Kyong-Tai Kim

G protein-coupled receptor signalling and cross-talk: Achieving rapidity and specificity

Activation of a given type of G protein-coupled receptor (GPCR) triggers a limited set of signalling events in a very rapid and specific manner. The classical paradigm of GPCR signalling was rather linear and sequential. Emerging evidence, however, has revealed that this is only a part of the complex signalling mediated by GPCR. Propagation of GPCR signalling involves cross-regulation of many but specific pathways, including cross-talks between different GPCRs as well as with other signalling pathways. Moreover, it is increasingly apparent that GPCRs can activate both heterotrimeric G protein-dependent and G protein-independent signalling pathways. In this review, we discuss how the signallings initiated by GPCRs achieve rapidity as well as specificity, and how the GPCRs can cross-regulate other specific signalling pathways at the same time. New concepts regarding GPCR signalling have been arising to address this issue, which include multiprotein signalling complex and signalling compartment in microdomain concepts that enable close colocalization or even contact among the proteins engaged in the specific signal transduction. The final outcome of a stimulation of GPCR will thus be the sum of its own specific set of intracellular signalling pathways it regulates.

Heterogeneous Nuclear Ribonucleoprotein C Modulates Translation of c-myc mRNA in a Cell Cycle Phase-Dependent Manner

ABSTRACT The c-myc proto-oncogene plays a key role in the proliferation, differentiation, apoptosis, and regulation of the cell cycle. Recently, it was demonstrated that the 5′ nontranslated region (5′ NTR) of human c-myc mRNA contains an internal ribosomal entry site (IRES). In this study, we investigated cellular proteins interacting with the IRES element of c-myc mRNA. Heterogeneous nuclear ribonucleoprotein C (hnRNP C) was identified as a cellular protein that interacts specifically with a heptameric U sequence in the c-myc IRES located between two alternative translation initiation codons CUG and AUG. Moreover, the addition of hnRNP C1 in an in vitro translation system enhanced translation of c-myc mRNA. Interestingly, hnRNP C was partially relocalized from the nucleus, where most of the hnRNP C resides at interphase, to the cytoplasm at the G2/M phase of the cell cycle. Coincidently, translation mediated through the c-myc IRES was increased at the G2/M phase when cap-dependent translation was partially inhibited. On the other hand, a mutant c-myc mRNA lacking the hnRNP C-binding site, showed a decreased level of translation at the G2/M phase compared to that of the wild-type message. Taken together, these findings suggest that hnRNP C, via IRES binding, modulates translation of c-myc mRNA in a cell cycle phase-dependent manner.

Phospholipase C-δ1 Is Activated by Capacitative Calcium Entry That Follows Phospholipase C-β Activation upon Bradykinin Stimulation

To characterize the regulatory mechanism of phospholipase C-δ1 (PLC-δ1) in the bradykinin (BK) receptor-mediated signaling pathway, we used a clone of PC12 cells, which stably overexpress PLC-δ1 (PC12-D1). Stimulation with BK induced a significantly higher Ca2+ elevation and inositol 1,4,5-trisphosphate (IP3) production with a much lower half-maximal effective concentration (EC50) of BK in PC12-D1 cells than in wild type (PC12-W) or vector-transfected (PC12-V) cells. However, BK-induced intracellular Ca2+ release and IP3 generation was similar between PC12-V and PC12-D1 cells in the absence of extracellular Ca2+, suggesting that the availability of extracellular Ca2+ is essential to the activation of PLC-δ1. When PC12-D1 cells were treated with agents that induce Ca2+ influx, more IP3 was produced, suggesting that the Ca2+ entry induces IP3production in PC12-D1 cells. Furthermore, the additional IP3 production after BK-induced capacitative calcium entry was detected in PC12-D1 cells, suggesting that PLC-δ1 is mainly activated by capacitative calcium entry. When cells were stimulated with BK in the presence of extracellular Ca2+, [3H]norepinephrine secretion was much greater from PC12-D1 cells than from PC12-V cells. Our results suggest that PLC-δ1 is activated by capacitative calcium entry following the activation of PLC-β, additively inducing IP3 production and Ca2+ rise in BK-stimulated PC12 cells.

