Phosphorylation of HSP25 during lens cell differentiation

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. (C) 1997 Academic Press Limited.