Salli U, Saito N, Stormshak F
Department of Biochemistry/Biophysics, Oregon State University, Corvallis, Oregon 97331, USA.
Biol Reprod. 2003 Dec;69(6):2053-8. doi: 10.1095/biolreprod.103.017640. Epub 2003 Aug 20.
In the bovine corpus luteum (CL) phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) protein in response to prostaglandin F2alpha (PGF2alpha) is correlated with the secretion of oxytocin. The present study was conducted to 1) examine the intracellular translocation characteristics of wild-type and mutant forms of a green fluorescent protein (GFP)-conjugated MARCKS (MARCKS-GFP) after PGF2alpha treatment and 2) evaluate PGF2alpha-induced temporal changes in MARCKS-GFP and actin cortex associated with exocytosis of oxytocin. In experiment 1, cells of the bovine CL were cultured on coverslips overnight. Then, wild-type and mutant MARCKS-GFP constructs were transfected separately into cells and expression was detected through fluorescence microscopy. Forty-eight hours after transfection, cells were treated with vehicle, PGF2alpha (56 nM), or a phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA], 1 microM). Treatment of cells expressing wild-type MARCKS-GFP with PGF2alpha and TPA resulted in translocation of MARCKS from the plasma membrane to the cytoplasm within 2.5 min. Phosphorylation mutant MARCKS-GFP (m3) protein was localized on the plasma membrane, and treatments did not cause its translocation to the cytoplasm. Myristoylation mutant MARCKS-GFP (G2A) was observed solely in the cytoplasm, and no changes were detected in the intracellular location of this mutant MARCKS after treatment. In experiment 2, luteal cells were transfected with one of the three MARCKS-GFP constructs. Cells were then fixed and probed sequentially for oxytocin and filamentous actin. Results revealed that only wild-type MARCKS-GFP transfected large luteal cells contained advanced signs of exocytosis (peripheral movement of oxytocin vesicles; shorter actin filaments) with translocation of MARCKS-GFP from membrane to cytoplasm in response to PGF2alpha treatment. These data demonstrate that phosphorylation of membrane-bound MARCKS protein is requisite for exocytosis of oxytocin to occur in bovine large luteal cells.
在牛黄体（CL）中，肉豆蔻酰化富含丙氨酸的C激酶底物（MARCKS）蛋白对前列腺素F2α（PGF2α）的磷酸化反应与催产素的分泌相关。本研究旨在：1）检测PGF2α处理后绿色荧光蛋白（GFP）偶联的MARCKS（MARCKS-GFP）野生型和突变型的细胞内转位特征；2）评估PGF2α诱导的与催产素胞吐相关的MARCKS-GFP和肌动蛋白皮质的时间变化。在实验1中，将牛CL细胞在盖玻片上培养过夜。然后，将野生型和突变型MARCKS-GFP构建体分别转染到细胞中，并通过荧光显微镜检测表达情况。转染后48小时，用溶剂、PGF2α（56 nM）或佛波酯（12-O-十四酰佛波醇-13-乙酸酯[TPA]，1 μM）处理细胞。用PGF2α和TPA处理表达野生型MARCKS-GFP的细胞，导致MARCKS在2.5分钟内从质膜转位到细胞质。磷酸化突变型MARCKS-GFP（m3）蛋白定位于质膜，处理未导致其转位到细胞质。肉豆蔻酰化突变型MARCKS-GFP（G2A）仅在细胞质中观察到，处理后该突变型MARCKS的细胞内位置未检测到变化。在实验2中，用三种MARCKS-GFP构建体之一转染黄体细胞。然后固定细胞，并依次检测催产素和丝状肌动蛋白。结果显示，只有转染野生型MARCKS-GFP的大黄体细胞含有胞吐的晚期迹象（催产素囊泡的外周移动；较短的肌动蛋白丝），并且响应PGF2α处理，MARCKS-GFP从膜转位到细胞质。这些数据表明，膜结合的MARCKS蛋白的磷酸化是牛大黄体细胞中催产素胞吐发生的必要条件。
