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유방의 양성종양과 악성종양에서 Phospholipase C-γ1의 발현양상
김희정,정기묵,김성원,문병인,안수정,노동영,유근영,최국진 대한외과학회 2002 Annals of Surgical Treatment and Research(ASRT) Vol.62 No.6
Purpose: The activation of phospholipase C (PLC) is one of the early events in various growth processes, including malignant transformation. Among PLC-isozymes, PLC-γ1 is activated through direct interaction with growth factor receptor tyrosine kinase. In this study, we evaluated the patterns of PLC-γ1 expression in benign and malignant tumors of the breast and compared the patterns with their normal counterpart. Methods: Using immunoblot assay, we evaluated the patterns of expression in PLC-γ1 in 20 breast cancer tissues, 13 fibroadenoma tissues, and normal tissues. The level of expression was analyzed by densitometry. Results: All of 20 breast cancer tissues and 13 fibroadenoma tissues showed overexpression of PLC-γ1 when compared with their normal counterparts. The level of the PLC-γ1 expression was 3.9-fold and 17.3-fold higher in fibroadenomas and breastcancers, respectively, than in normal tissues. Conclusion: The result suggested that the level of PLC-γ1 expression increases as the normal tissue undergoes progression to fibroadenoma, and finally to carcinoma. The pattern of expression of PLC-γ1 in breast tissue implies that the PLC-γ1-mediated signal transduction may play a significant role in the progression of breast cancer from normal tissue. (J Korean Surg Soc 2002;62:463-467)
Hwang, Sung Chul,Jhon, Deok-Young,Rhee, Sue Goo 아주대학교 의과학연구소 1996 아주의학 Vol.1 No.1
During the purification of phospholipase C (PLC)-rl which was over-expressed in HeLa cells transfected with a recombinant vaccinia virus carrying a complete cDNA sequence for PLC-γ₁ we noticed the presence of a heat-stable activator in crude cytosolic extract of HeLa cells which markedly stimulated phosphatidylinositol (PI) hydrolysis, when reconstituted with purified PLC-γ₁. Moreover, this putative factor was also found to be present in bovine brain cytosol. Subsequently, based on its ability to stimulate PI hydrolysis of PLC-γ₁ as an assay, this activation factor was purified to homogeneity from bovine brain cytosol. It was purified by heat treatment, trichloroacetic acid precipitation, and successive chromatographic steps on DEAE-5PW, phenyl-5PW, and heparin-5PW HPLC columns. The purified protein, as seen on SDS-PAGE, consisted of 4 or 5 closely spaced bands of apparent molecular weight between 48 and 62 kDa. However, on gel filtration chromatography on TSK-G3000SW HPLC, the estimated molecular weight was revealed to be approximately 350 kDa. The purified activator was identified as a microtubule-associated protein tau by electroelution from an SDS-polyacrylamide gel, partial peptide mapping by Staphylococcal V_(8) protease, and amino acid sequencing of cyanogen bromide cleaved peptides. The identity of the activator was re-confirmed by an immunoblotring using a specific anti-lau monoclonal antibody. In addition, reconstituting PLC-γ₁ with tau protein purified by an alternative method described elsewhere^ resulted in the same magnitude of activation. Tau protein mediated activation of PLC isozymes was calcium dependent. Isozyme specific activation of PLCs in 0.1% deoxycholate substrate appeared to be preferential toward PI hydrolysis. The magnitude of activation of PLC isozymes in PI substrate were PLC-γ₁ > PLC-r2 > PLC-δ₁ > PLC-β₁ in decreasing order. Approximately 200 nM concentration of tau-protein produced 15.5-, 5.4-, 4.2-, and 1.6-fold increase in activity of these enzymes, respectively, in the presence of 1 mM free calcium ion.
