Regulation of RCAN1 Protein Activity by Dyrk1A Protein-mediated Phosphorylation^*

Proteins and Antibodies

Mouse wild-type and Y321F kinase-inactive mutant Dyrk1A proteins with endogenous 13-histidine repeats were purified with Ni²⁺-NTA resin as described previously (10). Full-length human RCAN1-1S (RCAN1) and human Caln A cDNAs were cloned into pET29b and pET28b, respectively. The full-length human RCAN1 and an RCAN1 C-terminal (RCAN1Ct) cDNA corresponding to exon 7 were cloned into pGEX4T-3. The recombinant proteins were expressed in Escherichia coli BL21(DE3) strain RIL (Stratagene) and purified using Ni²⁺-NTA or glutathione-Sepharose 4B resin.

Anti-α-tubulin and HA antibodies were from Sigma. The anti-Caln antibody was from R&D Systems. The anti-Dyrk1A antibody was custom-made as described previously (16). The anti-RCAN1 antibodies were either from Abnova or custom-made using synthetic peptides, RPEYTPIHLS. A phosphospecific RCAN1 antibody to a synthetic phosphopeptide (¹⁸⁸RPEYpTPIHLS¹⁹⁷) was generated and affinity-purified first with a cognate nonphosphopeptide (RPEYTPIHLS) affinity column and then with a phosphopeptide column (Peptron, Inc., Daejeon, South Korea).

Plasmids, siRNAs, and Cell Transfection

The full-length wild-type and Y321F kinase-inactive Dyrk1A mutant cDNAs were cloned into pcDNA3.1 as described previously (10). The full-length human wild-type RCAN1-1S (RCAN1) and Caln A cDNAs were cloned into pcDNA3.1 (Invitrogen). Phosphorylation-defective mutants of RCAN1 cDNA were generated by DpnI-mediated site-directed mutagenesis (Stratagene), and the clones were verified by sequencing. For the siRNA experiment, the Dyrk1A-specific siRNA (5′-AUGGAGCUAUGGACGUUAA) with the TT overhang was designed and synthesized by Samchully Pharm Co. Ltd. (Seoul, South Korea). For siRNA delivery, Dyrk1A-specific duplex siRNA (100 pmol for a 6-well plate) was transfected into PC12 cells (7.5 × 10⁵ cells/well) using Lipofectamine 2000 (Invitrogen). Dyrk1A siRNA (5′-GUACAUCGUCAAGCUCAGGU) was used as an ineffective siRNA control. After 48–72 h of siRNA treatment, cell lysates were prepared for immunoblot analyses. HEK293T cells were transfected with the indicated plasmids using the calcium phosphate precipitation method. One day later, the cells were lysed in RIPA buffer (50 mm Tris, pH 8.0, 150 mm NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% deoxycholic acid) containing 1 mm PMSF, a phosphatase inhibitor mixture, and a protease inhibitor mixture (Calbiochem) and subjected to Western blot analysis.

Dyrk1A in Vitro Kinase Assay

For analysis by autoradiography, purified RCAN1 wild-type (WT) or mutant protein (100 ng) was incubated with recombinant full-length Dyrk1A protein (1 μg) for 1 h at 37 °C in kinase buffer (20 mm MOPS, pH 7.0, 10 mm MgCl₂, 1 mm DTT, and 20 μm sodium orthovanadate) containing 25 μm cold ATP and 5 μCi of [γ-³²P]ATP. The reaction mixtures were separated on SDS-polyacrylamide gels, and radioactive bands were detected with the Typhoon 9200 imaging system (Amersham Biosciences). The ability of Dyrk1A to prime RCAN1 WT or mutant proteins for GSK3β phosphorylation was examined by prephosphorylating GST fusion proteins bound to glutathione-Sepharose beads with 720 ng of Dyrk1A in the presence and absence of 1 mm non-radioactive ATP overnight at 30°C. After the non-radioactive priming step, the bead-bound GST fusion proteins were washed extensively to remove recombinant kinase and ATP. Phosphorylated GST fusion proteins were then incubated with 20 ng of GSK3β (Millipore) in a 25-μl final volume of the kinase buffer in the presence of 25 μm ATP and 10 μCi of [γ-³²P]ATP for 40 min at 37 °C and analyzed as described above.

