Four-and-a-half LIM Domains 1 (FHL1) Protein Interacts with the Rho Guanine Nucleotide Exchange Factor PLEKHG2/FLJ00018 and Regulates Cell Morphogenesis.

PLEKHG2/FLJ00018 is a Gßγ-dependent guanine nucleotide exchange factor for the small GTPases Rac and Cdc42 and has been shown to mediate the signaling pathways leading to actin cytoskeleton reorganization. Here we showed that the zinc finger domain-containing protein four-and-a-half LIM domains 1 (FHL1) acts as a novel interaction partner of PLEKHG2 by the yeast two-hybrid system. Among the isoforms of FHL1 (i.e. FHL1A, FHL1B, and FHL1C), FHL1A and FHL1B interacted with PLEKHG2. We found that there was an FHL1-binding region at amino acids 58-150 of PLEKHG2. The overexpression of FHL1A but not FHL1B enhanced the PLEKHG2-induced serum response element-dependent gene transcription. The co-expression of FHL1A and Gßγ synergistically enhanced the PLEKHG2-induced serum response element-dependent gene transcription. Increased transcription activity was decreased by FHL1A knock-out with the CRISPR/Cas9 system. Compared with PLEKHG2-expressing cells, the number and length of finger-like protrusions were increased in PLEKHG2-, Gßγ-, and FHL1A-expressing cells. Our results provide evidence that FHL1A interacts with PLEKHG2 and regulates cell morphological change through the activity of PLEKHG2.

Identification of a Rho family specific guanine nucleotide exchange factor, FLJ00018, as a novel actin-binding protein.

FLJ00018/PLEKHG2 is a guanine nucleotide exchange factor for the Rho family small GTPases. FLJ00018 is directly activated by heterotrimeric G protein Gßγ subunits. Using two-hybrid screening, we have identified non-muscle cytosolic actin as a binding partner of FLJ00018. We found that there were two actin-binding regions in FLJ00018 at the N-terminal region (150-283 amino acids) and at the C-terminal region (465-1386 amino acids). The overexpression of non-muscle cytosolic actin attenuated the FLJ00018-induced serum response element-dependent gene transcription. These results suggest that non-muscle cytosolic actin may be a negative regulator of FLJ00018 through its interaction with the Dbl homology domain.

Induction of peroxisomal lipid metabolism in mice fed a high-fat diet.

Peroxisomes catalyze a range of essential metabolic functions, mainly related to lipid metabolism. However, their roles in obesity have yet to be clarified. The aim of this study was to investigate the correlation between obesity and peroxisomal lipid metabolism, particularly very long-chain fatty acid (VLCFA) metabolism, gene expression of peroxisomal ß-oxidation enzymes, peroxisomal ATP-binding cassette (ABC) transporter adrenoleukodystrophy (ABCD1) gene and its related gene, ABCD2, the elongation of the VLCFA (ELOVL) gene family and the transcriptional factors involved in the regulation of these genes, including peroxisome proliferator-activated receptor α (PPARα) and sterol regulatory element-binding protein. These factors were analyzed in livers from mice fed a high-fat diet (HFD) or a regular diet (RD) for 20 weeks. Furthermore, the amounts of plasma saturated and unsaturated fatty acids, including VLCFAs, were measured. A HFD induced hepatic gene expression of not only hydroxysteroid 17-ß dehydrogenase 4 (HSD17b4) and sterol carrier protein 2 (SCP2) in peroxisomal ß-oxidation enzymes but also of ELOVL1, 2, 5 and 6, which are involved in the elongation of saturated and unsaturated VLCFAs. Furthermore, ABCD2 mRNA prominently increased in the HFD mice. The transcriptional regulator of these genes, PPARα, was also up-regulated in the HFD mice. VLCFA ratios including C24:0/C22:0, C25:0/C22:0 and C26:0/C22:0 are the most significant diagnostic markers of inherited peroxisomal diseases. These ratios were found to be low in the plasma of the HFD mice compared with the RD mice. The results suggest that HFD activates hepatic peroxisomal VLCFA metabolism, and may provide useful fundamental information to explain the role of peroxisomal function in obesity and lifestyle-related diseases.

Inhibition of Rho-associated coiled-coil containing protein kinase enhances the activation of epidermal growth factor receptor in pancreatic cancer cells.

