Activation of Phospholipase D2 through Phosphorylation of Tyrosine-470 in Antigen-stimulated Mast Cells

김영미,Kim Young Mi Korean Society of Life Science 2005 생명과학회지 Vol.15 No.3

PLDI 활성화 기전은 여러 보고가 있으나 PLD2 활성화에 대한 기전은 아직 연구의 대상이다. RBL-2H3 비만세포에서 HA-PLD2의 인산화 가능한 타이로신 잔기를 점돌연변이 시킨 DNA플라즈미드를 이용하여 11번, 14번, 470번의 타이로신이 항원자극에 의해 인산화 됨을 알아냈고 특히 470번 타이로신의 인산화가 PLD2 활성화에 중요하다는 결과를 얻었다. The mechanism of activation of phospholipase D2 (PLD2) remains undefined although mechanisms have been described for the activation of PLDI. By expression of mutated forms of haemaglutinnin-tagged PLD2 in a mast cell (RBL-2H3) line, we show that PLD2 is phosphorylated at tyrosines -11, -14, and -470 and that tyrosine-470 is critical for activation of PLD2 by antigen. Studies were performed with mutated-DNA constructs for haemaglutinnin-tagged PLD2 in which codons for tyrosine -11, -14, -165, and -470 were mutated to phenylalanine either individually or collectively. Transient expression of these constructs showed that mutation of tyrosine -11, -14, -470, or all tyrosines (all-mutated PLD2) suppressed antigen-induced tyrosine phosphorylation of PLD2 but only the tyrosine-470 mutant failed to be activated by antigen as assessed by in vitro assay of immunoprepitated PLD2 or by assay of PLD in intact cells. The critical role of tyrosine-470 was confirmed in studies with add-back mutants (phenylalanine back to tyrosine) of the all-mutated PLD. The findings provide the first description of a mechanism of activation of PLD2 in a physiological setting.

Phospholipase D Activity is Elevated in Hepatitis C Virus Core Protein-Transformed NIH 3T3 Mouse Fibroblast Cells

Kim, Joonmo,Jung, Eun-Young,Jang, Kyung-Lib,Min, Do-Sik Korean Society of Life Science 2003 생명과학회지 Vol.13 No.5

C형 간염바이러스는 간암을 야기하는 심각한 바이러스이다. C형 간염바이러스의 core 단백질의 과발현은 섬유아세포를 암화시키는 것으로 알려져 있다. Phospholipase D (PLD)의 효소활성이 세포증식 신호전달에 의해 활성화되어 있으며, 사람의 암조직에서 과발현 및 활성이 증가되어 있는 것으로 알려져 있다. 본 연구의 목적은, core 단백질에 의해 암화된 세포에서 PLD가 어떻게 조절되는지를 이해하고자 하는 것이다. 자극이 없는 상태에서뿐만 아니라 PMA에 의해 유도되는 PLD효소활성은, 암화된 세포에서 더 증가하였으며, control 세포와 core 단백질에 의해 암화된 세포에서 PLD와 PKC 단백질의 발현은 서로 유사하였다. PKC 특이적인 억제제와 PKC의 세포막으로의 이동에 관한 실험을 통해서, PKC-d가 암화된 세포에서 PMA에 의해 유도되는 PLD활성의 증가에 중요하게 관여하고 있음을 밝혔다. 이러한 결과는, PLD가 core 단백질에 의해 유도되는 세포의 암화과정에 관여하고 있을 것으로 추정된다. Hepatitis C Virus (HCV) is associated with a severe liver disease and increased frequency in the development of hepatocellular carcinoma. Overexpression of HCV core protein is known to transform fibroblast cells. Phospholipase D (PLD) activity is commonly elevated in response to mitogenic signals, and PLD has been also reported to be overexpressed and hyperactivated in some human cancer. The aim of this study was to understand how PLD can be regulated in HCV core protein-transformed NIH3T3 mouse fibroblast cells. We observed that in unstimulated state, basal PLD activity was higher in NIH3T3 cells overexpressing HCV core protein than in vector-transfected cells. Although expression of PLD and protein kinase C (PKC) in core protein-transformed cells was similar with that of control cells, phorbol 12-myristate 13-acetate (PMA), which is known to activate PKC, stimulated significantly PLD activity in core protein-transformed cells, compared with that of the control cells. PLD activity assay using PKC isozyme-specific inhibitor, and PKC translocation experiment showed that PKC-$\delta$ was mainly involved in the PMA-induced PLD activation in the core-transformed cells. Taken together, these results suggest that PLD might be implicated in core protein-induced transformation.

