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Biomedical subjects

Kiyoko Fukami

Publications and source records attributed to Kiyoko Fukami.

At least 19 recordsLinked to original sources
PubMed2006

Disruption of phospholipase Cdelta4 gene modulates the liver regeneration in cooperation with nuclear protein kinase C.

Phospholipase Cdelta4 (PLC delta4) gene has been cloned from the cDNA library of regenerating rat liver. Using PLC delta4 gene-disrupted mice (PLC delta4(-/-)), we studied a role of PLC delta4 during liver regeneration after partial hepatectomy (PH). In PLC delta4(-/-), liver regeneration occurred in an apparently normal way. However, BrdU-indices indicated that PLC delta4 gene disruption delayed the onset of DNA synthesis by 2 h. Noticeably, the BrdU-indices in PLC delta4(+/+) remained rather constant throughout S phase, 25-35%, whereas in PLC delta4(-/-), it fluctuated drastically from 25% at 34 h to 65% at late S, 42 h after PH. This fact showed that PLC delta4 gene disruption caused a higher synchronization of cell proliferation. The mRNA for PLC delta4 in PLC delta4(+/+) appeared at late G1, and the expression continued throughout S phase. PLC activity increased transiently in chromatin at the late G1 and S phases in only PLC delta4(+/+), but not in PLC delta4(-/-). The specific increases in PLC activity well correlated with the transient increases of protein kinase C (PKC) alpha in chromatin of PLC delta4(+/+). PKC epsilon also increased transiently in chromatin from PLC delta4(+/+) at late S. It is concluded that PLC delta4 regulates the liver regeneration in cooperation with nuclear PKC alpha and epsilon.

Animals↗
PubMed2006

Modulation of GABA(A) receptor phosphorylation and membrane trafficking by phospholipase C-related inactive protein/protein phosphatase 1 and 2A signaling complex underlying brain-derived neurotrophic factor-dependent regulation of GABAergic inhibition.

Brain-derived neurotrophic factor (BDNF) modulates several distinct aspects of synaptic transmission, including GABAergic transmission. Exposure to BDNF alters properties of GABA(A) receptors and induces changes in the expression level at the cell surface. Although phospholipase C-related inactive protein-1 (PRIP-1) plays an important role in GABA(A) receptor trafficking and function, its role in BDNF-dependent modulation of these receptors, together with the role of PRIP-2, was investigated using neurons cultured from PRIP double knock-out mice. The BDNF-dependent inhibition of whole cell GABA-evoked currents observed in wild type neurons was not detected in neurons cultured from knock-out mice. Instead, a gradual increase in GABA-evoked currents in these neurons correlated with a gradual increase in phosphorylation of GABA(A) receptor beta3 subunit in response to BDNF. To characterize the specific role(s) that PRIP plays as components of underlying molecular machinery, we examined the recruitment of protein phosphatase(s) to GABA(A) receptors. We demonstrate that PRIP associates with phosphatases as well as with beta subunits. PRIP was found to colocalize with GABA(A) receptor clusters in cultured neurons and with recombinant GABA(A) receptors when co-expressed in HEK293 cells. Importantly, a peptide mimicking a domain of PRIP involved in binding to beta subunits disrupted the co-localization of these proteins in HEK293 cells and potently inhibited the BDNF-mediated attenuation of GABA(A) receptor currents in wild type neurons. Together, the results suggest that PRIP plays an important role in BDNF-dependent regulation of GABA(A) receptors by mediating the specific association between beta subunits of these receptors with protein phosphatases.

Animals↗
PubMed2006

PIASy-mediated repression of the Ets-1 is independent of its sumoylation.

The transcription factor Ets-1 is involved in many physiological processes, including angiogenesis, hematopoietic development, and tumor progression, and its activity can be regulated by interactions with other proteins and post-translational modifications, such as phosphorylation. Here, we show that Ets-1 is a target for SUMO modification both in vivo and in vitro. Mutational analysis reveals that sumoylation of Ets-1 occurs at two lysine residues at amino acid positions 15 and 227, which lie within previously identified synergy control motifs. Replacement of sumoylation site lysines with arginine or overexpression of SENP1, a desumoylation enzyme, enhances the transactivation ability of Ets-1. Furthermore, we identify PIASy as a novel interaction partner and a specific SUMO-E3 ligase of Ets-1. PIASy represses the Ets-1-dependent transcription, and its repression is independent of the sumoylation status of Ets-1, but it is dependent on the sumoylation of other factors.

