NMDA receptors, leading to disinhibition of PP2A.