Haneji T, Morimoto H, Morimoto Y, Shirakawa S, Kobayashi S, Kaneda C, Shima H, Nagao M
First Department of Oral Anatomy, Kyushu Dental College, Kitakyushu, Japan.
Biochem Biophys Res Commun. 1998 Jul 9;248(1):39-43. doi: 10.1006/bbrc.1998.8913.
The cytolocalization of protein phosphatase type 1 catalytic subunits in exponentially growing mouse osteoblastic MC3T3-E1 cells was determined. Formaldehyde-fixed and alcohol-permeabilized cultured cells were reacted with the PP1 alpha, PP1 delta, PP1 gamma 1, and PP1 gamma 2 antibodies using immunohistochemical methods. With PP1 alpha antibody intense staining occurred in the nuclei, while with PP1 delta antibody nucleolus-like bodies were intensely stained. PP1 gamma 1 localized in the perinuclear region and in the nucleus of the cultured cells, with the staining reaction of the former being much stronger than that in the latter. An immunoreaction did not occur in the cells interacted with PP1 gamma 2 antibody or with the normal rabbit serum. Proteins were prepared from the exponentially growing cells and subconfluent cells. Cellular fractionation was also done with the exponentially growing cells and proteins were prepared from each fraction. Each protein preparation was subjected to SDS-PAGE followed by Western blot analysis with the antibodies. PP1 alpha recognized the 38 kDa proteins mainly present in the nucleus, whereas PP1 delta interacted with the proteins in the nucleolar fraction whose molecular weight was estimated as 37 kDa. PP1 gamma 1 antibody recognized a band corresponding to an estimated molecular weight of 36 kDa mainly in the cytosolic fraction. PP1 gamma 2 antibody and the normal rabbit serum did not interact with any proteins prepared from the cultured cells. Our observations show that four different isozymes of protein phosphatases occupy distinct compartments in MC3T3-E1 cells. This differential distribution suggests that these isozymes may play different roles in cellular functions.
测定了蛋白磷酸酶1催化亚基在指数生长期小鼠成骨细胞MC3T3-E1中的细胞定位。采用免疫组织化学方法，使甲醛固定并经乙醇通透处理的培养细胞与PP1α、PP1δ、PP1γ1和PP1γ2抗体反应。用PP1α抗体时，细胞核出现强烈染色；用PP1δ抗体时，核仁样小体被强烈染色。PP1γ1定位于培养细胞的核周区域和细胞核，前者的染色反应比后者强得多。与PP1γ2抗体或正常兔血清相互作用的细胞中未出现免疫反应。从指数生长期细胞和亚汇合细胞中制备蛋白质。对指数生长期细胞也进行了细胞分级分离，并从每个级分中制备蛋白质。每种蛋白质制备物都进行SDS-PAGE，然后用抗体进行蛋白质印迹分析。PP1α识别主要存在于细胞核中的38 kDa蛋白质，而PP1δ与核仁级分中分子量估计为37 kDa的蛋白质相互作用。PP1γ1抗体主要在细胞溶质级分中识别一条对应于估计分子量为36 kDa的条带。PP1γ2抗体和正常兔血清与从培养细胞中制备的任何蛋白质均不相互作用。我们的观察结果表明，蛋白磷酸酶的四种不同同工酶在MC3T3-E1细胞中占据不同的区室。这种差异分布表明这些同工酶可能在细胞功能中发挥不同作用。