Amino Acid Sequence↗
PubMed2006

A novel GTPase, CRAG, mediates promyelocytic leukemia protein-associated nuclear body formation and degradation of expanded polyglutamine protein.

Polyglutamine diseases are inherited neurodegenerative diseases caused by the expanded polyglutamine proteins (polyQs). We have identified a novel guanosine triphosphatase (GTPase) named CRAG that contains a nuclear localization signal (NLS) sequence and forms nuclear inclusions in response to stress. After ultraviolet irradiation, CRAG interacted with and induced an enlarged ring-like structure of promyelocytic leukemia protein (PML) body in a GTPase-dependent manner. Reactive oxygen species (ROS) generated by polyQ accumulation triggered the association of CRAG with polyQ and the nuclear translocation of the CRAG-polyQ complex. Furthermore, CRAG promoted the degradation of polyQ at PML/CRAG bodies through the ubiquitin-proteasome pathway. CRAG knockdown by small interfering RNA in neuronal cells consistently blocked the nuclear translocation of polyQ and enhanced polyQ-mediated cell death. We propose that CRAG is a modulator of PML function and dynamics in ROS signaling and is protectively involved in the pathogenesis of polyglutamine diseases.

Amino Acid Sequence↗
PubMed2005

Functional, biochemical, and chromatographic characterization of the complete [Ca2+]i oscillation-inducing activity of porcine sperm.

A cytosolic sperm protein(s), referred to as sperm factor (SF), is delivered into eggs by the sperm during mammalian fertilization to induce repetitive increases in the intracellular concentration of free Ca2+ ([Ca2+]i) that are referred to as [Ca2+]i oscillations. [Ca2+]i oscillations are essential for egg activation and early embryonic development. Recent evidence shows that the novel sperm-specific phospholipase C (PLC), PLCzeta, may be the long sought after [Ca2+]i oscillation-inducing SF. Here, we demonstrate the complete extraction of SF from porcine sperm and show that regardless of the method of extraction a single molecule/complex appears to be responsible for the [Ca2+]i oscillation-inducing activity of these extracts. Consistent with this notion, all sperm fractions that induced [Ca2+]i oscillations, including FPLC-purified fractions, exhibited high in vitro PLC activity at basal Ca2+ levels (0.1-5 microM), a hallmark of PLCzeta. Notably, we detected immunoreactive 72-kDa PLCzeta in an inactive fraction, and several fractions capable of inducing oscillations were devoid of 72-kDa PLCzeta. Nonetheless, in the latter fractions, proteolytic fragments, presumably corresponding to cleaved forms of PLCzeta, were detected by immunoblotting. Therefore, our findings corroborate the hypothesis that a sperm-specific PLC is the main component of the [Ca2+]i oscillation-inducing activity of sperm but provide evidence that the presence of 72-kDa PLCzeta does not precisely correspond with the Ca2+ releasing activity of porcine sperm fractions.

Animals↗
PubMed2005

A novel phospholipase C, PLC(eta)2, is a neuron-specific isozyme.

Twelve phospholipase C (PLC) isozymes have been cloned so far, and they are divided into six classes, beta-, gamma-, delta-, epsilon-, zeta-, and eta-type, on the basis of structure and activation mechanisms. Here we report the identification of a novel PLC isozyme, PLC(eta)2. PLC(eta)2 is composed of conserved domains including pleckstrin homology, EF-hand, X and Y catalytic, and C2 domains and the isozyme-specific C-terminal region. PLC(eta)2 consists of 1164 amino acids with a molecular mass of 125 kDa. The PLC activity of PLC(eta)2 was more sensitive to calcium concentration than the PLC activity of the PLCdelta-type enzyme, which is thought to be the most calcium-sensitive PLC. Immunofluorescence analysis showed that PLC(eta)2 was localized predominantly to the plasma membrane at resting state via the pleckstrin homology domain. This observation was supported by Western blot analysis of cytosol and membrane fractions. In addition, expression of PLC(eta)2 was detected after birth and showed a restricted distribution in the brain; it was particularly abundant in the hippocampus, cerebral cortex, and olfactory bulb. The pattern was similar to that of the neuronal marker microtubule-associated protein 2 by Western blot. Furthermore, in situ hybridization showed positive signals for PLC(eta)2 in pyramidal cells of the hippocampus. Finally, we found that PLC(eta)2 was expressed abundantly in neuron-containing primary culture but not in astrocyte-enriched culture. These results indicate that PLC(eta)2 is a neuron-specific isozyme that may be important for the formation and/or maintenance of the neuronal network in the postnatal brain.