Preparation of Lysates from Cell Cultures and Brains and Western Blot Analysis

HEK293T cells were harvested 24 h after transient transfection with the indicated plasmids. Cells were lysed in RIPA buffer with 1 mm PMSF and a protease inhibitor mixture. Transgenic mice overexpressing the human Dyrk1A gene, which was carried on a bacterial artificial chromosome, were generated and maintained as described previously (16). Experiments were performed in accordance with guidelines set forth by the Inje University Council directive for the proper care and use of laboratory animals. Mice were sacrificed by cervical dislocation. Brains were dissected, snap-frozen in liquid nitrogen, and Dounce-homogenized in RIPA buffer containing 1 mm PMSF, a protease inhibitor mixture, and 0.5 mm sodium orthovanadate. Protein concentration was determined using the BCA method (Sigma). Typically, 25–50 μg of the brain lysates was used for Western blotting. Densitometric quantification was carried out using ImageJ 1.42 software (National Institutes of Health, Bethesda, MD).

Co-immunoprecipitation and GST Pull-down Assay

For co-immunoprecipitation, brain lysates (2–6 mg) from the Dyrk1A TG mice or HEK293T cell lysates (500 μg) transfected with the indicated plasmids were incubated with control IgG (R&D Systems), anti-RCAN1, or anti-Dyrk1A antibodies overnight at 4 °C in RIPA buffer with protease inhibitors and 1 mm PMSF. The next day, after 1 h of incubation with protein A beads (Pierce), the bead mixture was gently washed with 1% Triton X-100 in RIPA buffer, and the bound proteins were subjected to immunoblot analysis with the indicated antibodies. For the GST pull-down assay, purified GST-RCAN1 WT or the phosphorylation-defective mutants were incubated with recombinant Dyrk1A protein (1 μg) or HEK293T cell lysates (500 μg) transfected with the plasmid encoding Caln for 1 h at 4 °C in binding buffer (50 mm Tris, pH 8.0, 100 mm NaCl, 10% glycerol, 2 mm β-mercaptoethanol, 0.01% Nonidet P-40, 0.5 mm EDTA, 1 mm PMSF). The beads were then washed with 300 mm NaCl-containing binding buffer three times, and the bound proteins were subjected to immunoblot analysis with the indicated antibodies.

Immunocytochemistry

PC12 cells were plated onto polyethyleneimine-coated coverslips, treated with nerve growth factor (NGF) for 6 days, and then fixed with 4% formaldehyde in phosphate-buffered saline (PBS). For primary embryonic rat cortical cultures, embryonic day 17 pregnant Sprague-Dawley rats were purchased from Orient Bio Inc. (Seongnam-si, South Korea). The cortices of the embryos were dissected and cultured as described previously (30, 31). Primary cortical cultures were plated onto poly-d-lysine-coated glass coverslips and grown in neurobasal growth medium for 5 days before fixing with 4% formaldehyde in PBS. The fixed cells were subsequently permeabilized with 0.1% Triton X-100 and blocked with 5% bovine serum albumin (BSA) and then incubated for 2 h at room temperature with primary antibodies (rabbit anti-Dyrk1A antibody (1:50), mouse anti-RCAN1 antibody (1:50), or rabbit phosphospecific RCAN1 antibody (1:2,000)). The cells were then incubated with secondary antibodies (Texas Red-conjugated goat anti-rabbit or Alexa Fluor 488-conjugated goat anti-mouse antibodies at a 1:500 dilution) for 1 h at room temperature, washed with PBS, and examined using a fluorescence microscope (Olympus BX61) or a confocal laser scan microscope (LSM 510 META, Carl Zeiss).

