AP2α is essential for MUC8 gene expression in human airway epithelial cells

Moon, Uk Yeol,Kim, Chang-Hoon,Choi, Jae Young,Kim, Yoon-Ju,Choi, Yeon Ho,Yoon, Ho-Geun,Kim, Hyeyoung,Yoon, Joo-Heon Wiley Subscription Services, Inc., A Wiley Company 2010 Journal of cellular biochemistry Vol.110 No.6

<P>Mucins are high molecular weight proteins that make up the major components of mucus. Hypersecretion of mucus is a feature of several chronic inflammatory airway diseases. MUC8 is an important component of airway mucus, and its gene expression is upregulated in nasal polyp epithelium. Little is known about the molecular mechanisms of MUC8 gene expression. We first observed overexpression of activator protein-2alpha (AP2α) in human nasal polyp epithelium. We hypothesized that AP2α overexpression in nasal polyp epithelium correlates closely with MUC8 gene expression. We demonstrated that phorbol 12-myristate 13-acetate (PMA) treatment of the airway epithelial cell line NCI-H292 increases MUC8 gene and AP2α expression. In this study, we sought to determine which signal pathway is involved in PMA-induced MUC8 gene expression. The results show that the protein kinase C and mitogen-activating protein/ERK kinase (MAPK) pathways modulate MUC8 gene expression. PD98059 or ERK1/2 siRNA and RO-31-8220 or PKC siRNA significantly suppress AP2α as well as MUC8 gene expression in PMA-treated cells. To verify the role of AP2α, we specifically knocked down AP2α expression with siRNA. A significant AP2α knock-down inhibited PMA-induced MUC8 gene expression. While dominant negative AP2α decreased PMA-induced MUC8 gene expression, overexpressing wildtype AP2α increased MUC8 gene expression. Furthermore, using lentiviral vectors for RNA interference in human nasal polyp epithelial cells, we confirmed an essential role for AP2α in MUC8 gene expression. From these results, we concluded that PMA induces MUC8 gene expression through a mechanism involving PKC, ERK1/2, and AP2α activation in human airway epithelial cells. J. Cell. Biochem. 110: 1386–1398, 2010. © 2010 Wiley-Liss, Inc.</P>

Cr₂O₃/AP 복합체 제조 및 그 열분해 특성

정재윤,김재경,심홍민,김현수,구기갑,Jung, Jae-Yun,Kim, Jae-Kyeong,Shim, Hong-Min,Kim, Hyoun-Soo,Koo, Kee-Kahb 한국공업화학회 2015 공업화학 Vol.26 No.2

Solvent/anti-solvent법으로 제조된 $Cr_2O_3$/과염소산암모늄 에너지 복합체의 X선 회절 분석 결과 $Cr_2O_3$이 내포된 과염소산암모늄 입자는 순수한 과염소산암모늄과 동일한 결정 구조로 확인되었으며 주사전자현미경 사진으로부터 측정된 입방체 형상 결정의 평균입도는 약 $2.5{\mu}m$이었다. 복합체의 열중량 분석으로부터 $Cr_2O_3$에 의해 과염소산암모늄의 고온 분해 영역 분해 온도가 낮아짐을 알 수 있었고, 복합체 분해 반응의 활성화 에너지는 Starlink 방법에 의해 계산되었다. 이와 같은 활성화 에너지의 변화로 인하여, 과염소산암모늄의 분해 반응 메카니즘은 전환율 약 0.25까지는 주로 핵생성에 의한 다공성 구조가 생성되면서 분해되는 것으로 보이며, 전환율 0.3 이상에서는 과염소산암모늄의 격렬한 분해 반응이 승화보다 우선하는 것으로 보인다. $Cr_2O_3/AP$ (ammonium perchlorate) energetic composites were prepared by a method of solvent/anti-solvent. XRD analysis revealed that the crystalline structure of AP in $Cr_2O_3/AP$ composites is the same as that of pure AP. SEM photomicrograph shows that an average size of cuboid $Cr_2O_3/AP$ composites is approximately $2.5{\mu}m$. TGA analysis shows that the addition of submicron $Cr_2O_3$ particles into AP lowers the HTD (high-temperature decomposition) compared to that of neat AP and the activation energy of the $Cr_2O_3/AP$ composites was calculated by the isoconversional Starlink method. Considering changes in the activation energy, the decomposition reaction mechanism of AP was suggested as follows; the decomposition with the formation of nucleation sites renders formation of porous structure in the composites up to conversion of about 0.25 and after further conversion of over 0.3, it seems that decomposition reaction vigorously takes place rather than sublimation of AP.