P2X7 Nucleotide Receptor Mediation of Membrane Pore Formation and Superoxide Generation in Human Promyelocytes and Neutrophils

The P2X7 receptor, which induces cation channel opening imparting significant permeability to Ca2+ and pore formation with changes in the plasma membrane potential, has been known to be rather restrictedly expressed in cells of the macrophage lineage including dendrites, mature macrophages, and microglial cells. However, we show here that the P2X7 receptor is also expressed in cells of granulocytic lineage such as HL-60 promyelocytes, granulocytic differentiated cells, and neutrophils. Exposure of these cells to 2′,3′-O-(4-benzoyl)benzoyl-ATP (BzATP) triggered intracellular Ca2+ rise through the mediation of phospholipase C-independent and suramin-sensitive pathways. BzATP also induced depolarization of the plasma membrane in the absence of extracellular Ca2+, whereas it hyperpolarized the cells in the presence of external Ca2+, probably in part through the activation of Ca2+-activated K+ channels. However, the hyperpolarization phenomenon was markedly attenuated in differentiated HL-60 cells and neutrophils. RT-PCR and Northern blot analysis revealed the presence of P2X7 receptors on both HL-60 and neutrophil-like cells. This was further confirmed by pore formation through which the uptake of Lucifer yellow and YO-PRO1 occurred on BzATP treatment. BzATP stimulated in a concentration-dependent manner the production of superoxide in differentiated HL-60 cells via a pathway partially dependent on extracellular Ca2+. Moreover, in human neutrophils, BzATP was a more effective inducer of superoxide generation than PMA. Taken together, this is a first demonstration of the expression of P2X7 receptors on neutrophils, which shows that the receptor is functionally involved in the defense mechanism by activation of the respiratory burst pathway.

Protein kinase Cδ-mediated proteasomal degradation of MAP kinase phosphatase-1 contributes to glutamate-induced neuronal cell death

Mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) is a dual-specificity phosphatase that is involved in the regulation of cell survival, differentiation and apoptosis through inactivating MAPKs by dephosphorylation. Here, we provide evidence for a role of MKP-1 in the glutamate-induced cell death of HT22 hippocampal cells and primary mouse cortical neurons. We suggest that, during glutamate-induced oxidative stress, protein kinase C (PKC) δ becomes activated and induces sustained activation of extracellular signal-regulated kinase 1/2 (ERK1/2) through a mechanism that involves degradation of MKP-1. Glutamate-induced activation of ERK1/2 was blocked by inhibition of PKCδ, confirming that ERK1/2 is regulated by PKCδ. Prolonged exposure to glutamate caused reduction in the protein level of MKP-1, which correlated with the sustained activation of ERK1/2. Furthermore, knockdown of endogenous MKP-1 by small interfering (si)RNA resulted in pronounced enhancement of ERK1/2 phosphorylation accompanied by increased cytotoxicity under glutamate exposure. In glutamate-treated cells, MKP-1 was polyubiquitylated and proteasome inhibitors markedly blocked the degradation of MKP-1. Moreover, inhibition of glutamate-induced PKCδ activation suppressed the downregulation and ubiquitylation of MKP-1. Taken together, these results demonstrate that activation of PKCδ triggers degradation of MKP-1 through the ubiquitin-proteasome pathway, thereby contributing to persistent activation of ERK1/2 under glutamate-induced oxidative toxicity.

O‐GlcNAcase is essential for embryonic development and maintenance of genomic stability

Dysregulation of O‐GlcNAc modification catalyzed by O‐GlcNAc transferase (OGT) and O‐GlcNAcase (OGA) contributes to the etiology of chronic diseases of aging, including cancer, cardiovascular disease, type 2 diabetes, and Alzheimer’s disease. Here we found that natural aging in wild‐type mice was marked by a decrease in OGA and OGT protein levels and an increase in O‐GlcNAcylation in various tissues. Genetic disruption of OGA resulted in constitutively elevated O‐GlcNAcylation in embryos and led to neonatal lethality with developmental delay. Importantly, we observed that serum‐stimulated cell cycle entry induced increased O‐GlcNAcylation and decreased its level after release from G2/M arrest, indicating that O‐GlcNAc cycling by OGT and OGA is required for precise cell cycle control. Constitutively, elevated O‐GlcNAcylation by OGA disruption impaired cell proliferation and resulted in mitotic defects with downregulation of mitotic regulators. OGA loss led to mitotic defects including cytokinesis failure and binucleation, increased lagging chromosomes, and micronuclei formation. These findings suggest an important role for O‐GlcNAc cycling by OGA in embryonic development and the regulation of the maintenance of genomic stability linked to the aging process.

Mouse period 2 mRNA circadian oscillation is modulated by PTB–mediated rhythmic mRNA degradation

Circadian mRNA oscillations are the main feature of core clock genes. Among them, period 2 is a key component in negative-feedback regulation, showing robust diurnal oscillations. Moreover, period 2 has been found to have a physiological role in the cell cycle or the tumor suppression. The present study reports that 3′-untranslated region (UTR)-dependent mRNA decay is involved in the regulation of circadian oscillation of period 2 mRNA. Within the mper2 3′UTR, both the CU-rich region and polypyrimidine tract-binding protein (PTB) are more responsible for mRNA stability and degradation kinetics than are other factors. Depletion of PTB with RNAi results in mper2 mRNA stabilization. During the circadian oscillations of mper2, cytoplasmic PTB showed a reciprocal expression profile compared with mper2 mRNA and its peak amplitude was increased when PTB was depleted. This report on the regulation of mper2 proposes that post-transcriptional mRNA decay mediated by PTB is a fine-tuned regulatory mechanism that includes dampening-down effects during circadian mRNA oscillations.

