• BMB Reports
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• v.38 no.3
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• pp.360-365
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• 2005

Membrane inositol glycerophospholipid (IGP) is metabolized to phosphatidylinositol-4-phosphate (PIP), phosphatidylinositol-4, 5-bisphosphate ($PIP_2$), and inositol triphosphate ($IP_3$) in signaling transduction. This study was carried out to determine the subclasses of IGP involved in signaling pathway. The acyl chain moieties of the phospholipids are easily modulated by dietary fatty acids. We analyzed acyl chain composition of IGP 3-subclasses, PIP and $PIP_2$ from rat brain after feeding sunflower seed oil enriched with linoleic acid or fish oil high in eicosapentaenoic acid and docosahexaenoic acid. Long chain polyunsaturated fatty acids (LCPUFA) as eicosapentaenoic acid and docosahexaenoic acid were not incorporated into ether-linked IGP (alkenylacylglycerophosphoinositol and alkylacyl-glycerophosphoinositol), PIP and $PIP_2$, while diacyl-glycerophosphoinositol (GPI) contained high LCPUFA. These results suggest that PIP might be phosphorylated from only the ether-linked IGP (alkenylacyl- and alkylacyl species) but not from diacyl subclass for signals to intracellular responses in the plasma membrane of rat brain.

• Journal of environmental and Sanitary engineering
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• v.16 no.4
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• pp.1-8
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• 2001

We have previously demonstrated that sodium molybdate(Mo) improved lead-intoxicated status by enhancing the metabolism of mao-inositol-related phospholipids in sciatic nerves isolated from rats. In this study, in order to address the reduction mechanism of Mo for lead toxicity, effects of Mo on cystidine-diglyceride transferase, phosphatidylinositol kinase, and phosphatidyl inositol-4-phosphate kinase, involved in mao-inositol metabolism of nerve, were investigated in vivo and in vitro. Mo significantly increased the activities of cystidine- diglyceride transferase and phosphatidylinositol kinase in lead-intoxicated rat, and the pattern of increase was dose-dependent manner. However, Mo did not affect the activity of phosp- hatidylinositiol-4-phosphate kinase in normal and lead-intoxicated rats. We also found that Mo affected the activities of phopholipid metabolism-related enzymes not by the indirect manner such as activation of another metabolic pathway but by the direct manner. These results suggest that the improvement mechanism of Mo for lead-intoxicated status might be a normalization of the activities of phospholipid metabolism-related enzymes in sciatic nerve.

• Molecules and Cells
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• v.22 no.1
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• pp.97-103
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• 2006

Phosphoinositides are critical regulators of ion channel and transporter activity. There are multiple isomers of biologically active phosphoinositides in the plasma membrane and the different lipid species are non-randomly distributed. However, the mechanism by which cells impose selectivity and directionality on lipid movements and so generate a non-random lipid distribution remains unclear. In the present study we investigated which structural elements of phosphoinositides are responsible for their subcellular location and movement. We incubated phosphatidylinositol (PI), phosphatidylinositol 4-monophosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate ($PI(4,5)P_2$) with short or long acyl chains in CHO and HEK cells. We show that phosphate number and acyl chain length determine cellular location and translocation movement. In CHO cells, $PI(4,5)P_2$ with a long acyl chain was released into the cytosol easily because of a low partition coefficient whereas long chain PI was released more slowly because of a high partition coefficient. In HEK cells, the cellular location and translocation movement of PI were similar to those of PI in CHO cells, whereas those of $PI(4,5)P_2$ were different; some mechanism restricted the translocation movement of $PI(4,5)P_2$, and this is in good agreement with the extremely low lateral diffusion of $PI(4,5)P_2$. In contrast to the dependence on the number of phosphates of the phospholipid head group of long acyl chain analogs, short acyl chain phospholipids easily undergo translocation movement regardless of cell type and number of phosphates in the lipid headgroup.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.34 no.4
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• pp.370-377
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• 2001

Flounder brain cytosol contains protein inhibitors that markedly inhibit the activity of partially purified brain membrane phospholipase D (PLD) which is dependent on phosphatidylinositol 4,5-bisphosphate ($PIP_2$) but insensitive to ADP-ribosylation factor (ARF), The PLD inhibitors have been enriched through several chromatographic steps and characterized with respect to size and mechanism of inhibition. Sequential chromatography of the brain cytosol yielded six inhibitor fractions, Two (IIA and IIB) of six inhibitor fractions showed the $PIP_2$-phosphatase activities. IIA was identified as synaptojanin, a nerve terminal protein that has known to be a member of the inositolpolyphosphate 5-phosphatase family, by immunoblot analysis. IIB showed an apparent molecular mass of 158 kDa by Superose 12 gel filtration chromatography and was immunologically distinct from synaptojanin. IIB hydrolyzed $PIP_2$, yielding only phosphatidylinositol phosphate (PIP) as product, suggesting that IIB hydrolyzes only one phosphate from either the 4- or 5-position of PI (4,5)$P_2$. These studies demonstrate that the existence of multiple $PIP_2$-phosphatases have been implicated in the negative regulation of $PIP_2$-dependent PLD activity within flounder brain.