CK2 phosphorylates AP-2α and increases its transcriptional activity

( Kai Qun Ren ),( Shuang Lin Xiang ),( Fang Li He ),( Wen Feng Zhang ),( Xiao Feng Ding ),( Yan Yang Wu ),( Li Ping Yang ),( Jian Lin Zhou ),( Xiang Gao ),( Jian Zhang ) 생화학분자생물학회 2011 BMB Reports Vol.44 No.7

Transcription factor AP-2α involves in the process of mammalian embryonic development and tumorigenesis. Many studies have shown that AP-2α functions in association with other interacting proteins. In a two-hybrid screening, the regulatory subunit β of protein casein kinase 2 (CK2β) was identified as an interacting protein of AP-2α; we confirmed this interaction using in-vitro GST pull-down and in-vivo co-immunoprecipitation assays; in an endogenous co-immunoprecipitation experiment, we further found the catalytic subunit α of protein casein kinase 2 (CK2α) also exists in the complex. Phosphorylation analysis revealed that AP-2α was phosphorylated by CK2 kinase majorly at the site of Ser429, and such phosphorylation could be blocked by CK2 specific inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) in a dose-dependent manner. Luciferase assays demonstrated that both CK2α and CK2β enhanced the transcription activity of AP-2α; moreover, CK2β increased the stability of AP-2α. Our data suggest a novel cellular function of CK-2 as a transcriptional co-activator of AP-2α.

Involvement of serine phosphorylation of spinal cord NR-2B subunit of the N-methyl-D-aspartate receptor following electroacupuncture stimulation

Kang Byeol-Rim,Choi Byung-Tae,Yoon Hyun-Min,Min Young-Kwang,Ahn Chang-Beohm 대한침구의학회 2007 대한침구의학회지 Vol.24 No.2