Phospholipase C γ1의 Src homology 3 (SH3)-domains에 결합하는 단백질의 특성규명
안수정,박태규,모효정,김찬길 건국대학교 자연과학연구소 1998 建國自然科學硏究誌 Vol.9 No.1
본 연구는 세포내 신호전달과정에서 중요한 구성요소인 Src Homology 3(SH3) domain 에 특이하게 결합하는 단백질을 분리하여, 그 특성을 규명함으로써 PLC γ1에 의한 신호전달과정을 규명하고자 하였다. SH3-domain 은 신호전달과정에 관련된 많은 단백질에서 발견되며 다수의 프롤린잔기를 인지함으로써 단백질간의 신호 전달을 매개하는 것으로 사료된다. 본 연구에서는 쥐 뇌의 추출물에서 PLC γ1의 SH3-domain에 특이하게 결합하는 110-kDa 와 100-kDa의 단백질을 순수 분리하여 펩티드 서열을 분석한 결과, 둘 다 시냅스소포 흡수과정에 필수적인 'dynamin' 단백질임을 확인하였다. Dynamin 이 PLC γ1의 SH3-domain 에 특이적으로 결합하는 것으로 보아 PLC γ1은 dynamin과 더블어 시냅스소포 흡수과정에 중요한 작용을 하는 것으로 사료된다. Src homology 3 (SH3) domains are found in a variety of proteins that are involved in signal transduction or represent components of the cytoskeleton. These domains are thought to serve as modules that mediate specific protein-protein interactions that include proline-rich sequences on the target protein. We have identified proteins of 110- and 100-kDa in rat brain extract that bind specifically to the SH3 domain of phospholipase C γ1(PLC γ1), a primary substrate of receptor tyrosine kinases, and characterized the both bands as the microtubule-activated GTPase dynamin. These results suggest that PLC γ1 might be involved in synaptic vesicle endocytosis by interacting with dynamin.
자궁근종에서의 Phospholipase C 발현에 관한연구
윤혜원 동국대학교 경주대학 1993 東國論集 Vol.12 No.-
Phosphoinositide-specific phospholipase C(PLC) is one of the key molecules in signal transduction for celular activity such as proliferation and differentiation. However, the biological significance of their molecules in carcinogenesis or tumor progression is not defined yet. Using PLC isozyme - specific antibodies, the relative contents of PLC isozyme have been examined in uterine myoma and normal myometrium. Immuno-reactive analysis revealed considerably hither levels of PLC-γ1 protein in all uterine myoma and little difference in PLC-β1, when compared to normal myometrial tissue. From Western blotting, PLC-γ1 showed 3 to 4 fold more expression in uterine myoma than that in normal myometrial tissue. There also is a linear relationship between activity and amount in PLC-γ1 increase of myoma. Taken together, the fact that the majority of uterine myoma displays elevated level of PLC-γ1 expression implies that PLC-γ1 mediated signal transduction may have significant role in proliferation and differentiation of myoma.
Point Mutations in the Split PLC-γ1 PH Domain Modulate Phosphoinositide Binding
( Sung Kuk Kim ),( Sung Mo Wee ),( Jong Soo Chang ),( Taeg Kyu Kwon ),( Do Sik Min ),( Young Han Lee ),( Pann Ghill Suh ) 생화학분자생물학회 2004 BMB Reports Vol.37 No.6
A number of signaling molecules contain small pleckstrin homology (PH) domains capable of binding phosphoinositides or proteins. Phospholipase C (PLC)-γ1 has two putative PH domains, an NH₂-terminal (PH1) and a split PH domain (nPH₂ and cPH₂). We previously reported that the split PH domain of PLC-γ1 binds to phosphatidγ1inositol 4-phosphate (PI(4)P) and phosphatidγ1inositol 4,5-bisphosphate (PI(4,5)P₂) (Chang et al., 2002). To identify the amino acid residues responsible for binding with PI(4)P and PI(4,5)P₂, we used site-directed mutagenesis to replace each amino acid in the variable loop-1 (VL-l) region of the PLC-γ1 nPH₂ 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-γ1 nPH2 domain mutants, P500A and HSO3A, showed reduced affinities for phosphoinositide binding. Furthermore, these mutant PLC-γ1 molecules showed reduced PI(4,5)P₂ hydrolysis. Using green fluorescent protein (GFP) fusion protein system, we showed that both PH₁ and nPH₂ domains are responsible for membrane-targeted translocation of PLC-γ1 upon serum stimulation. Together, our data reveal that the amino acid residues Pro^(500) and His^(503) are critical for binding of PLC-γ1 to one of its substrates, PI(4,5 )P₂ in the membrane.