Measurement of Caln Phosphatase Activity

The activity of Caln was determined using p-nitrophenyl phosphate as described previously (32). Phosphatase assays using 60 mm p-nitrophenyl phosphate (Sigma) were performed using 10 μg of purified recombinant Caln with and without purified recombinant RCAN1 WT or the phosphorylation-defective mutants (0.8 or 1.6 μg) and 200 nm calmodulin (CaM; Calbiochem) in assay buffer (100 mm Tris, pH 7.5, 100 mm NaCl, 0.5 mm DTT, 100 μg/ml BSA, 1 mm MnCl₂, 0.4 mm CaCl₂). In each assay, blank tubes containing buffer only were prepared to determine background levels of hydrolyzed p-nitrophenyl phosphate. Control tubes contained buffer and Caln/CaM. After 5 min of incubation at 30 °C, the reactions were initiated by the addition of 60 mm p-nitrophenyl phosphate. The tubes were incubated for an additional 60–90 min at 30 °C, and the liberation of p-nitrophenol was measured at 405 nm using a TECAN Infinite 200 plate reader.

NFAT-Luciferase Assay

For luciferase experiments, HEK293T cells grown in 24-well plates were transfected with 0.5 ng of pCMV-RL reporter (Renilla luciferase, internal standard), 200 ng of pGL3-Basic reporter (NFAT-luciferase, AP-1 promoter), and 50 ng of expression plasmids encoding empty vector, RCAN1 WT, or the phosphorylation-defective mutants. The total amount of plasmid was kept constant for all groups. After transfection, cells were treated with ionomycin (2.5 μm) for 48 h and analyzed for reporter gene activity using the Dual-Luciferase reporter assay (Promega) as recommended by the manufacturer. NFAT luciferase values were normalized to Renilla luciferase values.

Dyrk1A Interacts with RCAN1

To determine whether RCAN1 interacts with Dyrk1A, co-immunoprecipitation assays were carried out in HEK293T cells transfected with expression vectors encoding RCAN1 and Dyrk1A. RCAN1 co-immunoprecipitated with Dyrk1A, indicating that Dyrk1A interacts with RCAN1 (Fig. 1A). Furthermore, reverse co-immunoprecipitation experiments with the anti-RCAN1 antibody also revealed an interaction between Dyrk1A and RCAN1, as shown in Fig. 1A. To further determine whether endogenous RCAN1 and Dyrk1A interact in mammalian neurons, the brain lysates of Dyrk1A TG mice were immunoprecipitated with the anti-Dyrk1A or RCAN1 antibodies. RCAN1 associates with Dyrk1A in brain lysates (Fig. 1B). The direct interaction between purified Dyrk1A protein and GST-RCAN1 was further examined using a GST pull-down assay, which showed that GST-RCAN1 but not GST alone was able to bind directly to Dyrk1A as shown in Fig. 1C.

Dyrk1A Phosphorylates RCAN1 on Ser¹¹² and Thr¹⁹² Residues

To determine whether Dyrk1A can phosphorylate RCAN1 in vitro, purified RCAN1 was incubated with recombinant Dyrk1A WT or inactive Dyrk1A Y321F mutant in a kinase assay buffer containing [γ-³²P]ATP. A band that migrated to a position corresponding to the molecular size of RCAN1 was detected by autoradiography only when both Dyrk1A WT and RCAN1 were present in the reaction mixture (Fig. 2A), indicating that RCAN1 was phosphorylated by active Dyrk1A. In vitro kinase assays in the presence of [γ-³²P]ATP were also performed with the RCAN1-Dyrk1A immunocomplexes from HEK293T cells expressing RCAN1 and Dyrk1A. As shown in supplemental Fig. S1, phosphorylation of RCAN1 was detected in the RCAN1-Dyrk1A immunocomplex but not in control immunocomplex kinase assays. These data support the conclusion that Dyrk1A phosphorylates RCAN1.