Amino Acid Sequence↗
PubMed2005

The role of EF-hand domains and C2 domain in regulation of enzymatic activity of phospholipase Czeta.

Sperm-specific phospholipase C-zeta (PLCzeta) induces Ca2+ oscillations and egg activation when injected into mouse eggs. PLCzeta has such a high Ca2+ sensitivity of PLC activity that the enzyme can be active in resting cells at approximately 100 nM Ca2+, suitable for a putative sperm factor to be introduced into the egg at fertilization (Kouchi, Z., Fukami, K., Shikano, T., Oda, S., Nakamura, Y., Takenawa, T., and Miyazaki, S. (2004) J. Biol. Chem. 279, 10408-10412). In the present structure-function analysis, deletion of EF1 and EF2 of the N-terminal four EF-hand domains caused marked reduction of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-hydrolyzing activity in vitro and loss of Ca2+ oscillation-inducing activity in mouse eggs after injection of RNA encoding the mutant. However, deletion of EF1 and EF2 or mutation of EF1 or EF2 at the x and z positions of the putative Ca2+-binding loop little affected the Ca2+ sensitivity of the PLC activity, whereas deletion of EF1 to EF3 caused 12-fold elevation of the EC50 of Ca2+ concentration. Thus, EF1 and EF2 are important for the PLCzeta activity, and EF3 is responsible for its high Ca2+ sensitivity. Deletion of four EF-hand domains or the C-terminal C2 domain caused complete loss of PLC activity, indicating that both regions are prerequisites for PLCzeta activity. Screening of interactions between the C2 domain and phosphoinositides revealed that C2 has substantial affinity to PI(3)P and, to the lesser extent, to PI(5)P but not to PI(4,5)P2 or acidic phospholipids. PI(3)P and PI(5)P reduced PLCzeta activity in vitro, suggesting that the interaction could play a role for negative regulation of PLCzeta.

Animals↗
PubMed2005

A novel UbcH10-binding protein facilitates the ubiquitinylation of cyclin B in vitro.

UbcH10 is known to act as a ubiquitin-conjugating enzyme (E2) for anaphase-promoting complex/cyclosome. Since some E2s support different ubiquitin ligases (E3), it is possible that UbcH10 interacts with other proteins. We cloned a novel protein named H10BH by using a yeast two-hybrid screening method with UbcH10 as bait. The carboxyl terminus of H10BH showed a weak homology to the HECT (homologous to E6-AP carboxyl terminus) domain, which is conserved in one of the families of E3. H10BH bound UbcH10, and the amino acid sequence between 235 and 257 was necessary for this binding. H10BH showed a self-ubiquitinylation activity in a HECT-like sequence-dependent manner. The carboxyl terminal half (amino acids 188-389) showed stronger activity than the full-length H10BH. Furthermore, the carboxyl terminal half of H10BH was able to bind cyclin B and ubiquitinylate cyclin B in vitro. These results suggest that H10BH functions as an E3 using UbcH10 for its E2.

Amino Acid Sequence↗
PubMed2005

X-linked inhibitor of apoptosis functions as ubiquitin ligase toward mature caspase-9 and cytosolic Smac/DIABLO.