Hippocalcin increases phospholipase D2 expression through extracellular signal-regulated kinase activation and lysophosphatidic acid potentiates the hippocalcin-induced phospholipase D2 expression

Oh, Doo-Yi,Yon, Changsuek,Oh, Kyoung-Jin,Lee, Ki Sung,Han, Joong-Soo Wiley Subscription Services, Inc., A Wiley Company 2006 Journal of cellular biochemistry Vol.97 No.5

<P>We have previously isolated a 22 kDa protein from a rat brain which was found to be involved in activating phospholipsae D (PLD), and identified the protein as hippocalcin through sequence analysis. Nevertheless, the function of hippocalcin for PLD activation still remains to be resolved. Here, we proposed that hippocalcin was involved in extracellular signal-regulated kinase (ERK)-mediated PLD2 expression. To elucidate a role of hippocalcin, we made hippocalcin transfected NIH3T3 cells and showed that the expression of PLD2 and basal PLD activity were increased in hippocalcin transfected cells. We performed PLD assay with dominant negative PLD2 (DN-PLD2) and hippocalcin co-transfected cells. DN-PLD2 suppressed increase of basal PLD activity in hippocalcin transfected cells, suggesting that increased basal PLD activity is due to PLD2 over-expression. Hippocalcin is a Ca<SUP>2+</SUP>-binding protein, which is expressed mainly in the hippocampus. Since it is known that lysophosphatidic acid (LPA) increases intracellular Ca<SUP>2+</SUP>, we investigated the possible role of hippocalcin in the LPA-induced elevation of intracellular Ca<SUP>2+</SUP>. When the intracellular Ca<SUP>2+</SUP> level was increased by LPA, hippocalcin was translocated to the membrane after LPA treatment in hippocalcin transfected cells. In addition, treatment with LPA in hippocalcin transfected cells markedly potentiated PLD2 expression and showed morphological changes of cell shape suggesting that increased PLD2 expression acts as one of the major factors to cause change of cell shape by making altered membrane lipid composition. Hippocalcin-induced PLD2 expression potentiated by LPA in hippocalcin transfected cells was inhibited by a PI-PLC inhibitor, U73122 and a chelator of intracellular Ca<SUP>2+</SUP>, BAPTA–AM suggesting that activation of hippocalcin caused by increased intracellular Ca<SUP>2+</SUP> is important to induce over-expression of PLD2. However, downregulation of PKC and treatment of a chelator of extracellular Ca<SUP>2+</SUP>, EGTA had little or no effect on the inhibition of hippocalcin-induced PLD2 expression potentiated by LPA in the hippocalcin transfected cells. Interestingly, when we over-express hippocalcin, ERK was activated, and treatment with LPA in hippocalcin transfected cells significantly potentiated ERK activation. Specific inhibition of ERK dramatically abolished hippocalcin-induced PLD2 expression. Taken together, these results suggest for the first time that hippocalcin can induce PLD2 expression and LPA potentiates hippocalcin-induced PLD2 expression, which is mediated by ERK activation. J. Cell. Biochem. 97: 1052–1065, 2006. © 2005 Wiley-Liss, Inc.</P>

Genistein과 Calphostin C를 이용한 T98G 세포와 Hs 683 세포에서의 PLC활성과 세포증식의 관계에 대한 연구

최창명,김윤,김보연,양지호,이일우,정철구,강준기 대한신경외과학회 1998 Journal of Korean neurosurgical society Vol.27 No.8