An examination of the amino acid sequence of RCAN1 revealed the presence of five potential Dyrk1A phosphorylation sites at Ser¹⁰⁸, Ser¹¹², Thr¹²⁴, Thr¹⁵³, and Thr¹⁹², each of which is followed by a proline residue conserved in human, rat, and mouse sequences. To identify the specific RCAN1 residues phosphorylated by Dyrk1A, each potential RCAN1 phosphorylation site was replaced with alanine, resulting in S108A, S112A, T124A, T153A, T192A, and the double mutant S112A/T192A. Purified RCAN1 wild type and mutants were incubated with recombinant Dyrk1A in a kinase assay buffer containing [γ-³²P]ATP. This analysis revealed that mutation of serine 112 and threonine 192 to alanine strongly reduced the phosphorylation level of RCAN1. The S112A/T192A double mutation reduced the phosphorylation even further, whereas mutations at positions 108, 124, or 153 had little effect on Dyrk1A-dependent phosphorylation (Fig. 2B). As shown in Fig. 2C, the amounts of RCAN1 WT and mutants used in the kinase assay were similar. These results suggest that Dyrk1A selectively phosphorylates RCAN1 at Ser¹¹² and Thr¹⁹² residues.

Phosphorylation of RCAN1 by Dyrk1A at Ser¹¹² Primes the Protein for GSK3β-mediated Phosphorylation

Previous work showed that mitogen-activated protein kinase (MAPK) phosphorylates RCAN1 at serine 112 to prime it for subsequent phosphorylation at serine 108 by GSK3β (33). Dyrk1A occasionally plays a role as a GSK3β-priming kinase by phosphorylating the serine or threonine residue in target substrates located 4 amino acids downstream from the C terminus of another GSK3β-phosphorylated serine or threonine (4). Therefore, we anticipated that phosphorylation of Ser¹¹² by Dyrk1A would prime for the phosphorylation of Ser¹⁰⁸ by GSK3β. To investigate whether Dyrk1A could prime GSK3β-mediated phosphorylation of RCAN1, wild-type GST-RCAN1 protein bound to glutathione-Sepharose beads was prephosphorylated in the presence or absence of recombinant Dyrk1A. After the non-radioactive priming step, the bead-bound GST fusion proteins were washed extensively to remove recombinant Dyrk1A. Phosphorylated GST fusion proteins were then incubated with and without GSK3β in a kinase assay buffer containing [γ-³²P]ATP. Fig. 3A shows that RCAN1 prephosphorylated by Dyrk1A was strongly phosphorylated by GSK3β, although GSK3β and residual Dyrk1A alone slightly phosphorylated RCAN1.

We performed GSK3β mutation analysis to determine if Dyrk1A primes for phosphorylation at Ser¹⁰⁸ of RCAN1. GST-RCAN1 proteins, either wild-type or mutant forms (S108A, S112A, or T192A), bound to glutathione-Sepharose beads were first phosphorylated using recombinant Dyrk1A in the presence or absence of non-radioactive ATP. Phosphorylated GST fusion proteins were then incubated with GSK3β in a kinase assay buffer containing [γ-³²P]ATP. As shown in Fig. 3B, Dyrk1A can prime wild-type RCAN1 for subsequent phosphorylation by GSK3β only in the presence of ATP. RCAN1 phosphorylation by GSK3β did not occur with proteins carrying mutations at S108A and S112A, which are the sites of GSK3β and Dyrk1A phosphorylation, respectively. In the priming experiment with the T192A mutant, the Dyrk1A-prephosphorylated RCAN1 was as strongly phosphorylated by GSK3β as was the wild-type RCAN1, excluding the possibility of non-canonical GSK3 priming (the priming kinase phosphorylates a residue required for GSK3β phosphorylation not at the +4-position), which has been shown for mammalian Dyrk1A kinase on NFAT (5) and for the Dyrk1A orthologue MBK-2 in Caenorhabditis elegans (34). These results suggest that Dyrk1A-mediated phosphorylation of Ser¹¹² primes RCAN1 for subsequent phosphorylation by GSK3β at Ser¹⁰⁸ in vitro, supporting a role for Dyrk1A as a priming kinase for GSK3β.