Members of the IAP (inhibitor of apoptosis) family function as anti-apoptotic proteins by binding directly to caspase-3, -7, and -9 to inhibit their activities. During apoptosis, the activities of IAPs are relieved by a second mitochondria-derived caspase activator, named Smac/DIABLO. Some IAPs have a C-terminal RING finger domain that has been identified as the essential motif for the activity of ubiquitin ligase (E3). Here we show that X-linked IAP (XIAP) mediates the polyubiquitination of caspase-9 and Smac. The large subunit of mature caspase-9 was polyubiquitinated by XIAP in vitro, while procaspase-9 was not. Furthermore, the polyubiquitinated form of caspase-9 accumulated in an XIAP-dependent manner in intact cells. The ubiquitination of caspase-9 was significantly inhibited in the presence of mature Smac, whereas XIAP was also found to promote the polyubiquitination of cytosolic Smac both in vitro and in intact cells. These ubiquitination reactions require the RING finger domain of XIAP. These findings suggest that XIAP functions as ubiquitin ligase toward mature caspase-9 and Smac to inhibit apoptosis.

Apoptosis↗
PubMed2005

Phospholipase Cdelta4 associates with glutamate receptor interacting protein 1 in testis.

We reported previously that phospholipase C (PLC) delta4 is required for calcium mobilization in the zona pellucida-induced acrosome reaction in sperm. Here we focused on the function of the C2 domain of PLCdelta4 and report that glutamate receptor-interacting protein1 (GRIP1) was identified as a binding protein of the PLCdelta4-C2 domain on yeast two-hybrid screening. Physiological interaction of GRIP1 with PLCdelta4 in mouse testis was confirmed by immunoprecipitation with anti-PLCdelta4 antibodies and the association seemed to correlate with the maturation stage of sperm. We also determined that a PDZ-binding motif at the C-terminus of the PLCdelta4-C2 domain is responsible for GRIP1 binding, whereas the sixth or seventh PDZ domain of GRIP1 is essential and sufficient for association with the PLCdelta4-C2 domain. These results indicate that PLCdelta4 binds via its C2 domain to the PDZ6 or PDZ7 domain of GRIP1, and that this association may play a role in spermatogenesis.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Phospholipase C-delta1 and -delta3 are essential in the trophoblast for placental development.

Phosphoinositide-specific phospholipase C (PLC) is a key enzyme in phosphoinositide turnover and is involved in a variety of physiological functions. We analyzed PLCdelta1 knockout mice and found that PLCdelta1 is required for the maintenance of skin homeostasis. However, there were no remarkable abnormalities except hair loss and runting in PLCdelta1 knockout mice, even though PLCdelta1 is broadly distributed. Here, we report that mice lacking both PLCdelta1 and PLCdelta3 died at embryonic day 11.5 (E11.5) to E13.5. PLCdelta1/PLCdelta3 double-knockout mice exhibited severe disruption of the normal labyrinth architecture in the placenta and decreased placental vascularization, as well as abnormal proliferation and apoptosis of trophoblasts in the labyrinth area. Furthermore, PLCdelta1/PLCdelta3 double-knockout embryos supplied with a normal placenta by the tetraploid aggregation method survived beyond E14.5, clearly indicating that the embryonic lethality is caused by a defect in trophoblasts. On the basis of these results, we conclude that PLCdelta1 and PLCdelta3 are essential in trophoblasts for placental development.

Animals↗
PubMed2004

Phospholipase C delta-type consists of three isozymes: bovine PLCdelta2 is a homologue of human/mouse PLCdelta4.

To date, 12 phospholipase C (PLC) isozymes have been identified in mammals, and they are divided into five classes, beta-, gamma-, delta-, epsilon-, and zeta-type. PLCdelta-type is reported to be composed of four isozymes, PLCdelta1-delta4. Here we report that a screening for mouse PLCdelta2 from a BAC library with primers that amplify a specific region of bovine PLCdelta2 resulted in isolation of one clone containing the mouse PLCdelta4 gene. Furthermore, a database search revealed that there is only one gene corresponding to PLCdelta2 and PLCdelta4 in the mouse and human genomes, indicating that bovine PLCdelta2 is a homologue of human and mouse PLCdelta4. However, PLCdelta2 Western blot analysis with a widely used commercial anti-PLCdelta2 antibody showed an expression pattern distinct from that of PLCdelta4 in wild-type mice. In addition, an 80-kDa band, which was recognized by antibody against PLCdelta2, was smaller than an 85-kDa band detected by anti-PLCdelta4 antibody, and the 80-kDa band was detectable in lysates of brain, testis, and spleen from PLCdelta4-deficient mice. We also found that immunoprecipitates from brain lysates with this PLCdelta2 antibody contained no PLC activity. From these data, we conclude that bovine PLCdelta2 is a homologue of human and mouse PLCdelta4, and that three isozymes (delta1, delta3, and delta4) exist in the PLCdelta family.

