• 생명과학회지
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• 제21권1호
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• pp.31-35
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• 2011

Dual-specificity protein phosphatase (DUSP)들은 인산화된 티로신 잔기와 인산화된 세린 또는 트레오닌 잔기를 탈인산화시키는 단백질 탈인산화효소 군을 이루고 있으며, 대부분의 DUSP들은 세포의 생존이나 분화에 관여하고 있다. 본 연구에서는 잘 알려지지 않은 인간 유래의 dual-specificity protein phosphatase인 DUSP28을 인간신장 cDNA에서 분리하였다. 대장균에서 생산된 재조합단백질은 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP)에 대하여 좋은 활성을 보였다. 다양한 저해제와 2가 금속이온들이 DUSP28의 활성에 미치는 영향을 조사하였다. 다른 DUSP들에서와는 다르게, $Zn^{2+}$은 DUSP28의 탈인산화활성을 강하게 억제하였다. 이러한 결과로부터 DUSP28이 Zn과 연관된 신호전달경로에 관여할 것으로 추정된다. 더욱이, DUSP28은 인산화된 티로신잔기를 더욱 선호하는 경향이 있는 것으로 나타났고, 이는 세포 내에서도 비슷한 작용을 할 것으로 예상된다.

• BMB Reports
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• 제34권6호
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• pp.517-525
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• 2001

Six renaturable protein kinases that utilize the myelin basic protein (MBP) as a substrate were activated during prolonged exposure of cardiac myocytes to okadaic acid (OA). We characterized the substrate preference and activation of these kinases, with particular emphasis on 3 novel kinases-MBPK-55, MBPK-62 and MBPK-87. The transcription factors c-Jun, Elk, ATF2, and c-Fos that are used to assess mitogen-activated protein kinase activation were all poor substrates for these three kinases. MAPKAPK2 was also not phosphorylated. In contrast, Histone IIIS was phosphorylated by MBPK-55 and MBPK-62. These protein kinases were activated in cultured cardiac fibroblasts, H9c2 cardiac myoblasts, and Cos cells. High concentrations (0.5 to $1\;{\mu}M$) of OA were essential for the activation of the protein kinases in all of the cell types examined, whereas calyculin A [an inhibitor of protein phosphatase 1 (PP1) and PP2A], cyclosporin A (a PP2B inhibitor), and an inactive OA analog all failed to activate these kinases. The high dose of okadaic acid that is required for kinase activation was also required for phosphatase inhibition, as assessed by immunoblotting whole cell lysates with anti-phosphothreonine antibodies. A variety of chemical inhibitors, including PD98059 (MEK-specific), genistein (tyrosine kinase-specific) and Bisindolylmaleimide I (protein kinase C-specific), failed to inhibit the OA activation of these kinases. Thus, MBPK-55 and MBPK-62 are also Histone IIIS kinases that are widely expressed and specifically activated upon exposure to high OA concentrations.

• Bulletin of the Korean Chemical Society
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• 제18권4호
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• pp.428-432
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• 1997

The dephosphorylation specificity of protein phosphatase 2A (PP2A), calcineurin (PP2B) and protein phosphatase 2C (PP2C) were studied in vitro using myelin basic protein (MBP) as a model substrate which was fully phosphorylated at multiple sites by protein kinase C (PKC) or cyclic AMP-dependent protein kinase (PKA). In order to determine the site specificity of phosphates in myelin basic protein, the protein was digested with trypsin and the radioactive phosphopeptide fragments were isolated by high performance liquid chromatography (HPLC) on reversed-phase column. Subsequent analysis and/or sequential manual Edman degradation of the purified phosphopeptides revealed that Thr-65 and Ser-115 were most extensively phophorylated by PKA and Ser-55 by PKC. For the dephosphorylation kinetics, the phosphorylated MBP was treated with calcineurin or PP2C with various time intervals and the reaction was terminated by direct tryptic digest. Both Thr-65 and Ser-115 residues were dephosphorylated more rapidly than any other ones by phosphatases. However it can be differentiated further by first-order kinetics that the PP2B dephosphorylated both Thr-65 and Ser-115 with almost same manner, whereas PP2C dephosphorylated somewhat preferentially the Ser-115.

