Seon Sook Kim

Protein kinase A phosphorylates Down syndrome critical region 1 (RCAN1)

The Down syndrome critical region 1 (DSCR1) gene encodes a regulator of the calcineurin 1 (RCAN1) protein, and the elevated levels of RCAN1 are associated with Alzheimers disease (AD) and Down syndrome (DS). In this report, we found that protein kinase A (PKA) was able to phosphorylate RCAN1 in vitro and in vivo. In addition, we found that the phosphorylation of RCAN1 by PKA caused an increase of RCAN1 expression by increasing of the half-life of the protein. Consistently, the pharmacological inhibition of intracellular PKA using H-89 and the knockdown of the endogenous PKA catalytic subunit with siRNA decreased the expression of RCAN1. Furthermore, the phosphorylation of RCAN1 by PKA enhanced the inhibitory function of RCAN1 on calcineurin-mediated gene transcription. Our data provide the first evidence that PKA acts as an important regulatory component in the control of RCAN1 function through phosphorylation.

The Regulator of Calcineurin 1 (RCAN1/DSCR1) Activates the cAMP Response Element-binding Protein (CREB) Pathway

Background: The regulator of the calcineurin 1 (RCAN1) gene is located on human chromosome 21, the trisomy of which causes Down syndrome (DS). Results: RCAN1 increases the phosphorylation of transcription factor CREB. Conclusion: RCAN1 activates CREB signaling by inhibition of calcineurin activity. Significance: Identification of RCAN1 as a CREB signaling regulator could contribute to the understanding of DS pathogenesis. cAMP response element-binding protein (CREB) is one of the best known transcription factors in the development and function of the nervous system. In this report, we found that the regulator of calcineurin 1 (RCAN1), which is overexpressed in the brain of patients with Down syndrome, increased the phosphorylation of CREB and cAMP response element-mediated gene transcription in response to the activation of the intracellular cAMP pathway. Furthermore, we found that the increased activation of CREB signaling by RCAN1 depended on the ability of RCAN1 to inhibit calcineurin activity. Our data provide the first evidence that RCAN1 acts as an important regulatory component in the control of CREB signaling.

Carmustine induces ERK- and JNK-dependent cell death of neuronally-differentiated PC12 cells via generation of reactive oxygen species

Accumulation of reactive oxygen species (ROS) caused by the inhibition of glutathione reductase (GR) has been proposed as one of the mechanisms responsible for carmustine (1,3-bis(2-chloroethyl)-1-nitrosourea, BCNU)-induced cytotoxicity. Since mitogen-activated protein kinases (MAPKs) are known to mediate ROS-dependent cell death in multiple cell types, we examined whether redox-sensitive MAPK activation mediated the carmustine-induced cell death of neuronally differentiated PC12 cells. Carmustine induced a concentration- and time-dependent cell death, which was associated with increased caspase-3 activation, a reduction in GR activity accompanied by a concomitant decrease in reduced glutathione levels, and accumulation of ROS. Carmustine-induced caspase-3 activation and cell death were prevented by pretreatment with anti-oxidants or a reducing agent, indicating that carmustine-induced caspase-3 activation and cell death occur via redox-dependent processes. Carmustine induced phosphorylation of the MAPKs, such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. The activation of these kinases was inhibited by pretreatment with N-acetyl-L-cysteine (NAC). Although all the MAPKs were activated by carmustine, only the inhibitors of JNK and ERK prevented carmustine-induced cell death and caspase-3 activation. Our data suggest that carmustine-induced neurotoxicity is, at least in part, due to the activation of ROS-dependent JNK and ERK signaling.

Activation of adenylate cyclase by forskolin increases the protein stability of RCAN1 (DSCR1 or Adapt78)

MINT‐7262390: PKA (uniprotkb:P22694) phosphorylates (MI:0217) RCAN1 (uniprotkb:P53805) by protein kinase assay (MI:0424)

Activation of Rac1-dependent redox signaling is critically involved in staurosporine-induced neurite outgrowth in PC12 cells.

Abstract Staurosporine, a non-specific protein kinase inhibitor, has been shown to induce neurite outgrowth in PC12 cells, but the mechanism by which staurosporine induces neurite outgrowth is still obscure. In the present study, we investigated whether the activation of Rac1 was responsible for the neurite outgrowth triggered by staurosporine. Staurosporine caused rapid neurite outgrowth independent of the ERK signaling pathways. In contrast, neurite outgrowth in response to staurosporine was accompanied by activation of Rac1, and the Rac1 inhibitor NSC23766 attenuated the staurosporine-induced neurite outgrowth in a concentration-dependent manner. In addition, suppression of Rac1 activity by expression of the dominant negative mutant Rac1N17 also blocked the staurosporine-induced morphological differentiation of PC12 cells. Staurosporine caused an activation of NADPH oxidase and increased the production of reactive oxygen species (ROS), which was prevented by NSC23766 and diphenyleneiodonium (DPI), an NADPH oxidase inhibitor. Staurosporine-induced neurite outgrowth was attenuated by pretreatment with DPI and exogenous addition of sublethal concentration of H2O2 accelerated neurite outgrowth triggered by staurosporine. These results indicate that activation of Rac1, which leads to ROS generation, is required for neurite outgrowth induced by staurosporine in PC12 cells.