BACKGROUND: Rho-associated coiled-coil containing protein kinase (Rho-kinase/ROCK) is involved in various cellular functions including cell proliferation, and is generally considered to be oncogenic, while some studies show that ROCK functions as a negative regulator of cancer progression. As a result, the precise role of ROCK remains controversial. We have previously reported that Rho-kinase/ROCK negatively regulates epidermal growth factor (EGF)-induced cell proliferation in SW480 colon cancer cells. In the present study, we investigated the role of ROCK in EGF receptor (EGFR) signaling in the pancreatic cancer cell lines, Panc1, KP3 and AsPc1. RESULTS: In these cells, Y27632, a specific ROCK inhibitor, enhanced EGF-induced BrdU incorporation. The blockade of EGF stimulation utilizing anti-EGFR-neutralizing antibodies suppressed Panc1 cell proliferation. EGF induced RhoA activity, as well as the phosphorylation of cofilin and myosin light chain (MLC), both targets of ROCK signaling, and Y27632 suppressed both of these processes, indicating that the phosphorylation of cofilin and MLC by EGF occurs through ROCK in Panc1 cells. EGF-induced phosphorylation of EGFR at tyrosine residues was augmented when the cells were pretreated with Y27632 or were subjected to gene silencing using ROCK-siRNA. We also obtained similar results using transforming growth factor-α. In addition, EGF-induced phosphorylation of p44/p42 mitogen-activated protein kinase and Akt were also enhanced by Y27632 or ROCK-siRNA. Moreover, an immunofluorescence microscope study revealed that pretreatment with Y27632 delayed EGF-induced internalization of EGFR. Taken together, these data indicate that ROCK functions to switch off EGFR signaling by promoting the internalization of the EGFR. CONCLUSIONS: While EGF first stimulates the activation of the EGFR and subsequently increases cancer cell proliferation, EGF concurrently induces the activation of ROCK, which then turns off the activated EGFR pathway via a negative feedback system.

Suppression by heat shock protein 20 of hepatocellular carcinoma cell proliferation via inhibition of the mitogen-activated protein kinases and AKT pathways.

Heat shock protein (HSP) 20, one of the low-molecular weight HSPs, is known to have versatile functions, such as vasorelaxation. However, its precise role in cancer proliferation remains to be elucidated. While HSP20 is constitutively expressed in various tissues including the liver, we have previously reported that HSP20 protein levels in human hepatocellular carcinoma (HCC) cells inversely correlate with the progression of HCC. In this study, we investigated the role of HSP20 in HCC proliferation. The activities of extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and AKT were negatively correlated with the HSP20 protein levels in human HCC tissues. Since HSP20 proteins were hardly detected in HCC-derived cell lines, the effects of HSP20 expression were evaluated using human HCC-derived HuH7 cells that were stably transfected with wild-type human HSP20 (HSP20 overexpressing cells). In HSP20 overexpressing cells, cell proliferation was retarded, and the activation of the mitogen-activated protein kinases (MAPKs) signaling pathways, including the ERK and JNK, and AKT pathways, as well as cyclin D1 accumulation induced by either transforming growth factor-α (TGFα) or hepatocyte growth factor, were significantly suppressed compared with the empty vector-transfected cells (control cells). Taken together, our findings strongly suggest that HSP20 suppresses the growth of HCC cells via the MAPKs and AKT signaling pathways, thus suggesting that the HSP20 could be a new therapeutic target for HCC.

UVC radiation induces downregulation of EGF receptor via phosphorylation at serine 1046/1047 in human pancreatic cancer cells.