Animals↗
PubMed2004

Human mitotic spindle-associated protein PRC1 inhibits MgcRacGAP activity toward Cdc42 during the metaphase.

Although many proteins have been shown to participate in mitotic events, including cytokinesis, their specific roles and interactions remain unclear. A novel interaction of proteins is demonstrated in this report. Yeast two-hybrid screening using PRC1 (protein-regulating cytokinesis 1) cDNA, a human mitotic spindle-associated cyclin-dependent kinase (CDK) substrate, which is involved in cytokinesis, as bait was performed. Data show that the PRC1 bait bound to MgcRacGAP, which is a GTPase-activating protein (GAP) for the Rho family GTPases also involved in cytokinesis. In addition, the two proteins showed similar localization during the M phase. PRC1 was shown to bind to the COOH-terminal GAP-conserved domain of MgcRacGAP and to inhibit its GAP activity toward Cdc42. This binding and/or inhibition of MgcRacGAP GAP activity was found to depend on further binding of PRC1 to the basic region (125-285 amino acids) of MgcRacGAP. Furthermore, the basic region was phosphorylated with Aurora B kinase, and this phosphorylation prevented the inhibition of GAP activity by PRC1. Cells overexpressing a phosphorylation mimic mutant of MgcRacGAP exhibited an abnormality of spindle morphology in the metaphase. Cdc42 showed high activity and was localized to the mitotic spindles and centrosomes during the metaphase. We propose that PRC1 down-regulates the GAP activity of MgcRac-GAP during the metaphase and thereby contributes to the correct formation of the spindle.

Cell Cycle Proteins↗
PubMed2004

Evidence that activation of Src family kinase is not required for fertilization-associated [Ca2+]i oscillations in mouse eggs.

Recent evidence in marine invertebrate, frog, and zebrafish eggs suggests the involvement of a Src family kinase (SFK) in fertilization-induced Ca2+ release. In the present study, we have investigated whether activation of an SFK is required for initiation of intracellular Ca2+ ([Ca2+]i) oscillations in mouse fertilization. We detected a Hck-like protein and tyrosine-phosphorylated proteins in soluble and insoluble sperm fractions, respectively. However, the presence of these proteins did not correspond to the active fractions of porcine sperm extracts (pSE). Moreover, [Ca2+]i oscillations induced by pSE in mouse eggs were unaltered by pre-incubation of pSE with specific SFK inhibitors such as 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazol[3,4-d]-pyrimidine (PP2) or lavendustin A, despite the fact that the inhibitors were shown to be active both in vivo and in vitro. Another SFK inhibitor, peptide A, blocked oscillations when incubated with pSE prior to injection into eggs, but this inhibition required more than ten times the concentration reportedly required to inhibit SFK activity. In addition, pre-injection or pre-incubation of eggs with these inhibitors did not affect the ability of pSE to trigger [Ca2+]i oscillations in mouse eggs. Microinjection of a recombinant c-Src protein or mRNAs encoding constitutively active Src proteins did not induce [Ca2+]i release. Finally, when sperm and eggs, both of which were pre-treated with PP2, were fertilized, [Ca2+]i oscillations occurred normally. We can therefore conclude that activation of an SFK is neither necessary nor sufficient for triggering fertilization-induced [Ca2+]i oscillations.

Animals↗
PubMed2003

Recombinant phospholipase Czeta has high Ca2+ sensitivity and induces Ca2+ oscillations in mouse eggs.