• Journal of Life Science
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• v.24 no.1
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• pp.14-19
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• 2014

Kinesin-1 consists of two heavy chains (KHCs), also called KIF5s, and two light chains (KLCs) that form a heterotetrameric complex. Here, we demonstrate the binding of a neuronal KHC, KIF5A, to the carboxyl (C)-terminal tail region of Fig4 (also known as Sac3), a phosphatase that removes the 5-phosphate from phosphatidylinositol-3,5-bisphosphate ($PtdIns(3,5)P_2$). Fig4 bound to the C-terminal region of KIF5A but not to other KHCs (KIF5B and KIF5C) and KLC1 in yeast two-hybrid assays. The interaction was further confirmed in a glutathione S-transferase pull-down assay and by co-immunoprecipitation. Anti-KIF5A antibody co-immunoprecipitated Fig4 with KIF5A from mouse brain extracts. These results suggest that kinesin-1 could transport the Fig4-associated protein complex or cargo in cells.

• Molecules and Cells
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• v.39 no.4
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• pp.322-329
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• 2016

Voltage-gated $Ca^{2+}$ ($Ca_V$) channels are dynamically modulated by Gprotein-coupled receptors (GPCR). The $M_1$ muscarinic receptor stimulation is known to enhance $Ca_V2.3$ channel gating through the activation of protein kinase C (PKC). Here, we found that $M_1$ receptors also inhibit $Ca_V2.3$ currents when the channels are fully activated by PKC. In whole-cell configuration, the application of phorbol 12-myristate 13-acetate (PMA), a PKC activator, potentiated $Ca_V2.3$ currents by ~two-fold. After the PMA-induced potentiation, stimulation of $M_1$ receptors decreased the $Ca_V2.3$ currents by $52{\pm}8%$. We examined whether the depletion of phosphatidylinositol 4,5-bisphosphate ($PI(4,5)P_2$) is responsible for the muscarinic suppression of $Ca_V2.3$ currents by using two methods: the Danio rerio voltage-sensing phosphatase (Dr-VSP) system and the rapamycin-induced translocatable pseudojanin (PJ) system. First, dephosphorylation of $PI(4,5)P_2$ to phosphatidylinositol 4-phosphate (PI(4)P) by Dr-VSP significantly suppressed $Ca_V2.3$ currents, by $53{\pm}3%$. Next, dephosphorylation of both PI(4)P and $PI(4,5)P_2$ to PI by PJ translocation further decreased the current by up to $66{\pm}3%$. The results suggest that $Ca_V2.3$ currents are modulated by the $M_1$ receptor in a dual mode-that is, potentiation through the activation of PKC and suppression by the depletion of membrane $PI(4,5)P_2$. Our results also suggest that there is rapid turnover between PI(4)P and $PI(4,5)P_2$ in the plasma membrane.

• Molecules and Cells
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• v.21 no.3
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• pp.428-435
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• 2006

Specific interaction of the epsin N-terminal homology(ENTH) domain with the plasma membrane appears to bridge other related proteins to the specific regions of the membrane that are invaginated to form endocytic vesicles. An additional $\alpha$-helix, referred to as helix 0 (H0), is formed in the presence of the soluble ligand inositol-1,4,5-trisphosphate [$Ins(1,4,5)P_3$] at the N terminus of the ENTH domain (amino acid residues 3-15). The ENTH domain alone and full-length epsin cause tubulation of liposomes made of brain lipids. Thus, it is believed that H0 is membrane-inserted when it is coordinated with the phospholipid phosphatidylinositol-4,5-bisphosphate [$PtdIns(4,5)P_2$], resulting in membrane deformation as well as recruitment of accessory factors to the membrane. However, formation of H0 in a real biological membrane has not been demonstrated. In the present study, the membrane structure of H0 was determined by measurement of electron paramagnetic resonance (EPR) nitroxide accessibility. H0 was located at the phosphate head-group region of the membrane. Moreover, EPR line-shape analysis indicated that no pre-formed H0-like structure were present on normal acidic membranes. $PtdIns(4,5)P_2$ was necessary and sufficient for interaction of the H0 region with the membrane. H0 was stable only in the membrane. In conclusion, the H0 region of the ENTH domain has an intrinsic ability to form H0 in a $PtdIns(4,5)P_2$-containing membrane, perhaps functioning as a sensor of membrane patches enriched with $PtdIns(4,5)P_2$ that will initiate curvature to form endocytic vesicles.