Epidermal growth factor receptor (EGFR) is overexpressed in human pancreatic cancer and is one of the clinical targets in its treatment. In the present study we investigated the mechanism underlying ultraviolet C (UVC)-radiation-induced cell growth inhibition and downregulation of EGFR in human pancreatic cancer cells (Panc1 and KP3). The cell proliferation assay indicated that Panc1 and KP3 cells were more sensitive to UVC radiation, and their growth was significantly inhibited compared to cells of the normal human pancreatic epithelial cell line, PE. Although EGFR levels was extremely low in PE cells, EGFR were highly overexpressed in Panc1 and KP3 cells, and UVC radiation downregulated the expression of EGFR in a time-dependent manner and induced phosphorylation of EGFR at Ser1046/1047 (S1046/7) in Panc1 and KP3 cells. UVC radiation induced activation of p38 mitogen-activated protein kinase (MAPK), and EGFR phosphorylation at S1046/7 induced by UVC radiation was markedly attenuated by the inhibition of p38 MAPK. Moreover, fluorescence microscopy revealed that p38 MAPK activated by UVC radiation triggered EGFR internalization and that this was not correlated with c-Cbl, an ubiquitin ligase, which plays an important role in EGF-induced EGFR downregulation. Taken together, our results suggest that in pancreatic cancer cells UVC radiation induced desensitization of the cells to EGFR stimuli via phosphorylation of EGFR at S1046/7 by activation of p38 MAPK, independent of c-Cbl.

Plk1 is negatively regulated by RNF8.

RNF8 is a nuclear protein having an N-terminal forkhead-associated (FHA) domain and a C-terminal RING-finger (RF) domain. Depletion of RNF8 caused cell growth inhibition and cell cycle arrest at not only S but also G2/M phases. In addition, cell death was frequently observed in RNF8-depleted cells. Analyses of time-lapse microscopy revealed that the cells died in mitosis and interphase. To elucidate the RNF8 function in M phase, the Plk1 content in RNF8-depleted cells was examined. The amount of RNF8 decreased time-dependently, whereas Plk1 reciprocally increased by transfection of RNF8 siRNA. Protein contents of RNF8 and Plk1 among various cell lines were also compared. RNF8 in normal cell lines was much higher than that in many cancer cell lines. Conversely, Plk1 in normal cell lines was lower than in cancer cell lines. These results suggest that RNF8 is downregulated in many cancer cells and inversely correlated with Plk1.

Ultraviolet irradiation can induce evasion of colon cancer cells from stimulation of epidermal growth factor.

Receptor down-regulation is the most prominent regulatory system of EGF receptor (EGFR) signal attenuation and a critical target for therapy against colon cancer, which is highly dependent on the function of the EGFR. In this study, we investigated the effect of ultraviolet-C (UV-C) on down-regulation of EGFR in human colon cancer cells (SW480, HT29, and DLD-1). UV-C caused inhibition of cell survival and proliferation, concurrently inducing the decrease in cell surface EGFR and subsequently its degradation. UV-C, as well as EGFR kinase inhibitors, decreased the expression level of cyclin D1 and the phosphorylated level of retinoblastoma, indicating that EGFR down-regulation is correlated to cell cycle arrest. Although UV-C caused a marked phosphorylation of EGFR at Ser-1046/1047, UV-C also induced activation of p38 MAPK, a stress-inducible kinase believed to negatively regulate tumorigenesis, and the inhibition of p38 MAPK canceled EGFR phosphorylation at Ser-1046/1047, as well as subsequent internalization and degradation, suggesting that p38 MAPK mediates EGFR down-regulation by UV-C. In addition, phosphorylation of p38 MAPK induced by UV-C was mediated through transforming growth factor-ß-activated kinase-1. Moreover, pretreatment of the cells with UV-C suppressed EGF-induced phosphorylation of EGFR at tyrosine residues in addition to cell survival signal, Akt. Together, these results suggest that UV-C irradiation induces the removal of EGFRs from the cell surface that can protect colon cancer cells from oncogenic stimulation of EGF, resulting in cell cycle arrest. Hence, UV-C might be applied for clinical strategy against human colon cancers.

Effects of AZD1152, a selective Aurora B kinase inhibitor, on Burkitt's and Hodgkin's lymphomas.