• BMB Reports
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• 제30권3호
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• pp.222-228
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• 1997

Protein phosphatase 2C (PP2C) is one of the four major serine/threonine phosphatases which is dependent on $Mg^{2+}$ for its activity. PP2C was purified from rat liver cytosol and its characteristics were investigated. The substrate employed for routine assay was $[^{32}P]casein$ phosphorylated by PKA. The purification process involved DEAE chromatography, ammonium sulfate fractionation, phenyl sepharose chromatography, sephacryl 5-200 gel filtration, and histone agarose chromatography. The SDS-PAGE of PP2C showed one major single protein band at a position corresponding to a molecular mass of 43 kd and the purification fold was 637. The enzyme showed a pH optimum of 8 and $K_M$ value was $1.9\;{\mu}M$. However, when the substrate was changed to $[^{32}P]histone$, the pH optimum was shifted to 7 and $K_M$ value was $2.3\;{\mu}M.\;Mg^{2+}$ was essential to the enzyme activity and okadaic acid did not exert any inhibitory effect on the enzyme. To examine residue in the active site of PP2C effects of some protein-modifying reagents were tested.

• Molecules and Cells
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• 제42권7호
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• pp.546-556
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• 2019

Protein phosphatase 4 (PP4) is a crucial protein complex that plays an important role in DNA damage response (DDR), including DNA repair, cell cycle arrest and apoptosis. Despite the significance of PP4, the mechanism by which PP4 is regulated remains to be elucidated. Here, we identified a novel PP4 inhibitor, protein phosphatase 4 inhibitory protein (PP4IP) and elucidated its cellular functions. PP4IP-knockout cells were generated using the CRISPR/Cas9 system, and the phosphorylation status of PP4 substrates (H2AX, KAP1, and RPA2) was analyzed. Then we investigated that how PP4IP affects the cellular functions of PP4 by immunoprecipitation, immunofluorescence, and DNA double-strand break (DSB) repair assays. PP4IP interacts with PP4 complex, which is affected by DNA damage and cell cycle progression and decreases the dephosphorylational activity of PP4. Both overexpression and depletion of PP4IP impairs DSB repairs and sensitizes cells to genotoxic stress, suggesting timely inhibition of PP4 to be indispensable for cells in responding to DNA damage. Our results identify a novel inhibitor of PP4 that inhibits PP4-mediated cellular functions and establish the physiological importance of this regulation. In addition, PP4IP might be developed as potential therapeutic reagents for targeting tumors particularly with high level of PP4C expression.

• BMB Reports
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• 제28권4호
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• pp.331-335
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• 1995

Simian virus 40 (SV40) small-t antigen (small-t) has been known to regulate the activity of a cellular enzyme, protein phosphatase 2A (PP2A), composed of A. B, and C subunits, via binding to the A subunit In the study presented here, the stoichiometry of the binding of small-t to PP2A was determined to be 1: 1. It was also shown that small-t binds to the AC form of PP2A with a higher apparent affinity than it binds to the free A subunit. We also characterized the interaction of PP2A with wild-type and various mutant small-ts. A single-point mutant (Val134Met) and a double-point mutant (Trp147Gly;Leu152 Pro) of small-t exhibited 3-fold and 5-fold lower potencies in inhibiting PP2A activity. respectively. This suggests that the region around amino acids between 134 and 152 of small-t might be important in regulating the enzyme activity of PP2A.

• 생명과학회지
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• 제27권3호
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• pp.370-381
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• 2017

단백질 탈 인산화는 단백질 탈 인산화 효소에 의해 매개되는 과정으로 세포 생존에 매우 중요하다. 단백질 탈 인산화 효소 중에서 최근 CTD (carboxy-terminal domain) 탈 인산화 효소들이 등장하고 있으며 이들에 대한 새로운 생물학적 역할이 밝혀지고 있다. 이 효소의 그룹에는CTD 탈 인산화 효소 1(CTDP1), CTD 소형 탈 인산화 효소 1(CTDSP1), CTD 소형 탈 인산화 효소 2(CTDSP2), CTD 소형 탈 인산화 효소 유사(CTDSPL), CTD 소형 탈 인산화 효소 유사 2(CTDSPL2), CTD 핵 탈 인산화 효소(CTDNEP1) 및 유비퀴틴 유사 도메인 함유CTD 탈 인산화 효소 1(UBLCP1)들이 존재한다. CTDP1은 RNA 중합 효소 II (RNAPII)의 CTD의 두 번째 인산화 된 세린을 탈 인산화 시키고, CTDSP1, STDSP2 및 CTDSPL은 RNAPII의 CTD의 다섯 번째 인산화 된 세린을 탈 인산화 시킨다. 그리고 CTDSP1은 SMAD들, CDCA3, Twist1, 종양억제 단백질인 PML, c-Myc과 같은 새로운 기질을 탈 인산화 시키는 것으로 밝혀지고 있다. CTDP1은 유사 분열 조절 및 암세포 성장과 관련이 있다. CTDSP1, CTDSP2 및 CTDSPL은 종양 억제 기능 및 줄기 세포 분화와 관련이 있다. CTDNEP1은 LIPIN1을 탈 인산화 시키고 핵막 형성과 관련이 있다. CTDSPL2는 조혈 줄기 세포 분화와 관련이 있다. UBLCP1은 26S 프로테아좀을 탈 인산화 시키고 핵 프로테아좀 활성 조절과 관련이 있다. 결론적으로, CTD 탈 인산화 효소의 새로운 기능과 역할은 최근의 연구에서 밝혀지고 있으며, 이 리뷰는 CTD 탈 인산화 효소의 새롭게 밝혀진 역할들을 요약하고자 정리한 것이다.

