Phosphorylation of MRP14, an S100 protein expressed during monocytic differentiation, modulates Ca(2+)-dependent translocation from cytoplasm to membranes and cytoskeleton.
Phosphorylation of MRP14, an S100 protein expressed during monocytic differentiation, modulates Ca(2+)-dependent translocation from cytoplasm to membranes and cytoskeleton.
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DOI：
10.4049/jimmunol.156.3.1247
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发表时间：
1996-02
影响因子：
4.4
通讯作者：
C. V. D. Bos;Johannes Roth;Hans-Georg Koch;M. Hartmann;C. Sorg
中科院分区：
文献类型：
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作者：
C. V. D. Bos;Johannes Roth;Hans-Georg Koch;M. Hartmann;C. Sorg
MRP 8和MRP 14是在骨髓单核细胞中表达的两种Ca（2+）结合蛋白。MRP 8和MRP 14的复合物以Ca（2+）依赖的方式与膜和中间丝共定位。与MRP 8不同，MRP 14以两种同种型存在，其中较小的（MRP 14 '）已被证明缺乏前四个氨基酸; MRP 14和MRP 14'也以磷酸化形式存在。如本工作所示，通过用[35 S]甲硫氨酸代谢标记单核细胞，MRP 14和MRP 14 '同时翻译。通过PCR分析，我们没有发现存在不同mRNA种类的证据。由于MRP 14是由单拷贝基因编码的，我们的数据表明，MRP 14 '的形成是由于单个mRNA种类的选择性翻译。[32 P]正磷酸盐标记的单核细胞蛋白质的二维电泳，然后通过Western印迹和放射自显影显示，两个磷酸化的MRP 14亚型纳入了大部分的放射性单核细胞蛋白质。使用差速离心，我们证明了在不同的亚细胞位置存在不同的亚型模式。此外，在响应升高的Ca 2+浓度，我们观察到一个优先易位的磷酸化MRP 14亚型从细胞质向膜和细胞骨架。这可能是由钙结合改变引起的，实际上，使用等电聚焦和45 Ca 2+覆盖，与含有其他MRP 14同种型的条带相比，含有磷酸化MRP 14的MRP 14条带显示出增加的Ca 2+结合。这是磷酸化MRP 14亚型与非磷酸化MRP 14亚型相比功能差异的第一个证据。这些功能差异表明，MRP 14代表了MRP 8/MRP 14复合物的调节亚基。
MRP8 and MRP14 are two Ca(2+)-binding proteins expressed in myelomonocytic cells. Complexes of MRP8 and MRP14 colocalize with membranes and intermediate filaments in a Ca(2+)-dependent manner. MRP14, unlike MRP8, exists in two isoforms, the smaller of which (MRP14') has been shown to lack the first four amino acids; both MRP14 and MRP14' are also present as phosphorylated forms. As shown in the present work by metabolic labeling of monocytes with [35S]methionine, MRP14 and MRP14' are translated simultaneously. By PCR analysis we found no evidence for the presence of different mRNA species. Since MRP14 is encoded by a single copy gene, our data indicate that MRP14' formation is due to alternative translation of a single mRNA species. Two-dimensional electrophoresis of [32P]orthophosphate-labeled monocyte proteins followed by Western blotting and autoradiography revealed that the two phosphorylated MRP14 isoforms incorporated the bulk of the radioactivity found in monocytic proteins. Using differential centrifugation we demonstrated the presence of distinct isoform patterns in different subcellular locations. Further, in response to elevated Ca2+ concentrations we observed a preferential translocation of phosphorylated MRP14 isoforms from the cytosol toward membranes and the cytoskeleton. This might be caused by altered calcium binding, and indeed, using isoelectric focusing and 45Ca2+ overlay the MRP14 band containing phosphorylated MRP14 revealed increased Ca2+ binding compared with bands containing other MRP14 isoforms. This represents the first evidence for functional differences in phosphorylated MRP14 isoforms compared with nonphosphorylated MRP14 isoforms. These functional differences suggest that MRP14 represents the regulatory subunit of MRP8/MRP14 complexes.