We studied the effects of AZD1152, an Aurora B kinase inhibitor, on Burkitt's lymphoma (BL) and Hodgkin's lymphoma (HL) in human tissues and cell cultures and in a murine xenograft model of lymphoma. Aurora kinase A and B levels were assessed by RT-PCR and immunohistochemistry. They were aberrantly expressed in BL and HL cell lines, and in lymph nodes from patients with BL and HL. Next, activation of the Aurora B promoter was detected by reporter gene assays. The promoter activity of Aurora B kinase was high in BL cell lines and the Aurora B promoter contained a positive regulatory region between -74 and -104 from the transcription initiation site. AZD1152-hQPA had antiproliferative effects in the BL and HL cell lines studied; inhibited the phosphorylation of histone H3 and retinoblastoma proteins, and resulted in cells with > 4N DNA content. AZD1152-hQPA induced caspase-dependent apoptosis of some cell lines, demonstrated by loss of mitochondrial membrane potential, activation of caspase-9, followed by activation of caspase-3. This effect was accompanied by the inhibition of survivin expression. In vivo efficacy was determined in NOD/SCID/γc(null) mice implanted with the Ramos human BL cell line. AZD1152 had anti-tumour effects in this murine xenograft model. There preclinical data suggest that the inhibition of Aurora B kinase is a potentially useful therapeutic strategy in BL and HL.

Evolutionary features and intracellular behavior of the PRTB protein.

Human PRTB encodes a proline-rich protein of 168 amino acids (PRTB). We analyzed the evolutionary patterns of PRTB from various vertebrate species. Maximum likelihood analyses indicated that while mammalian PRTB has been very well conserved and underwent a significantly slower rate of evolution, only the branch leading to fish PRTB has undergone adaptive evolution. We generated several mutant PRTBs fused to the GFP variant, Venus, and found that the degradation of PRTB was enhanced by the transfection of an E2, UbcH5. Since mutation of the K153 site in PRTB was refractory to its degradation, proteolysis was suggested to be mediated by ubiquitination of K153. The subcellular localization of PRTB was also investigated, which showed that mutation of the K4 site completely prevented the nuclear localization of this protein. Together, these results suggest that Lys residues might play important roles in regulating the intracellular dynamics of the PRTB protein.

Rho-kinase inhibitor upregulates migration by altering focal adhesion formation via the Akt pathway in colon cancer cells.

Although Rho-kinase is reportedly implicated in carcinogenesis and the progression of human cancers, its precise mechanism has not been fully elucidated. We recently reported that Rho-kinase negatively regulates epidermal growth factor (EGF)-stimulated cancer progression in SW480 colon cancer cells. In the present study, we investigated the effect of Rho-kinase on the migration of SW480 colon cancer cells and the mechanism underlying the involvement of Rho-kinase. Interestingly, (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, 2HCl (Y27632), a specific inhibitor of Rho-kinase, dose-dependently enhanced cell migration. SW480 cells spontaneously release vascular endothelial growth factor (VEGF), however, Y27632 had little effect on its release. While Rho-kinase, which is generally phosphorylated in unstimulated cells, was clearly suppressed by Y27632, exogenous VEGF did not affect its phosphorylation. Immunofluorescence microscopy revealed that Y27632 caused a dramatic change in the localization of focal adhesion components, vinculin, phosphorylated caveolin-1 and tyrosine-phosphorylated proteins in SW480 cells. Furthermore, Akt inhibitor restored the loss of vinculin-stained focal adhesion formation induced by Y27632. We also observed similar effects for Y27632 on the migration and localization of focal adhesion components such as vinculin in another colon cancer cell line, HT29. Taken together, these results strongly suggest that Rho-kinase negatively regulates the migration of colon cancer cells by altering focal adhesion formation via the Akt pathway.

Aberrant promoter hypermethylation of the CHFR gene in oral squamous cell carcinomas.

Recent studies have shown that promoter hypermethylation of tumor suppressor genes is an important factor in carcinogenesis of several human organs. The purpose of this study was to examine the methylation status of CHFR, a novel cell cycle regulatory gene, in both primary oral cancer tumors and the adjacent normal mucosa, and to clarify the relation between the methylation status and expression of the CHFR-related chromosomal passenger protein Aurora-A. The methylation status of the CHFR gene was examined by the methylation-specific PCR (MSP) in 49 primary oral squamous cell carcinomas (OSCC) and 6 OSCC cell lines. In 13 cases, the adjacent normal oral mucosal tissues were also examined. Normal oral mucosa from 18 healthy volunteers was used as the control. The mRNA level of Aurora-A and CHFR in OSCC cell lines was investigated by real-time RT PCR and the protein expression of Aurora-A in certain tumor samples was confirmed by immunohistochemistry. Aberrant promoter methylation of the CHFR gene was detected in 34.7% (17 of 49) of OSCC cases. As for the 13 OSCC cases with paired cancerous and adjacent normal tissues, promoter hypermethylation of the CHFR gene was detected in 46.1% (6 of 13) of the cancerous tissues. In contrast, promoter hypermethylation of the CHFR gene was recognized in only 7.7% (1 of 13) of the surrounding normal mucosa. No hypermethylation of the CHFR gene was detected in healthy volunteers. Only one OSCC cell line shows hypermethylation of the CHFR gene with concurrently silenced mRNA expression, however, Aurora-A was expressed abundantly in all cell lines. Furthermore, there is no significant relationship between methylation status of the CHFR gene and Aurora-A protein expression in OSCC. Hypermethylation of the CHFR gene was detected in a certain part of OSCC cases whereas it had very low frequency in adjacent normal oral tissues. Although further study is needed, Aurora-A gene expression seems to be independent from methylation status of the CHFR gene in OSCC.