Phosphorylation at Thr¹⁹² by Dyrk1A Enhances the Activity of RCAN1 as a Caln Inhibitor

The effect of Dyrk1A-mediated phosphorylation of RCAN1 on the activity of RCAN1 as an inhibitor of Caln was examined. Prior to performing the Caln activity assay, purified GST-RCAN1 bound to glutathione-Sepharose beads was incubated with and without Dyrk1A in the presence and absence of 10 mm non-radioactive ATP for 90 min at 30°C, and the bead-bound GST-RCAN1 was washed extensively to remove recombinant kinase and ATP. As shown in Fig. 4A, RCAN1 phosphorylated by Dyrk1A inhibited Caln activity by 59%, whereas nonphosphorylated RCAN1 without ATP and without Dyrk1A inhibited Caln activity by 52 and 53%, respectively. In five independent experiments, the phosphorylation of RCAN1 enhanced its ability to inhibit Caln phosphatase activity by 27 ± 6% relative to that of nonphosphorylated RCAN1 without ATP (p < 0.05) and by 14 ± 3% relative to that of nonphosphorylated RCAN1 without Dyrk1A (p < 0.05) (Fig. 4A, right), suggesting that Dyrk1A-mediated phosphorylation of RCAN1 enhances its ability to inhibit Caln.

The effect of Ser¹¹² and Thr¹⁹² phosphorylation on RCAN1 inhibition of Caln was assessed using RCAN1 WT and the S112A and T192A mutants. We first performed RCAN1 dosage-dependent Caln assays and found that RCAN1 inhibits Caln activity in a dose-dependent manner (supplemental Fig. S2A). We used 0.8 or 1.6 μg of RCAN1 WT and the phosphorylation-defective mutants in Fig. 4B with similar results at both concentrations. Both RCAN1 WT and RCAN1(S112A) inhibited Caln activity by 55%, whereas RCAN1(T192A) inhibited Caln activity by 51% (Fig. 4B). In six independent experiments, the RCAN1(S112A) and RCAN1(T192A) mutants exhibited reduced inhibition of Caln phosphatase activity (3 ± 1% (p < 0.05) and 9 ± 1% (p < 0.001), respectively) relative to that of RCAN1 WT (Fig. 4B, right). These results suggest that RCAN1 inhibition of Caln activity is mostly regulated by Dyrk1A-mediated phosphorylation of RCAN1 at Thr¹⁹², although there could be a small contribution by phosphorylation at Ser¹¹².

A previous report showed that RCAN1 inhibited NFAT transcriptional activity in a dose-dependent manner in HEK293T cells, and Dyrk1A showed a synergistic effect with RCAN1 in blocking NFAT-dependent transcription in cortical neurons (25). The effect of RCAN1 WT and the S112A and T192A mutants on NFAT transcriptional activity was therefore investigated. We first determined the dosage dependence of RCAN1 in NFAT assays and found that RCAN1 inhibits NFAT transcriptional activity in a dose-dependent manner (supplemental Fig. S2B). In HEK293T cells, RCAN1 WT inhibited NFAT transcriptional activity by 61%, whereas the RCAN1(S112A) and RCAN1(T192A) mutants inhibited NFAT-dependent transcription by 60 and 53%, respectively (Fig. 4C, left). The expression of RCAN1 WT and mutants was similar (Fig. 4C). In 11 independent experiments, the RCAN1(T192A) mutant showed a 10 ± 2% reduction in the ability to inhibit NFAT-dependent transcription in comparison with RCAN1 WT (p < 0.001) (Fig. 4C, right). These results suggest that Dyrk1A-mediated phosphorylation of RCAN1 at Thr¹⁹² regulates the transcriptional activity of NFAT, possibly through the inhibition of Caln activity.