Objective : This study was to examine the low frequency EA is associated with NMDAR and phosphorylation of NMDAR NR-2B subunits in the spinal cordMethods : We investigated that only those rats without overt signs of spinal cord or root damage such as paralysis or lameness were used for experimentation. The NMDA antagonist, D-2-amino-5-phosphonopentanoic acid dissolved in sterile saline and injected intrathecally. Two stainless-steel needles were inserted in each hind leg at those acupoints corresponding to Zusanli(S36) and Sanyinjiao(Sp6) in man and wereconnected to an electric stimulator The EA wit 2 ㎐ stimulation started immediately after AP-5 injection for 30 min. Results : EA analgesia was slightly reduced by intrathecal injection of NMDAR antagonist AP-5, but analgesic effects of EA still showed at 60 minutes after termination of stimulation in all EA-treated group. In the Western blot analysis, the levels of NMDAR NR-2B and phospho-NR-2B were slightly induced by EA stimulation in the spinal cord. These expressions were significantly inhibited by spinal blockage of AP-5. As for regional reaction of NMDAR NR-2B and phospho-NR-2B, immunoreactions were observed throughout all laminae of the dorsal horn of spinal cord and weaker ones showed in the neck region. The mean IOD of phospho-NR-2B in the superficial laminae and nucleus proprius of EA-treated rats were significantly increased compared with normal ones, these expressions were decreased in EA-treated with AP-5 groupsConclusion : It is concluded that EA stimulation may be involved NMDAR activation through phosphorylation of spinal NMDAR NR-2B subunit. 목적 : 저주파에 해당하는 2 Hz 전침 자극이 척수 N-methyl-D-aspartate receptor (NMDAR)의 NR-2B subunit의 발현 및 인산화에 미치는 영향을 조사하였다. 방법 : Sprague-Dawley계 흰쥐를 Størkson 등의 방법에 의해 척수막의 지주막하강에 catheter를 삽입하는 수술을 행한 후 마비 등의 척수 손상을 나타내지 않는 개체를 대상으로 하였다. N-methyl-D-aspartate (NMDA) antagonist인 D-2-amino-5- phosphonopentanoic acid (AP-5)를 투여한 후 족삼리와 삼음교에 해당하는 부위에 30분간 전침 자극하였다. 무통각 여부는 hot plate test를 시행하였으며 NMDAR NR-2 subunit 발현과 인산화 여부는 Western blot과 면역조직화학적으로 살펴보았다. 결과 : 전침 무통각은 전침 자극 후 180분 후까지 지속되었으며 NMDA antagonist인 AP-5를 투여하였을 때 전침 무통각이 저하되었으나 유의성은 나타내지 않았다. Western blot 분석으로 보아 NMDAR NR-2B 및 인산화 NR-2B의 발현은 전침자극에 의해 미약한 증가를 보이나 AP-5투여에 의해 현저한 저해를 보였다. 면역조직화학에 의한 척수배각 구역별 발현을 보면 NMDAR NR-2B 및 인산화 NR-2B는 전 배각에 걸쳐 관찰되나 경부 (층판 Ⅴ-Ⅵ)에서 약한 반응을 보였다. 전침 자극에 의한 각 군별 NR-2B 발현은 유의한 차이를 보여 주지 않았으나 인산화 NR-2B는 천층 (층판 Ⅰ-Ⅱ)및 고유핵 (층판 Ⅲ-Ⅳ)에서 유의성 있는 증가를 보였다. 전침 자극시 AP-5 투여는 유의성은 보이지 않았으나 인산화 NR-2B발현을 저해하였다. 결론 : 저주파 2 Hz 전침에 의한 무통각은 NMDA antagonist인 AP-5 투여에 의해 저해될 뿐아니라 NMDAR NR-2B subunit의 인산화를 저해하는 것으로 보아 전침 무통각의 과정에 NMDAR 및 NMDAR NR-2B의 인산화가 관여함을 알 수 있다.

ATF2 전사인자의 발현과 AP-1 전사인자인 BATF, c-Fos, c-Jun과의 이량체 형성

장혜영,김재호,Jang Hye-Young,Kim Jae-Ho 한국생명과학회 2005 생명과학회지 Vol.15 No.6

ATF2는 c-Fos와 c-Jun과 같은 전사인자이며 이들은 루신지퍼 단백질이다. 루신지퍼 단백질은 동형이합체 혹은 이형이합체를 형성하며 promoter 영역에 결합하여 전사조절에 중요한 역할을 한다. 세포의 전사인자 ATF2는 ATF/CRE site에 결합하며 특히 선택적인 interfamily 이형이량체를 형성함으로써 전사 조절에 있어서 다양한 메카니즘을 제공할 수 있다. 본 연구에서는 ATF2 cDNA를 6xHis를 가진 expression vector에 subcloning하여 E.cnli BL2l에서 발현시켰다. 6xHis tag은 nickel-chelating chromatography를 가능하게 하였다 발현된 ATF2는 In vitro binding pull-down assay에서 동형이합체를 이를 뿐만 아니라 AP-1 그룹의 인자들과 선택적인 이형이합체를 형성함을 보여 주었다. BATF와는 강하게 결합하였으며 c-Jun과도 안정된 이합체를 형성하였다. 그러나 c-Fos와는 이합체를 형성하지 않으므로서 AP-1그룹 내에서도 이합체 형성이 선택적으로 이루어짐을 알 수 있다. ATF2 is a cellular transcription factor which belongs to the CREB/ATF class and it is leucine zipper protein which generally binds to DNA as dimers. This paper presents the procedure for subcloning the ATF2 gene and the results of experiment used the expressed ATF2. The pET expression vector was used since it produced 6xHis fusion protein for easy purification using affinity column. The Nickel chelating chromatography was used for Purifying the expressed ATE2 from E- codi BL2l. Subsequen시y In vitro binding pull-down assay showed the binding specificity of ATF2 with AP-1 family factors such as BATF, c-Fos, c-Jun and ATF2 itselgf. ATF2 forms homodimer as well as strong heterodimer with BATF. It also forms stable dimer with c-Jun but barely binds with c-Fos.