• BMB Reports
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• v.37 no.6
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• pp.720-725
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• 2004

A number of signaling molecules contain small pleckstrin homology (PH) domains capable of binding phosphoinositides or proteins. Phospholipase C (PLC)-${\gamma}1$ has two putative PH domains, an $NH_2$-terminal (PH1) and a split PH domain ($nPH_2$ and $cPH_2$). We previously reported that the split PH domain of PLC-${\gamma}1$ binds to phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)$P_2$) (Chang et al., 2002). To identify the amino acid residues responsible for binding with PI(4)P and PI(4,5)$P_2$, we used site-directed mutagenesis to replace each amino acid in the variable loop-1 (VL-1) region of the PLC-${\gamma}1$ $nPH_2$ domain with alanine (a neutral amino acid). The phosphoinositide-binding affinity of these mutant molecules was analyzed by Dot-blot assay followed by ECL detection. We found that two PLC-${\gamma}1$ nPH2 domain mutants, P500A and H503A, showed reduced affinities for phosphoinositide binding. Furthermore, these mutant PLC-${\gamma}1$ molecules showed reduced PI(4,5)$P_2$ hydrolysis. Using green fluorescent protein (GFP) fusion protein system, we showed that both $PH_1$ and $nPH_2$ domains are responsible for membrane-targeted translocation of PLC-${\gamma}1$ upon serum stimulation. Together, our data reveal that the amino acid residues $Pro^{500}$ and $His^{503}$ are critical for binding of PLC-${\gamma}1$ to one of its substrates, PI(4,5)$P_2$ in the membrane.

• Animal cells and systems
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• v.13 no.4
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• pp.363-370
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• 2009

Since vitamin $D_3$ is an important regulator of osteoblastic differentiation, a presently-established vitamin $D_3$-entrapped calcium phosphate film (VCPF) was evaluated for hard tissue engineering. The entrapped vitamin $D_3$ more rapidly induced bone nodule formation. To characterize the cellular events leading to regulations including faster differentiation, signal transduction pathways were investigated in osteoblastic MG63 cells at a molecular level. Major signaling pathways for MG63 cell proliferation including phosphatidylinositol-3-kinase, extracellular signal-regulated kinase, c-Jun N-terminal kinase and focal adhesion kinase pathways were markedly down-regulated when cells were cultured on calcium phosphate film (CPF) and VCPF. This agreed with our earlier observations of the immediate delay in proliferation of MG63 cells upon culture on CPF and VCPF. On the other hand, the p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase A (PKA) pathways were significantly up-regulated on both CPF and VCPF. CPF alone could simulate differential behaviors of MG63 cells even in the absence of osteogenic stimulation and entrapment of vitamin $D_3$ within CPF further amplified the signal pathways, resulting in continued promotion of MG63 cell differentiation. Interplay of p38 MAPK and PKA signaling pathways likely is a significant event for the promotion of differentiation and mineralization of MG63 cells.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.95-95
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• 2003

Inositol은 세포의증식 및 정보전달과정에 관여하는 phosphatidylinositol (PI)의 구성성분으로서 중요한 세포내 기능을 수행한다. P1는 세포내에서 특이적 인산화효소에 의하여 Pl4-phosphate(PIP), Pl4,5-phosphate($PIP_2$)로 변환되며 PIP$_2$는 phospholipase C(PLC)에 의하여 세포내 second messengers인 1,2-diacylglycerol (DAG)와 inositol 1,4,5-triphosphate ($IP_3$)로 변환된다. 이렇게 생산된 DAG와 IP3는 각각 protein kinase C의 활성과 $Ca^{2++}$의 동원에 관여하여 다양한 세포내 신호전달에 관여하는 것으로 보고되고 있다. 또한 mouse에서 $IP_3$의 작용에 의한 $Ca^{2++}$의 상승은 난모세포의 성숙분열이 촉진되고 돼지 난포세포에 있어서도 PI대사가 일어나고 있는 것으로 보고되고 있다. 본 연구에서는 돼지 미성숙 난모세포의 성숙과 단위발생에 미치는 inositol의 영향을 확인하기 위하여 실시하였다. inositol의 농도가 난포란의 성숙에 미치는 영향을 검토하기 위하여 난포란을 각각 $150 \mu mole$, $250 \mu mole$, $350 \mu mole$, inositol을 포함하는 Whitten's 배양액에서 44시간 성숙시킨 결과 $93.57 \pm 4.21, 93.91 \pm 2.71, 92.96 \pm 3.58%$가 성숙되어 대조구의 $87.10 \pm 4.21$보다 유의적(P<0.05)으로 차이를 나타내었다. 난포란의 등급에 따른 inositol의 영향을 확인하기 위하여 형태적으로 난구세포가 치밀한 난포란과 난구세포가 치밀하지 않은 난포란을 $250 \mu mole$ inositol을 포함하는 Whitten's 배양액에서 성숙을 유도한 결과 난구세포가 치밀한 난포란과 난구세포가 치밀하지 않은 난포란에서 inositol을 첨가하였을 때 성숙율은 각각 $95.35 \pm 2.22와 63.55 \pm 8.12$로 inositol을 첨가하지 않은 대조구보다($89.21 \pm 3.69와 48.56 \pm 8.99$) 유의적인 차이를 보였다. 난구세포가 inositol에 의한 성숙에 미치는 영향을 확인하기 위하여 난구세포를 제거한 난포란을 inositol을 포함하는 배지에서 성숙을 유도한 결과 inositol을 첨가하지 않은 처리구보다 양호한 성숙율을 보였다.