• Journal of Applied Biological Chemistry
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• 제50권2호
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• pp.58-62
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• 2007

Identification of Dual-specificity protein phosphatase (DSP) substrates is essential in revealing physiological roles of DSPs. We isolated VHZ-interacting proteins from extracts of 293T cells overexpressing a VHZ (C95S, D65A) mutant known to be substrate- trapping mutant. Analysis of specific proteins bound to VHZ by 2D gel electrophoresis and mass spectroscopy revealed that these proteins contained Chaperonin containing TCP1, Type II phosphatidylinositol phosphate kinase ${\gamma}$, Intraflagellar transport 80 homolog, and Kinesin superfamily protein 1B. VHZ-interacting proteins showed that VHZ is involved in many important cellular signal pathways such as protein folding, molecular transportation, and tumor suppression.

• The Korean Journal of Physiology and Pharmacology
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• 제18권2호
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• pp.155-161
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• 2014

Overexpression of amyloid precursor protein with the Swedish mutation causes abnormal hyperphosphorylation of the microtubule-associated protein tau. Hyperphosphorylated isoforms of tau are major components of neurofibrillary tangles, which are histopathological hallmarks of Alzheimer's disease. Protein phosphatase 2A (PP2A), a major tau protein phosphatase, consists of a structural A subunit, catalytic C subunit, and a variety of regulatory B subunits. The B subunits have been reported to modulate function of the PP2A holoenzyme by regulating substrate binding, enzyme activity, and subcellular localization. In the current study, we characterized regulatory B subunit-specific regulation of tau protein phosphorylation. We showed that the PP2A B subunit PPP2R2A mediated dephosphorylation of tau protein at Ser-199, Ser-202/Thr-205, Thr-231, Ser-262, and Ser-422. Down-regulation of PPP2R5D expression decreased tau phosphorylation at Ser-202/Thr-205, Thr-231, and Ser-422, which indicates activation of the tau kinase glycogen synthase kinase 3 beta ($GSK3{\beta}$) by PP2A with PPP2R5D subunit. The level of activating phosphorylation of the $GSK3{\beta}$ kinase Akt at Thr-308 and Ser-473 were both increased by PPP2R5D knockdown. We also characterized B subunit-specific phosphorylation sites in tau using mass spectrometric analysis. Liquid chromatography-mass spectrometry revealed that the phosphorylation status of the tau protein may be affected by PP2A, depending on the specific B subunits. These studies further our understanding of the function of various B subunits in mediating site-specific regulation of tau protein phosphorylation.

• 생명과학회지
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• 제24권1호
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• pp.14-19
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• 2014

Kinesin-1은 2개의 장쇄(KHCs, 또는 KIF5s)와 2개의 단쇄(KLCs)가 결합한 복합체로 되어 있다. 본 연구에서 효모 two-hybrid system을 이용하여 중추신경계의 신경세포에서 주로 발현되는 KIF5A와 결합하는 단백질을 탐색한 결과 phosphatidylinositol-3,5-bisphosphate ($PI(3,5)P_2$)의 5번 위치 인산을 제거하는 탈인산화효소 Fig4(Sac3)를 분리하였다. KIF5A는 Fig4의 C-말단과 결합함을 효모 two-hybrid assay로 확인하였다. Fig4는 KIF5A의 C-말단과 결합하지만, 두 개의 다른 장쇄인 KIF5B와 KIF5C 그리고 KLC1와는 결합하지 않았다. 단백질 간 결합을 glutathione S-transferase pull-down assay와 공동면역침강으로 추가 검증하였다. 생쥐의 뇌 파쇄액을 KIF5A 항체로 면역 침강한 결과 Fig4가 같이 침강하였다. 이러한 결과들은 kinesin-1이 Fig4와 결합한 단백질 복합체 혹은 운반체를 세포 내에서 운반함을 시사한다.