Endocytosis of somatodendritic NCKX2 is regulated by Src family kinase-dependent tyrosine phosphorylation

Lee, Kyu-Hee,Ho, Won-Kyung,Lee, Suk-Ho Frontiers Media S.A. 2013 Frontiers in cellular neuroscience Vol.7 No.-

<P>We have previously reported that the surface expression of K<SUP>+</SUP>-dependent Na<SUP>+</SUP>/Ca<SUP>2+</SUP> exchanger 2 (NCKX2) in the somatodendritic compartment is kept low by constitutive endocytosis, which results in the polarization of surface NCKX2 to the axon. Clathrin-mediated endocytosis is initiated by interaction of the μ subunit of adaptor protein complex 2 (AP-2) with the canonical tyrosine motif (YxxΦ) of a target molecule. We examined whether endocytosis of NCKX2 involves two putative tyrosine motifs (<SUP>365</SUP>YGKL and <SUP>371</SUP>YDTM) in the cytoplasmic loop of NCKX2. Coimmunoprecipitation assay revealed that the <SUP>365</SUP>YGKL motif is essential for the interaction with the μ subunit of AP-2 (AP2M1). Consistently, either overexpression of NCKX2-Y365A mutant or knockdown of AP2M1 in cultured hippocampal neurons significantly reduced the internalization of NCKX2 from the somatodendritic surface and thus abolished the axonal polarization of surface NCKX2. Next, we tested whether the interaction between the tyrosine motif and AP2M1 is regulated by phosphorylation of the 365th tyrosine residue (Tyr-365). Tyrosine phosphorylation of heterologously expressed NCKX2-WT, but not NCKX2-Y365A, was increased by carbachol (CCh) in PC-12 cells. The effect of CCh was inhibited by PP2, a Src family kinase (SFK) inhibitor. Moreover, PP2 facilitated the endocytosis of NCKX2 in both the somatodendritic and axonal compartments, suggesting that tyrosine phosphorylation of NCKX2 by SFK negatively regulates its endocytosis. Supporting this idea, activation of SFK enhanced the NCKX activity in the proximal dendrites of dentate granule cells (GCs). These results suggest that endocytosis of somatodendritic NCKX2 is regulated by SFK-dependent phosphorylation of Tyr-365.</P>

Cr2O3/AP 복합체 제조 및 그 열분해 특성

정재윤 ( Jae Yun Jung ),김재경 ( Jae Kyeong Kim ),심홍민 ( Hong Min Shim ),김현수 ( Hyoun Soo Kim ),구기갑 ( Kee Kahb Koo ) 한국공업화학회 2015 공업화학 Vol.26 No.2

Cr2O3/AP (ammonium perchlorate) energetic composites were prepared by a method of solvent/anti-solvent. XRD analysis revealed that the crystalline structure of AP in Cr2O3/AP composites is the same as that of pure AP. SEM photomicrograph shows that an average size of cuboid Cr2O3/AP composites is approximately 2.5 μm. TGA analysis shows that the addition of submicron Cr2O3 particles into AP lowers the HTD (high-temperature decomposition) compared to that of neat AP and the activation energy of the Cr2O3/AP composites was calculated by the isoconversional Starlink method. Considering changes in the activation energy, the decomposition reaction mechanism of AP was suggested as follows; the decomposition with the formation of nucleation sites renders formation of porous structure in the composites up to conversion of about 0.25 and after further conversion of over 0.3, it seems that decomposition reaction vigorously takes place rather than sublimation of AP.

Phosphorylation on TRPV4 824 serine residue regulates Association between TRPV4 and AP2mu1