파노라마 촬영시 눈과 갑상선에 미치는 표면선량에 관한 연구

동경래(Dong Kyung-Rae) 한국콘텐츠학회 2009 한국콘텐츠학회 종합학술대회 논문집 Vol.7 No.1

파노라마 촬영 시 눈과 갑상선의 표면선량 실험은 광주지역 10개 병원을 대상으로 열형광선량계(Thermoluminescent dosimeter, TLD)와 형광유리선량계(Photoluminescent dosimeter, PLD)를 이용하여 각각 병원에서 사용하는 조건으로 측정(measurement)하였다. ICRP 60과 ICRP 73에서 권고한 눈에 대한 허용기준은 15mSv, 갑상선에 대한 허용기준은 연간 1mSv이다. 왼쪽 눈(Left Eye)의 TLD와 PLD값은 각각 0.19mSv와 0.24mSv, 오른쪽 눈(Right Eye)의 TLD와 PLD의 값은 0.23mSv와 0.25mSv, 갑상선의 TLD와 PLD의 값은 0.08mSv와 0.25mSv로 허용기준치를 초과하지 않았다. 또한 각 장기에 대한 TLD와 PLD의 비교에서는 왼쪽 눈과 갑상선이 유의한 차이가 있다고 볼 수 있고(p<0.01), 오른쪽 눈은 유의한 차이가 없다고 볼 수 있다(p>0.05). 각 병원에서 사용하는 파노라마 기기로 눈과 갑상선에 미치는 선량을 TLD와 PLD로 측정 하였을 때 눈과 갑상선의 표면선량은 ICRP 60에서 권고한 선량을 넘지 않았지만, 확률적 영향이 일어날 수 있으므로 모든 준위의 선량에 대해서 고려되어야 한다. Ten hospitals from the Gwangju area were used to examine shallow dose to eyes and thyroid from panoramagraphy. Thermoluminescent dosimeter (TLD) and Photoluminescent dosimeter (PLD) were used as measurement devices at each hospital. ICRP 60 and ICRP 73 set standards for acceptability for eyes at 15mSv and thyroid at 1mSv per year. Left eye measures with TLD and PLD resulted in 0.19mSv and 0.24mSv respectively. Right eye measures with TLD and PLD resulted in 0.23mSv and 0.25mSv respectively. Thyroid measures with TLD and PLD resulted in 0.08mSv and 0.25mSv respectively with both measures not exceeding standards for acceptance. There was a significant difference in comparing the left eye and thyroid for TLD and PLD (p<0.01). There was no significant difference with the right eye (p>0.05). The absorbed dose measurements for eyes and thyroid using TLD and PLD in regards to panorama devices at each hospital were within the ICRP 60 recommendations; however, with the possibility of stochastic effect, all dose levels were taken into consideration.

Phospholipase D in Guinea Pig Lung Tissue Membrane is Regulated by Cytosolic ARF Proteins

( Yean Jun Chung ),( Jin Rak Jeong ),( Byung Chul Lee ),( Ji Young Kim ),( Young In Park ),( Jai Youl Ro ) 한국미생물생명공학회 2003 Journal of microbiology and biotechnology Vol.13 No.6

Over-Expression of Phospholipase D Isozymes Down-Regulates Protein Kinase CKII Activity via Proteasome-Dependent CKII Degradation in NIH3T3 Cells

윤수현,민도식,Young-Seuk Bae 한국분자세포생물학회 2009 Molecules and cells Vol.27 No.3

Over-expression of phospholipase D (PLD) 1 or PLD2 downregulated CKII activity in NIH3T3 cells. The same results were found with catalytically inactive mutants of PLD isozymes, indicating that the catalytic activity of PLD is not required for PLD-mediated CKII inhibition. Consistent with this, 1-butanol did not alter CKII activity. The reduction in CKII activity in PLD-over-expressing NIH3T3 cells was due to reduced protein level, but not mRNA level, of the CKIIβ subunit. This PLD-induced CKIIβ degradation was mediated by ubiquitin-proteasome machinery, but MAP kinase and mTOR were not involved in CKIIβ degradation. PLD isozymes interacted with the CKIIβ subunit. Immunocytochemical staining revealed that PLD and CKIIβ colocalize in the cytoplasm of NIH3T3 cells, especially in the perinuclear region. PLD binding to CKIIβ inhibited CKIIβ autophosphorylation, which is known to be important for CKIIβ stability. In summary, the current data indicate that PLD isozymes can down-regulate CKII activity through the acceleration of CKIIβ degradation by ubiquitin-proteasome machinery.