Human astrocytes and aortic endothelial cells actively convert the oxidized form of albumin to the reduced form: reduced albumin might participate in redox regulation of nerve and blood vessel systems.

Human serum albumin (HSA) is a mixture of mercaptalbumin (HMA, reduced form) and nonmercaptalbumin (HNA, oxidized form), i.e., a protein-thiol redox couple in the extracellular fluid (ECF), and it might have antioxidant properties. Forty-two patients with orthopedic disorders participated in this study and were divided into two groups according to their age (young and older groups). By using HPLC to separate HSA into HMA and HNA, we analyzed the percentages of HMA and HNA in serum and lumbar cerebrospinal fluid (CSF). We also examined the redox activity of cultured normal human astrocytes, aortic endothelial cells, and dermal fibroblasts for HSA-thiol. The mean HMA value from the serum of the older group was significantly lower than that of the young group, whereas that from CSF was not significantly different between the two groups; CSF albumin is almost completely in the reduced form, and no age-related differences were observed. Cultured astrocytes and aortic endothelial cells showed conversion of HNA to HMA, whereas dermal fibroblasts showed no such redox activity. From the results obtained from in-vivo and in-vitro studies, HMA is considered to participate in redox regulation in the ECF, for example in the CSF that surrounds the central nervous system (CNS), and in blood serum.

Sphingosine kinase isoforms regulate oxaliplatin sensitivity of human colon cancer cells through ceramide accumulation and Akt activation.

The relationship between sphingosine kinase (SPHK), cellular ceramide concentration and chemosensitivity was investigated in human colon cancer cell lines. Among nine colon cancer cell lines, SPHK1 and SPHK2 activity and protein expression was highest in RKO cells and lowest in HCT116 cells. A viability assay revealed that HCT116 cells were sensitive to the effects of oxaliplatin (l-OHP), whereas RKO cells were resistant to those of l-OHP. Treatment with 5microg/ml l-OHP induced a marked time-dependent increase in various ceramides (C16, C24, C24:1) in HCT116 cells but not in RKO cells, as indicated by liquid chromatography/mass spectrometry. The increase in ceramide and caspase activation induced by l-OHP in the sensitive HCT116 cells was abolished by pretreatment with a neutral sphingomyelinase inhibitor, suggesting that the ceramide formation was due to the activation of neutral, rather than acid, sphingomyelinase. In contrast, in l-OHP-resistant RKO cells, treatment with an SPHK inhibitor or SPHK1 and SPHK2 silencing by RNA interference suppressed cell viability and increased caspase activity and cellular ceramide formation after l-OHP treatment. The elevated ceramide formation induced by SPHK inhibition and l-OHP was inhibited by fumonisin B1 but not myriocin, suggesting that ceramide formation was through the salvage pathway. Endogenous phosphorylated Akt levels were much higher in the resistant RKO cells than in the sensitive HCT116 cells. Either SPHK1 or SPHK2 silencing in RKO cells decreased phosphorylated Akt levels and increased p53 and p21 protein levels as well as poly(ADP-ribose) polymerase cleavage in response to l-OHP treatment. These findings indicate that SPHK isoforms and neutral sphingomyelinase contribute to the regulation of chemosensitivity by controlling ceramide formation and the downstream Akt pathway in human colon cancer cells.

EWS-Fli1 up-regulates expression of the Aurora A and Aurora B kinases.