( Sang Sun Kang ) 충북대학교 과학교육연구소 2016 과학교육연구논총 Vol.31 No.2

The TRPV4 cation channel, a member of the TRP vanilloid subfamily, is expressed in a broad range of tissues and participates in the generation of a Ca2+ signal and/ or depolarization of membrane potential. Here, I identified the medium (μ) subunits of clathrin adaptor (AP) complexes (AP2 mu1) which lap the pit of plasma membrane during the endocytosis, as the auxiliary protein of TRPV4 Ca2+ channels, using coimmunoprecipitation, confocal microscopy assays. TRPV4 forms a complex with AP2mul; this interaction seems to be mediated by the C-terminal cytoplasmic domain of TRPV4. Previously, I demonstrated that TRPV4 serine residue 824 (S824) is phosphorylated by serum/glucocorticoid regulated kinase 1. I elucidate here the effect of TRPV4 S824 phosphorylation on its association with AP2mu1, the regulatory protein for endocytosis. An inactivated mutant version of TRPV4, S824A, exhibited a decreased ability to bind AP2mul, whereas an activated mutant version of TRPV4, S824D, exhibited enhanced binding to AP2mul. Thus, association of the TRPV4 by AP2mu1 and localization of the channel to the plasma membrane appear to be regulated by the phosphorylation status of residue S824 in TRPV4. The identified protein appears to play a role in the molecular pathways modulating transport activity. Therefore, the interaction between these two proteins (TRPV4 and AP2 mu1) seems to explain how TRPV4 is localized in the Plasma membrane through endocytosis.

No Role of Protected Region B of Human Cytochrome P4501A2 Gene (CYP1A2) As an AP-1 Response Element

Chung, Injae,Jung, Kihwa 德成女子大學校 藥學硏究所 2002 藥學論文誌 Vol.13 No.1

Cytochrome P4501A2 (CYP1A2) is a member of the cytochrome P450 family of isozymes involved in the phase I drug metabolism of vertebrates. CYP1A2 is responsible for the activation of a number of aromatic amines to mutagenic and carcinogenic forms. Thus, the level of CYP1A2, which varies among different populations, may determine an individual's susceptibility to these chemicals. We have previously reported on the importance of a cis element named PRB (protected region B) in the regulation of human Cytochrome P4501A2 (CYP1A2) gene, sequence(-2218 to -2187 bp) revealed a putative AP-1 binding site, TGACTAA, at -2212 bp(Chung and Brenick, 1997). To elucidate the role of AP-1 in CYP1A2 regulation, we transiently overexpressed c-Jun and c-Fos transcription factors in human hepatoma HepG2 cells, and examined their influence on the CYP1A2 promoter activity by reporter gene assays. Cotransfection of the c-Jun and the c-Fos expression vectors increased the induced transactivation by five to six fold from the CYP1A2 promoter constructs. However, deletion of the PRB element did not affect the degree of activation by the c-Jun and the c-Fos. Therefore, it is unlikely that the c-Jun and the c-fos activate the CYP1A2 promoter through this AP-1 consensus-like sequence in the PRB region.

No Role of Protected Region B of Human Cytochrome P4501A2 Gene (CYP1A2) As an AP-1 Response Element

Chung, In-Jae,Jung, Ki-Hwa The Pharmaceutical Society of Korea 2002 Archives of Pharmacal Research Vol.25 No.3

Cytochrome P4501A2 (CYP1A2) is a member of the cytochrome P450 family of isozymes involved in the phase I drug metabolism of vertebrates. CYP1A2 is responsible for the activation of a number of aromatic amines to mutagenic and carcinogenic forms. Thus, the level of CYP1A2, which varies among different populations, may determine an individual's susceptibility to these chemicals. We have previously reported on the importance of a cis element named PRB (protected region B) in the regulation of human Cytochrome P4501A2 (CYP1A2) gene, which appeared to act as a positive regulatory element. Closer examination of the PRB sequence (-2218 to -2187 bp) revealed a putative AP-1 binding site, TGACTAA, at -2212 bp (Chung and Bresnick, 1997). To elucidate the role of AP-1 in CYP1A2 regulation, we transiently overexpressed c-Jun and c-Fos transcription factors in human hepatoma HepG2 cells, and examined their influence on the CYP1A2 promoter activity by reporter gene assays. Cotransfection of the c-Jun and the c-Fos expression vectors increased the induced transactivation by five to six fold from the CYP1A2 promoter constructs. However, deletion of the PRB element did not affect the degree of activation by the c-Jun and the c-Fos. Therefore, it is unlikely that the c-Jun and the c-Fos activate the CYP1A2 promoter through this AP-1 consensus-like sequence in the PRB region.