PKA regulates calcineurin function through the phosphorylation of RCAN1: Identification of a novel phosphorylation site

Calcineurin is a calcium/calmodulin-dependent phosphatase that has been implicated in T cell activation through the induction of nuclear factors of activated T cells (NFAT). We have previously suggested that endogenous regulator of calcineurin (RCAN1, also known as DSCR1) is targeted by protein kinase A (PKA) for the control of calcineurin activity. In the present study, we characterized the PKA-mediated phosphorylation site in RCAN1 by mass spectrometric analysis and revealed that PKA directly phosphorylated RCAN1 at the Ser 93. PKA-induced phosphorylation and the increase in the half-life of the RCAN1 protein were prevented by the substitution of Ser 93 with Ala (S93A). Furthermore, the PKA-mediated phosphorylation of RCAN1 at Ser 93 potentiated the inhibition of calcineurin-dependent pro-inflammatory cytokine gene expression by RCAN1. Our results suggest the presence of a novel phosphorylation site in RCAN1 and that its phosphorylation influences calcineurin-dependent inflammatory target gene expression.

Nedd4-2 ubiquitin ligase interacts with RCAN1 (DSCR1) for the proteasomal degradation

Regulator of Calcineurin 1 (RCAN1/DSCR1/Adapt78) gene is located in the Down syndrome (DS) region of chromosome 21, and critical for the phenotype of DS and Alzheimer disease (AD). In this report, we found that expression of Nedd4-2 E3 ubiquitin ligase decreased the protein level of RCAN1. Decrease of RCAN1 protein expression by Nedd4-2 was blocked by proteasome inhibitor MG132, indicating that this decrease was mediated by the ubiquitin-proteasome pathway. Furthermore, we found that the ability of Nedd4-2 to degrade RCAN1 depended on the direct binding with RCAN1. Consistently, Nedd4-2 enhanced the ubiquitination of RCAN1 protein. Our data provide the first evidence that Nedd4-2 acts as an important regulatory component in the control of RCAN1 protein stability.

Pituitary Adenylate Cyclase-activating Polypeptide (PACAP) Targets Down Syndrome Candidate Region 1 (DSCR1/RCAN1) to control Neuronal Differentiation

Background: Pituitary adenylate cyclase-activating peptide (PACAP) is a neurotrophic peptide involved in the development and function of the nervous system. Results: PACAP targets RCAN1, a Down syndrome-related gene, via activation of the PKA-CREB pathway. Conclusion: Proper expression of RCAN1 is necessary for neuronal differentiation. Significance: Identification of RCAN1 as a PACAP target gene could contribute to the understanding of mechanisms in neurodevelopment. Pituitary adenylate cyclase-activating peptide (PACAP) is a neurotrophic peptide involved in a wide range of nervous functions, including development, differentiation, and survival, and various aspects of learning and memory. Here we report that PACAP induces the expression of regulator of calcineurin 1 (RCAN1, also known as DSCR1), which is abnormally expressed in the brains of Down syndrome patients. Increased RCAN1 expression is accompanied by activation of the PKA-cAMP response element-binding protein pathways. EMSA and ChIP analyses demonstrate the presence of a functional cAMP response element in the RCAN1 promoter. Moreover, we show that PACAP-dependent neuronal differentiation is significantly disturbed by improper RCAN1 expression. Our data provide the first evidence of RCAN1, a Down syndrome-related gene, as a novel target for control of the neurotrophic function of PACAP.

Pyrrolidine dithiocarbamate (PDTC) inhibits inflammatory signaling via expression of regulator of calcineurin activity 1 (RCAN1): Anti-inflammatory mechanism of PDTC through RCAN1 induction

Pyrrolidine dithiocarbamate (PDTC) is a thiol compound that elicits anti-inflammatory effects by inhibiting NF-κB signaling. In this study, we report that regulator of calcineurin activity 1 (RCAN1) expression is induced by PDTC treatment and that increased RCAN1 expression is dependent on the generation of reactive oxygen species (ROS) and activation of p38 MAPK and JNK signaling. We also report that the ability of PDTC to induce RCAN1 is mediated by activator protein-1 (AP-1)-dependent gene transcription, and identified a functional AP-1 binding site in the RCAN1 promoter by producing mutations and conducting chromatin immunoprecipitation (ChIP) analyses. Moreover, we show that the PDTC-mediated inhibitory effect on NF-κB signaling is significantly perturbed by knocking out RCAN1. Our data provide the first evidence that PDTC prevents in vivo expression of pro-inflammatory cytokines by inducing RCAN1 expression.

Hydrogen peroxide-induced MAPK activation causes the increase of RCAN1 (DSCR1) protein expression

The Down syndrome critical region 1 (DSCR1) gene encodes a regulator of calcineurin 1 (RCAN1), which is overexpressed in the patients with Down syndrome. In this study, we found that the protein expression of RCAN1 was increased by the hydrogen peroxide (H2O2). The increase of RCAN1 expression by H2O2 was blocked by the treatment with anti-oxidants and inhibitors of mitogen-activated protein kinases (MAPKs), indicating that this increase was caused by the generation of reactive oxygen species and activation of MAPKs. In addition, we found that the phosphorylation of RCAN1 by H2O2 caused an increase of RCAN1 expression by increasing of the half-life of the protein. Our results provide the evidence that H2O2 acts as an important regulator in the control of RCAN1 protein expression through phosphorylation.