공유결합으로 다공성 막에 고정화된 PLD에 의한 포스퍼티딕산 생산

박진원(Jin-Won Park) 한국청정기술학회 2015 청정기술 Vol.21 No.4

포스포라이페이즈디를 공유결합을 통해서 초미세다공성막에 고정화하였다. 고정화는 폴리에틸렌이민, 글루타알데하이드, 포스포라이페이즈디를 순차적으로 처리함으로써 수행되었다. X선 광전자 분광기를 이용하여 고정화가 확인되었다. 포스퍼티딜콜린이 분산된 버퍼용액의 pH값을 시간에 따라 모니터링하여 고정화된 경우와 그렇지 않은 경우에 대해 촉매활성을 산출하였다. 속도상수는 폴리스타이렌나노입자에 고정화된 포스포라이페이즈디에서는 0.64 s<SUP>-1</SUP>, 다공성 셀룰로스아세테이트막에 고정화된 포스포라이페이즈디에서는 0.52 s<SUP>-1</SUP>, 그리고 고정화되지 않은 포스포라이페이즈디에서는 0.75 s<SUP>-1</SUP>의 결과가 도출되었다. 재사용에 대한 연구가 10차례까지 수행되었으며, 초기 사용시의 활성대비로 95%가 유지되었다. 열과 저장성에 대한 안정성도 고찰되었으며, 다공성막에 고정화된 포스포라이페이즈디의 경우에 활성손실이 가장 적은 것으로 관찰되었다. 이 연구결과들로부터, 포스퍼티딕산의 생산용 포스포라이페이즈디의 고정화에 대한 지지체로 다공성 막을 사용할 수 있음을 알 수 있다. Phospholipase D (PLD) was immobilized on a submicro-porous membrane through covalent immobilization. The immobilization was conducted on the porous membrane surface with the treatment of polyethyleneimine, glutaraldehyde, and the anhydrase, in sequence. The immobilization was confirmed using X-ray photon spectrometer. The pH values of phosphatidylcholine (PC) dispersion solution with buffer were monitored with respect to time to calculate the catalytic activities of PC for free and immobilized PLD. The catalytic rate constant values for free PLD, immobilized PLD on polystyrene nanoparticles, and immobilized PLD on a porous cellulose acetate membrane were 0.75, 0.64, and 0.52 s<SUP>-1</SUP>, respectively. Reusability was studied up to 10 cycles of PC hydrolysis. The activity for the PLD immobilized on the membrane was kept to 95% after 10 cycles, and comparable to the PLD on the nanoparticles. The stabilities for heat and storage were also investigated for the three cases. The results suggested that the PLD immobilized on the membrane had the least loss rate of the activity compared to the others. From these studies, the porous membrane was feasible as a carrier for the PLD immobilization in the production of phosphatidic acid.

Comparative Analysis of Phospholipase D2 Localization in the Pancreatic Islet of Rat and Guinea Pig

Ryu, Gyeong-Ryul,Kim, Myung-Jun,Song, Chan-Hee,Min, Do-Sik,Rhie, Duck-Joo,Yoon, Shin-Hee,Hahn, Sang-June,Kim, Myung-Suk,Jo, Yang-Hyeok The Korean Society of Pharmacology 2003 The Korean Journal of Physiology & Pharmacology Vol.7 No.4

To examine the localization pattern of phospholipase D2 (PLD2) in the pancreatic islet (the islet of Langerhans) depending on species, we conducted a morphological experiment in the rat and guinea pig. Since individual islets display a typical topography with a central core of B cell mass and a peripheral boundary of A, D, and PP cells, double immunofluorescent staining with a panel of antibodies was performed to identify PLD2-immunoreactive cells in the islets PLD2 immunoreactivity was mainly present in A and PP cells of the rat pancreatic islets. And yet, in the guinea pig, PLD2 immunoreactivity was exclusively localized in A cells, and not in PP cells. These findings suggest a possibility that PLD2 is mainly located in A cells of rodent pancreatic islets, and that the existence of PLD2 in PP cells is not universal in all species. Based on these results, it is suggested that PLD2 may play a significant role in the function of A and/or PP cells via a PLD-mediated signaling pathway.

Phospholipase D1 Signaling: Essential Roles in Neural Stem Cell Differentiation

Park, Shin-Young,Han, Joong-Soo Springer US 2018 Journal of molecular neuroscience Vol.64 No.3

<P>Phospholipase D1 (PLD1) is generally accepted as playing an important role in the regulation of multiple cell functions, such as cell growth, survival, differentiation, membrane trafficking, and cytoskeletal organization. Recent findings suggest that PLD1 also plays an important role in the regulation of neuronal differentiation of neuronal cells. Moreover, PLD1-mediated signaling molecules dynamically regulate the neuronal differentiation of neural stem cells (NSCs). Rho family GTPases and Ca<SUP>2+</SUP>-dependent signaling, in particular, are closely involved in PLD1-mediated neuronal differentiation of NSCs. Moreover, PLD1 has a significant effect on the neurogenesis of NSCs via the regulation of SHP-1/STAT3 activation. Therefore, PLD1 has now attracted significant attention as an essential neuronal signaling molecule in the nervous system. In the current review, we summarize recent findings on the regulation of PLD1 in neuronal differentiation and discuss the potential role of PLD1 in the neurogenesis of NSCs.</P>