The finding that the overexpression of RCAN1 caused the hyperphosphorylation of Tau (35), and our previous observation that Dyrk1A phosphorylated Tau at Thr²¹² and Ser⁴⁰⁴ residues prompted us to examine the effect of RCAN1 and Caln on Tau phosphorylation. Western blot analysis was performed with lysates of HEK293T cells that had been transiently transfected with a Tau expression plasmid, either in the presence or absence of a plasmid encoding RCAN1 or Caln A. Phosphorylation of Thr²¹² and Ser⁴⁰⁴ residues was detected with phospho-Thr²¹²-Tau (p-Tau212)-specific and phospho-Ser⁴⁰⁴-Tau (p-Tau404)-specific antibodies, respectively. Although the amounts of Tau were similar, the amounts of p-Tau212 and p-Tau404 were increased in cells expressing RCAN1 but decreased in cells expressing Caln (Fig. 4D), suggesting that these Tau residues are dephosphorylated by Caln. To further examine whether the expression of p-Tau212 and p-Tau404 is sensitive to Caln, HEK293T cells expressing Tau and Caln were treated with a Caln inhibitor, FK506 (1 μm). As shown in supplemental Fig. S3, the amounts of p-Tau212 and p-Tau404 were decreased in cells expressing Caln but recovered in cells treated with FK506, supporting the conclusion that p-Tau212 and p-Tau404 are dephosphorylated by Caln.

Examination of the effect of RCAN1 WT and the S112A and T192A mutants on Tau phosphorylation revealed that the RCAN1(T192A) mutant reduced the phosphorylation of Tau at Thr²¹² and Ser⁴⁰⁴ residues compared with RCAN1 WT (Fig. 4E). The effect of RCAN1(S112A) on these Tau residues was nearly the same as that of RCAN1 WT (Fig. 4E). In six independent experiments, the RCAN1(T192A) mutant inhibited Tau phosphorylation at Thr²¹² and Ser⁴⁰⁴ by 55 ± 5% and 36 ± 8%, respectively, relative to RCAN1 WT (p < 0.01) (Fig. 4F). These results suggest that the increased phosphorylation of Tau mediated by the inhibition of its dephosphorylation by Caln is regulated by Dyrk1A-mediated phosphorylation of RCAN1 at Thr¹⁹².

Dyrk1A-mediated Phosphorylation of RCAN1 Increases Binding of RCAN to Caln

Because the C-terminal region of RCAN1 is reported to bind to Caln (29, 36, 37), we tested whether the effect of Dyrk1A-mediated phosphorylation of RCAN1 on its ability to inhibit Caln activity is linked to its ability to bind Caln. Purified GST-RCAN1 bound to glutathione-Sepharose beads was incubated with Dyrk1A in the presence and absence of 10 mm non-radioactive ATP for 1 h at 37 °C, and the bead-bound GST fusion proteins were washed to remove recombinant kinase and ATP prior to performing the binding assay. As shown in Fig. 5A, RCAN1 phosphorylated by Dyrk1A showed enhanced binding to Caln compared with nonphosphorylated RCAN1. In six independent experiments, the binding of phosphorylated RCAN1 to Caln was enhanced by 16 ± 6% relative to nonphosphorylated RCAN1 (p < 0.05) (Fig. 5A). To examine the role of Ser¹¹² and Thr¹⁹² phosphorylation in the binding of RCAN1 to Caln, the interaction between Caln and either GST-RCAN1 WT or the phosphorylation-defective mutant was compared using a GST pull-down assay. As shown in Fig. 5B, the GST-RCAN1, but not GST, was able to bind to Caln overexpressed in HEK293T cell lysates, and the RCAN1(T192A) mutant showed inhibited Caln binding compared with that of RCAN1 WT. There was little difference between RCAN1 WT and RCAN1(S112A) in their effect on Caln binding. In 16 independent experiments, the binding of the RCAN1 T192A mutant to Caln was reduced by 13 ± 4% relative to the RCAN1 WT (p < 0.01) (Fig. 5B). These results suggest that Dyrk1A-mediated phosphorylation enhances the ability of RCAN1 to inhibit Caln, potentially through increased binding to Caln.