EWS-Fli1, a fusion gene resulting from the chromosomal translocation t(11;22, q24;q12), encodes a transcriptional activator, promotes cellular transformation, and is often found in Ewing sarcoma and primitive neuroectodermal tumor. The Aurora A and Aurora B kinases belong to a highly conserved family of serine/threonine protein kinases, are tightly regulated during the cell cycle, and are overexpressed in many carcinomas. Because the relationship between the Aurora A and/or Aurora B genes and the EWS-Fli1 fusion gene is unknown, we investigated the regulatory mechanism(s) by which Aurora kinases are controlled. Knockdown of EWS-Fli1 by small interfering RNA reduced mRNA levels not only of EWS-Fli1 but also of Aurora A and Aurora B. Luciferase assay using Aurora A and Aurora B promoters showed up-regulated activities compared with those of an empty vector. Experiments with deletion and point mutants showed positive regulatory Ets-binding sites located -84 and -71 bp upstream of the transcription initiation sites in Aurora A and Aurora B, respectively. Moreover, chromatin immunoprecipitation assay revealed that EWS-Fli1 gene products interact with both the Aurora A and Aurora B promoters. These results strongly suggest that the mitotic kinases Aurora A and Aurora B are regulated by EWS-Fli1 fusion protein in Ewing sarcoma cells.

An evolutionarily conserved leucine-rich repeat protein CLERC is a centrosomal protein required for spindle pole integrity.

The centrosome functions as the microtubule-organizing center (MTOC) and plays a vital role in organizing spindle poles during mitosis. The pair of centrioles, which are the core components of the centrosome, duplicate once per cell cycle, and this feature is essential for the establishment of spindle bipolarity. Here we describe the molecular characterization of a novel protein called CLERC (Centrosomal leucine-rich repeat and coiled-coil containing protein) which is a human ortholog of Chlamydomonas Vfl1 protein. CLERC is a protein of 1032 amino acids with a calculated molecular mass of 120 kDa and possesses leucine-rich repeat and coiled-coil domains. Database searches revealed that CLERC has homologs in a wide variety of eukaryotes and is evolutionarily conserved. Endogenous CLERC protein associated with the centrosomes throughout the cell cycle and accumulated during mitosis. RNAi-mediated depletion of CLERC blocked formation of normal mitotic spindles and led to multipolar spindles. Moreover, many of the spindle poles in CLERC depleted cells contained only one centriole, indicating that centrosomes split into fractions containing a single centriole. These data indicate that the major function of CLERC during mitosis is to maintain the structural integrity of centrosomes, thereby contributing to spindle bipolarity.

Depolarization-induced differentiation of PC12 cells is mediated by phospholipase D2 through the transcription factor CREB pathway.

The present study examined the role of phospholipase D2 (PLD2) in the regulation of depolarization-induced neurite outgrowth and the expression of growth-associated protein-43 (GAP-43) and synapsin I in rat pheochromocytoma (PC12) cells. Depolarization of PC12 cells with 50 mmol/L KCl increased neurite outgrowth and elevated mRNA and protein expression of GAP-43 and synapsin I. These increases were suppressed by inhibition of Ca2+-calmodulin-dependent protein kinase II (CaMKII), PLD, or mitogen-activated protein kinase kinase (MEK). Knockdown of PLD2 by small interfering RNA (siRNA) suppressed the depolarization-induced neurite outgrowth, and the increase in GAP-43 and synapsin I expression. Depolarization evoked a Ca2+ rise that activated various signaling enzymes and the cAMP response element-binding protein (CREB). Silencing CaMKIIdelta by siRNA blocked KCl-induced phosphorylation of proline-rich protein tyrosine kinase 2 (Pyk2), Src kinase, and extracellular signal-regulated kinase (ERK). Inhibition of Src or MEK abolished phosphorylation of ERK and CREB. Furthermore, phosphorylation of Pyk2, ERK, and CREB was suppressed by the PLD inhibitor, 1-butanol and transfection of PLD2 siRNA, whereas it was enhanced by over-expression of wild-type PLD2. Depolarization-induced PLD2 activation was suppressed by CaMKII and Src inhibitors, but not by MEK or protein kinase A inhibitors. These results suggest that the signaling pathway of depolarization-induced PLD2 activation was downstream of CaMKIIdelta and Src, and upstream of Pyk2(Y881) and ERK/CREB, but independent of the protein kinase A. This is the first demonstration that PLD2 activation is involved in GAP-43 and synapsin I expression during depolarization-induced neuronal differentiation in PC12 cells.

