Conformational changes resulting from pseudophosphorylation of mammalian small heat shock proteins--a two-hybrid study.

The human genome codes for 10 so-called mammalian small heat shock or stress proteins (sHsp) with the various tissues expressing characteristic sets of sHsps. Most sHsps interact with each other and form homo- and heterooligomeric complexes. Some of the sHsps are phosphoproteins in vivo, and phosphorylation has been implicated in the regulation of complex size and composition. In this study, we analyze, by the 2-hybrid method, the reporter gene activation pattern of several sHsp pairs that previously have been demonstrated to interact. We show that pseudophosphorylation (mimicry of phosphorylation) of the homologous phosphorylation sites Ser15 and Ser16 in Hsp27 and Hsp20, respectively, modulates characteristics of these sHsps that can be detected by their ability to activate reporter genes in suitable 2-hybrid assays. Pseudophosphorylation of the separated N-terminus of Hsp27 alone is not sufficient for the activation of the reporter genes, whereas the separated C-terminus is sufficient. We conclude that pseudophosphorylation of Hsp27 and Hsp20 at their N-termini results in conformational changes that can be detected by their interaction with other sHsps. Pseudophosphorylation of alphaB-crystallin at Ser19, in contrast, had no detectable consequences.