We then determined whether a similar effect of Dyrk1A-mediated phosphorylation is observed with the 57 C-terminal amino acids of RCAN1 (RCAN1Ct) composed of exon 7. As shown in supplemental Fig. S4, RCAN1Ct phosphorylated by Dyrk1A showed enhanced binding to Caln in comparison with nonphosphorylated RCAN1. In 4–9 independent experiments, the binding of phosphorylated RCAN1 to Caln was enhanced by 19 ± 4% relative to nonphosphorylated RCAN1 without Dyrk1A (p < 0.05) and by 12 ± 5% relative to nonphosphorylated RCAN1 without ATP (p < 0.05) (supplemental Fig. S4, A and B). When the interaction between Caln and either GST-RCAN1Ct WT or the T192A mutant was compared using a GST pull-down assay, the RCAN1Ct(T192A) mutant showed inhibited Caln binding compared with that of RCAN1Ct WT. In five independent experiments, the binding of the RCAN1 T192A mutant to Caln was reduced by 32 ± 9% relative to the RCAN1Ct WT (p < 0.05) (supplemental Fig. S4C). These results support the hypothesis that Dyrk1A-mediated phosphorylation at Thr¹⁹² enhances the ability of RCAN1 to bind to Caln, resulting in the inhibition of Caln activity.

Dyrk1A-mediated Phosphorylation of RCAN1 Leads to an Extended Half-life

In a previous study, Lee et al. (38) reported that the NF-κB-inducing kinase-mediated phosphorylation of the RCAN1 C-terminal region, spanning amino acids 90–197, increased the half-life of RCAN1. We examined whether Dyrk1A-mediated phosphorylation affects the protein stability of RCAN1. HEK293T cells were transfected with a plasmid encoding Myc-tagged RCAN1 alone or together with either WT Dyrk1A or the kinase-inactive mutant K188R or transfected with a plasmid encoding the Myc-tagged RCAN1(S112A or T192A) mutant alone or together with Dyrk1A as indicated in the legend to Fig. 5C and supplemental Fig. S5. The transfected cells were incubated with cycloheximide, a protein synthesis inhibitor, for the indicated time, and the expression level of RCAN1 was measured by Western blot analysis using an anti-Myc antibody (Fig. 5C). Compared with the half-life of RCAN1 in cells expressing RCAN1 alone or together with Dyrk1A K188R, the half-life of RCAN1 expressed with Dyrk1A WT was significantly increased (Fig. 5, C and D).

The half-life of RCAN1(T192A) was slightly shortened compared with that of RCAN1 WT and was not affected by co-expression with Dyrk1A (Fig. 5, C and D, and supplemental Fig. S5A). The half-life of the S112A mutant of RCAN1 was similar to or a little shorter than that of RCAN1 WT (supplemental Fig. S5B). The half-life of the S112A mutant was extended by Dyrk1A co-expression (supplemental Fig. S5C). These results suggest that Dyrk1A-mediated phosphorylation at Thr¹⁹² extends the half-life of RCAN1, potentially contributing to the enhanced ability of RCAN1 to inhibit Caln.