Phosphorylated retinoid X receptor alpha loses its heterodimeric activity with retinoic acid receptor beta.

A malfunction in retinoid X receptor (RXR) alpha due to phosphorylation is associated with the development of hepatocellular carcinoma. However, the precise mechanisms by which phosphorylated RXRalpha loses its physiological function remain unclear. In the present study we examined whether phosphorylation of RXRalpha affects its dimeric activity. Fluorescence resonance energy transfer studies and immunoprecipitation assays showed that the physical interaction between RXRalpha and retinoic acid receptor beta was impaired when 293T cells were transfected with phosphomimic mutant RXRalpha (T82D/S260D), whereas this interaction was activated at a level similar to wild-type RXRalpha-transfected cells when the cells were transfected with an unphosphorylated mutant RXRalpha (T82A/S260A). Treating the T82A/S260A-transfected cells with retinoid resulted in a significant increase in the transcriptional activities of the retinoic acid receptor responsive element and RXR responsive element promoters, whereas these transcriptional activities did not increase in the T82D/S260D-transfected cells. Transfection with T82A/S260A enhanced both the inhibition of cell growth and the induction of apoptosis caused by retinoid, although the T82D/S260D-transfected cells lost their responsiveness to retinoid. Moreover, transfection with T82A/S260A caused an inhibition of cell growth and a reduction of colony-forming ability in soft agar in HuH7 human hepatocellular carcinoma cells. These findings suggest that phosphorylation of RXRalpha abolishes its ability to form homodimers and heterodimers with RXR and retinoic acid receptor beta, thus resulting in the loss of cell growth control and the acceleration of cancer development. In conclusion, the inhibition of RXRalpha phosphorylation and the restoration of its original function as a master regulator of nuclear receptors might therefore be an effective strategy for controlling cancer cell growth.

Human Misato regulates mitochondrial distribution and morphology.

Misato of Drosophila melanogaster and Saccharomyces cerevisiae DML1 are conserved proteins having a homologous region with a part of the GTPase family that includes eukaryotic tubulin and prokaryotic FtsZ. We characterized human Misato sharing homology with Misato of D. melanogaster and S. cerevisiae DML1. Tissue distribution of Misato exhibited ubiquitous distribution. Subcellular localization of the protein studied using anti-Misato antibody suggested that it is localized to the mitochondria. Further experiments of fractionating mitochondria revealed that Misato was localized to the outer membrane. The transfection of Misato siRNA led to growth deficiencies compared with control siRNA transfected HeLa cells, and the Misato-depleted HeLa cells showed apoptotic nuclear fragmentation resulting in cell death. After silencing of Misato, the filamentous mitochondrial network disappeared and fragmented mitochondria were observed, indicating human Misato has a role in mitochondrial fusion. To examine the effects of overexpression, COS-7 cells were transfected with cDNA encoding EGFP-Misato. Its overexpression resulted in the formation of perinuclear aggregations of mitochondria in these cells. The Misato-overexpressing cells showed low viability and had no nuclei or a small and structurally unusual ones. These results indicated that human Misato has a role(s) in mitochondrial distribution and morphology and that its unregulated expression leads to cell death.

The RING finger protein RNF8 recruits UBC13 for lysine 63-based self polyubiquitylation.

The heterodimeric ubiquitin conjugating enzyme (E2) UBC13-UEV mediates polyubiquitylation through lysine 63 of ubiquitin (K63), rather than lysine 48 (K48). This modification does not target proteins for proteasome-dependent degradation. Searching for potential regulators of this variant polyubiquitylation we have identified four proteins, namely RNF8, KIA00675, KF1, and ZNRF2, that interact with UBC13 through their RING finger domains. These domains can recruit, in addition to UBC13, other E2s that mediate canonical (K48) polyubiquitylation. None of these RING finger proteins were known previously to recruit UBC13. For one of these proteins, RNF8, we show its activity as a ubiquitin ligase that elongates chains through either K48 or K63 of ubiquitin, and its nuclear co-localization with UBC13. Thus, our screening reveals new potential regulators of non-canonical polyubiquitylation.