Sperm-specific phospholipase Czeta (PLCzeta) is known to induce intracellular Ca(2+) oscillations and subsequent early embryonic development when expressed in mouse eggs by injection of RNA encoding PLCzeta (Saunders, C. M., Larman, M. G., Parrington, J., Cox, L. J., Royse, J., Blayney, L. M., Swann, K., and Lai, F. A. (2002) Development 129, 3533-3544). The present study addressed characteristics of purified mouse PLCzeta protein that was synthesized using the baculovirus/Sf9 cell expression system. Microinjection of recombinant PLCzeta protein into mouse eggs induced serial Ca(2+) spikes quite similar to those produced by the injection of sperm extract, probably because of repetitive Ca(2+) release from the endoplasmic reticulum caused by continuously produced inositol 1,4,5-trisphosphate. Recombinant PLCdelta1 also induced Ca(2+) oscillations, but a 20-fold higher concentration was required compared with PLCzeta. In the enzymatic assay of phosphatidylinositol 4,5-bisphosphate hydrolyzing activity in vitro at various calcium ion concentrations ([Ca(2+)]), PLCzeta exhibited a significant activity at [Ca(2+)] as low as 10 nm and had 70% maximal activity at 100 nm [Ca(2+)] that is usually the basal intracellular calcium ion concentration level of cells. On the other hand, the activity of PLCdelta1 increased at a [Ca(2+)] between 1 and 30 microm. EC(50) was 52 nm for PLCzeta and 5.7 microm for PLCdelta1. Thus, PLCzeta has an approximately 100-fold higher Ca(2+) sensitivity than PLCdelta1. The ability of purified PLCzeta protein to induce Ca(2+) oscillations qualifies PLCzeta as a proper candidate of the mammalian egg-activating sperm factor. Furthermore, such a high Ca(2+) sensitivity of PLC activity as PLCzeta that can be active in cells at the resting state is thought to be an appropriate characteristic of the sperm factor, which is introduced into the ooplasm upon sperm-egg fusion, triggers Ca(2+) release first, and maintains Ca(2+) oscillations.

Animals↗
PubMed2003

Phospholipase Cdelta1 is required for skin stem cell lineage commitment.

Phosphoinositide-specific phospholipase C (PLC) is a key enzyme in phosphoinositide turnover and is involved in a variety of physiological functions. Here we report that PLCdelta(1)-deficient mice undergo progressive hair loss in the first postnatal hair cycle. Epidermal hyperplasia was observed, and many hairs in the skin of PLCdelta(1)-deficient mice failed to penetrate the epidermis and became zigzagged owing to occlusion of the hair canal. Two major downstream signals of PLC, calcium elevation and protein kinase C activation, were impaired in the keratinocytes and skin of PLCdelta(1)-deficient mice. In addition, many cysts that had remarkable similarities to interfollicular epidermis, as well as hyperplasia of sebaceous glands, were observed. Furthermore, PLCdelta(1)-deficient mice developed spontaneous skin tumors that had characteristics of both interfollicular epidermis and sebaceous glands. From these results, we conclude that PLCdelta(1) is required for skin stem cell lineage commitment.

Animals↗
PubMed2003

Phospholipase Cdelta4 is required for Ca2+ mobilization essential for acrosome reaction in sperm.

Zona pellucida (ZP)-induced acrosome reaction in sperm is a required step for mammalian fertilization. However, the precise mechanism of the acrosome reaction remains unclear. We previously reported that PLCdelta4 is involved in the ZP-induced acrosome reaction in mouse sperm. Here we have monitored Ca2+ responses in single sperm, and we report that the [Ca2+]i increase in response to ZP, which is essential for driving the acrosome reaction in vivo, is absent in PLCdelta4-/- sperm. Progesterone, another physiological inducer of the acrosome reaction, failed to induce sustained [Ca2+]i increases in PLCdelta4-/- sperm, and consequently the acrosome reaction was partially inhibited. In addition, we observed oscillatory [Ca2+]i increases in wild-type sperm in response to these acrosome inducers. Calcium imaging studies revealed that the [Ca2+]i increases induced by exposure to ZP and progesterone started at different sites within the sperm head, indicating that these agonists induce the acrosome reaction via different Ca2+ mechanisms. Furthermore, store-operated channel (SOC) activity was severely impaired in PLCdelta4-/- sperm. These results indicate that PLCdelta4 is an important enzyme for intracellular [Ca2+]i mobilization in the ZP-induced acrosome reaction and for sustained [Ca2+]i increases through SOC induced by ZP and progesterone in sperm.

Acrosome↗