Phospho-Thr¹⁹²-RCAN1 (pT192-RCAN1) Is Increased in Dyrk1A TG Mice

To further explore the link between Dyrk1A and RCAN1 through the phosphorylation of RCAN1 at Thr¹⁹² in vivo, the brains of transgenic mice overexpressing the human Dyrk1A protein (Dyrk1A TG mice) were analyzed. A rabbit polyclonal antibody to the phosphopeptide RPEYT¹⁹²(PO₄)PIHLS was generated and affinity-purified. To determine the specificity of the phosphospecific RCAN1 (pT192-RCAN1) antibody, Western blot analysis was performed with lysates of HEK293T cells that had been transiently transfected with RCAN1 WT or RCAN1(T192A) expression plasmids either alone or in the presence of plasmids encoding Dyrk1A. The phosphospecific antibody was able to detect the phospho-RCAN1 band only in the presence of both RCAN1 WT and Dyrk1A (Fig. 6A). The specificity of the pT192-RCAN1 antibody was further demonstrated by peptide competition experiments showing that the antibody signal in Western blots of mouse brain lysates was blocked by preincubation with the RCAN1-phosphopeptide but not with the RCAN1-nonphosphopeptide (Fig. 6B). When mouse brain lysates were treated with λ-protein phosphatase and Western blotting was performed with the pT192-RCAN1 antibody, the antibody signal disappeared, confirming that the phosphospecific RCAN1 antibody recognizes the phosphorylated forms of RCAN1 (Fig. 6C).

To determine whether Dyrk1A phosphorylates RCAN1 at Thr¹⁹² in cells, PC12 cells were transfected with Dyrk1A-specific siRNA. An ineffective Dyrk1A siRNA was used as a negative control. After 48–72 h of siRNA treatment, Dyrk1A siRNA reduced the endogenous Dyrk1A expression (Fig. 6D). Correspondingly, the level of phospho-Thr¹⁹²-RCAN1 was also reduced, whereas the expression of RCAN1 was similar for the siRNA-transfected cells. This result suggests that Dyrk1A is the kinase responsible for phosphorylating RCAN1 at Thr¹⁹².

To determine whether the expression of phospho-RCAN1 is increased in Dyrk1A TG mice, immunoblot analyses were performed with brain lysates prepared from the hippocampus of Dyrk1A TG mice and the control littermates (Fig. 6E). When compared with the control littermates, Dyrk1A TG mice show a 1.5–2-fold increase in Dyrk1A activity and expression in the hippocampus (10). The level of phospho-RCAN1 (normalized by α-tubulin) in the Dyrk1A TG mice was increased by 34 ± 13% (p < 0.05) in the hippocampus relative to that of the controls. After normalization to the levels of total RCAN1, the amount of phospho-RCAN1 in the Dyrk1A TG mice was increased by 41 ± 13% (p < 0.05) in the hippocampus, compared with that of the controls; in contrast, the amount of RCAN1 itself did not differ significantly between Dyrk1A TG and control littermate mice (Fig. 6F). These results demonstrate that the levels of phospho-RCAN1 are enhanced when Dyrk1A levels are increased, suggesting that overexpression of Dyrk1A may be functionally linked to RCAN1 activity in vivo through phosphorylation.

RCAN1 Partially Colocalizes with Dyrk1A

To determine whether endogenous Dyrk1A and RCAN1 co-localize in the cell, double immunocytochemistry was performed on NGF-differentiated PC12 cells and primary rat neuronal cells. Dyrk1A and RCAN1 proteins are primarily found in the cytoplasm, although Dyrk1A and RCAN1 staining are also present in the nucleus (Fig. 7, A and B). In the merged image, Dyrk1A was partially co-localized with RCAN1 in the cytosol, in the nucleus, and near the end of the neuron-like processes in NGF-treated PC12 cells (Fig. 7A) or synaptic boutons in the primary neuronal cells (Fig. 7B), suggesting that Dyrk1A potentially phosphorylates RCAN1 in these areas.

We then examined the expression of endogenous phospho-RCAN1. The specificity of the pT192-RCAN1 antibody was demonstrated by peptide competition experiments showing that the antibody signal of NGF-differentiated PC12 cells was completely blocked by preincubation with the RCAN1-phosphopeptide, but not with the RCAN1-nonphosphopeptide (Fig. 7C). Notably, the phospho-RCAN1 antibody labeled the neuritic terminals and nuclei of NGF-differentiated PC12 cells (Fig. 7, C and D) and synaptic boutons in the primary neuronal cells (Fig. 7E), consistent with a merged image of Dyrk1A and RCAN1 staining (Fig. 7, A and B). This finding was further supported by the co-localization of RCAN1 and pT192-RCAN1 (supplemental Fig. S6).