Mitotic Histone H3 Phosphorylation by Vaccinia-Related Kinase 1 in Mammalian Cells†

ABSTRACT Mitotic chromatin condensation is essential for cell division in eukaryotes. Posttranslational modification of the N-terminal tail of histone proteins, particularly by phosphorylation by mitotic histone kinases, may facilitate this process. In mammals, aurora B is believed to be the mitotic histone H3 Ser10 kinase; however, it is not sufficient to phosphorylate H3 Ser10 with aurora B alone. We show that histone H3 is phosphorylated by vaccinia-related kinase 1 (VRK1). Direct phosphorylation of Thr3 and Ser10 in H3 by VRK1 both in vitro and in vivo was observed. Loss of VRK1 activity was associated with a marked decrease in H3 phosphorylation during mitosis. Phosphorylation of Ser10 by VRK1 is similar to that by aurora B. Moreover, expression and chromatin localization of VRK1 depended on the cell cycle phase. Overexpression of VRK1 resulted in a dramatic condensation of nuclei. Our findings collectively support a role of VRK1 as a novel mitotic histone H3 kinase in mammals.

Stabilization and activation of p53 induced by Cdk5 contributes to neuronal cell death

The p53 tumor suppressor protein is a key regulator of cellular functions including responses to numerous stress signals, and triggers apoptosis in many cell types, including neurons. The major mechanisms known to regulate p53 stabilization and activation include phosphorylation and ubiquitin ligase-mediated proteasomal degradation. Cyclin-dependent kinase 5 (Cdk5), a proline-directed serine/threonine kinase, is most active in the central nervous system and plays a variety of roles in neuronal degeneration. Here, we demonstrate for the first time that Cdk5 interacts with p53 and increases its stability through posttranslational regulation, leading to accumulation of p53, particularly in the nucleus. We show that Cdk5 phosphorylates p53 on Ser15, Ser33 and Ser46 in vitro, and that increased Cdk5 activity in the nucleus mediates these phosphorylation events in response to genotoxic and oxidative stresses. Cdk5 mediates disruption of the interaction between p53 and Hdm2 (also known as Mdm2), and prevents Hdm2-induced p53 ubiquitylation and downregulation. Cdk5 additionally enhances phosphorylation-dependent binding of the p300 coactivator, inducing acetylation of p53. Cdk5-stabilized p53 protein is transcriptionally active, resulting in the induction of pro-apoptotic genes and subsequent mitochondria-mediated apoptosis in response to genotoxic or oxidative stress. Collectively, these novel findings help define the mechanisms underlying neuronal apoptosis occurring as a result of Cdk5-mediated p53 stabilization and transcriptional activation.

Induction of cyclin-dependent kinase 5 and its activator p35 through the extracellular-signal-regulated kinase and protein kinase A pathways during retinoic-acid mediated neuronal differentiation in human neuroblastoma SK-N-BE(2)C cells

Cyclin‐dependent kinase 5 (Cdk5), a neuronal Cdc2‐like kinase, exhibits a variety of functions in neuronal differentiation and neurocytoskeleton dynamics, as well as neuronal degeneration. However, its role and induction mechanisms in retinoic acid (RA)‐induced neuronal differentiation have not been well understood. In this study we newly found that RA treatment of SK‐N‐BE(2)C, human neuroblastoma cells, increased the expression of Cdk5 and its neuron specific activator p35 through the extracellular‐signal‐regulated kinase1/2 (ERK1/2) and cAMP‐dependent protein kinase A (PKA) pathway. Inhibition of Cdk5 activity either by an inhibitor, roscovitine, or by transfection with a dominant negative form of Cdk5 caused a dramatic decrease in RA‐induced differentiation, suggesting the requirement of Cdk5 kinase activity for the RA‐induced neurite outgrowth. Furthermore, Cdk5 and p35 expression was decreased by ERK1/2 inhibition with PD98059 and increased by overexpression of a constitutive active mitogen‐activated protein kinase kinase 1 (MEK1) mutant, suggesting the critical role of ERK1/2 in the induction of Cdk5 and p35. In addition, a transcription factor early growth response 1 (Egr‐1) was induced by RA through the ERK1/2 pathway, suggesting its possible involvement in the p35 induction. RA treatment also induced c‐fos mediated AP‐1 binding, and cAMP‐responsive element binding protein (CREB) mediated CRE binding via ERK1/2 and PKA pathway, respectively, in the Cdk5 promoter region, resulting in the induction of Cdk5. Our results suggest that ERK1/2 and PKA‐induced regulation of Cdk5 activity possibly through Egr‐1, c‐fos, and CREB plays a critical role in the RA‐induced neuronal differentiation.