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N., Zhang Z., Williams R. during development and in adults. RCAN1?/? mice show impairments in spatial learning and memory (21), and knockdown or overexpression of the RCAN1 homolog leads to severe learning defects (22). RCAN1 has therefore been proposed as a candidate protein responsible for mental retardation in DS. RCAN1 has also been implicated in oxidative stress and exocytosis (23, 24). Increased expression of certain genes on chromosome 21, alone or in cooperation, is thought to be responsible for the DS phenotype, including mental retardation, congenital heart defects, gastrointestinal malformations, immune and endocrine system defects, and early onset of dementia of the Alzheimer type. Increased expression of RCAN1 and Dyrk1A in individuals with DS destabilizes the NFAT genetic circuit (25). Increased Dyrk1A or RCAN1 expression has been reported in both human DS and AD brains (12, 26C29). The present study investigated the possible direct link between Dyrk1A and RCAN1 and examined the effect of Dyrk1A-mediated phosphorylation on RCAN1 activity Kobe0065 in relation to the pathological traits of DS. EXPERIMENTAL PROCEDURES 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 BL21(DE3) strain RIL IFNB1 (Stratagene) and purified using Ni2+-NTA or glutathione-Sepharose 4B resin. Anti–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 Kobe0065 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 siRNA (5-GUACAUCGUCAAGCUCAGGU) was used as an Kobe0065 ineffective siRNA control. After 48C72 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 g) for 1 h at 37 C in kinase buffer (20 mm MOPS, pH 7.0, 10 mm MgCl2, 1 mm DTT, and 20 m sodium orthovanadate) containing 25 m cold ATP and 5 Ci of [-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 GSK3 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 30C. After the nonradioactive 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 GSK3 (Millipore) in a 25-l final volume of the kinase buffer in the presence of 25 m ATP and 10 Ci of [-32P]ATP.