Chiesa R, Noguera I, Sredy J
Department of Ophthalmology, Columbia University, New York, NY 10032, USA.
Exp Eye Res. 1997 Aug;65(2):223-9. doi: 10.1006/exer.1997.0324.
Small heat shock proteins and alpha-crystallins are related proteins with several common structural and functional properties including homologous amino acid sequences and similar chaperone-like activity. Furthermore, small heat shock proteins and alpha-crystallins are phosphorylated in vivo at serine residues within homologous amino acid sequences. During the differentiation of lens epithelial cells to fiber cells, significant changes in the patterns of expression and phosphorylation of alpha-crystallins take place, leading to the accumulation of phosphorylated forms of these proteins in lens fiber cells. To determine whether the small heat shock protein HSP25 undergoes phosphorylation in lens cells and to ascertain whether its phosphorylation state changes during lens cell differentiation, a comparative analysis of the HSP25 phosphorylation pattern in epithelial and fiber cells was undertaken. Analysis of phosphorylated and non-phosphorylated forms of HSP25 was carried out in cell extracts from rat lens epithelium and cortex by isoelectric focusing and Western blot using an antibody specific for the recombinant murine protein. The phosphorylated forms were identified by their isoelectric points and the characteristic shift upon in vitro dephosphorylation with phosphoprotein phosphatase 2B. HSP25 accounted for up to 2.4% of the protein content of rat lens extracts where it was present predominantly in mono- and bi-phosphorylated forms. Compared to epithelial cells extracts, the fiber cells extracts contained 67% more total HSP25 and a significantly higher proportion of bi-phosphorylated form. Phosphorylated HSP25 was sensitive to dephosphorylation by phosphoprotein phosphatase 2B in both cell extracts but the apparent dephosphorylation rate was significantly slower in the fiber cell extracts. The results demonstrate that HSP25 is phosphorylated in the lens in vivo. Furthermore, synthesis and phosphorylation of HSP25 change with lens cell differentiation resulting in a significant accumulation of bi-phosphorylated form in the fiber cells. These findings indicate that HSP25 and its phosphorylation may have important roles in lens cell differentiation.
小分子热休克蛋白和α-晶体蛋白是相关蛋白，具有多种共同的结构和功能特性，包括同源氨基酸序列和相似的伴侣样活性。此外，小分子热休克蛋白和α-晶体蛋白在体内同源氨基酸序列中的丝氨酸残基处发生磷酸化。在晶状体上皮细胞向纤维细胞分化的过程中，α-晶体蛋白的表达和磷酸化模式发生了显著变化，导致这些蛋白的磷酸化形式在晶状体纤维细胞中积累。为了确定小分子热休克蛋白HSP25在晶状体细胞中是否发生磷酸化，并确定其磷酸化状态在晶状体细胞分化过程中是否发生变化，对上皮细胞和纤维细胞中HSP25的磷酸化模式进行了比较分析。使用针对重组鼠蛋白的特异性抗体，通过等电聚焦和蛋白质印迹法，对大鼠晶状体上皮和皮质的细胞提取物中磷酸化和非磷酸化形式的HSP25进行了分析。通过其等电点和用蛋白磷酸酶2B进行体外去磷酸化后的特征性迁移来鉴定磷酸化形式。HSP25占大鼠晶状体提取物蛋白质含量的2.4%，主要以单磷酸化和双磷酸化形式存在。与上皮细胞提取物相比，纤维细胞提取物中的总HSP25多67%，双磷酸化形式的比例明显更高。两种细胞提取物中的磷酸化HSP25对蛋白磷酸酶2B的去磷酸化敏感，但纤维细胞提取物中的表观去磷酸化速率明显较慢。结果表明，HSP25在体内晶状体中发生磷酸化。此外，HSP25的合成和磷酸化随晶状体细胞分化而变化，导致双磷酸化形式在纤维细胞中显著积累。这些发现表明，HSP25及其磷酸化可能在晶状体细胞分化中起重要作用。
