Analysis of the chaperone function of Xenopus laevis small heat shock protein hsp30C.

摘要

Small heat shock proteins protect cells from stress presumably by acting as molecular chaperones. Here we report on the functional characterization of a developmentally regulated, heat-inducible member of the Xenopus small heat shock protein family, hsp30C. An expression vector containing the open reading frame of hsp30C was expressed in E. coli. These bacterial cells displayed greater thermoresistance than wild type or plasmid-containing cells. Purified recombinant protein, 30C, was recovered as multimeric complexes which inhibited heat-induced aggregation of either citrate synthase or luciferase as determined by light scattering assays. Additionally, 30C attenuated but did not reverse heat-induced inactivation of enzyme activity. In contrast to an N-terminal deletion mutant, removal of the last 25 amino acids from the C-terminal end of 30C severely impaired its chaperone activity. Furthermore, heat-treated concentrated solutions of the C-terminal mutant formed non-functional complexes and precipitated from solution. Similar results were observed with mutant 30C proteins containing specific amino acid substitutions at the C-terminus. Immunoblot and gel filtration analysis indicated that 30C binds with and maintains the solubility of luciferase preventing it from forming heat-induced aggregates. Co-immunoprecipitation experiments suggested that the carboxyl region is necessary for 30C to interact with target proteins. These results clearly indicate a molecular chaperone role for Xenopus hsp30C and provide evidence that its activity requires the carboxyl terminal region.; Additional studies were performed that examined the role of phosphorylation on the physical properties and chaperone function hsp30C. Both heat stress and sodium arsenite treatment of A6 cells rapidly activated the p38 MAP kinase pathway and the downstream effector MAPKAP kinase-2 as demonstrated by in vitro kinase analyses. However, co-immunoprecipitation and Western blot analyses indicated that the association of MAPKAP kinase-2 with hsp30C and the subsequent phosphorylation of hsp30C occurred primarily during recovery after heat stress. SB203580 attenuated the activation of p38 in A6 cells and markedly inhibited hsp30C phosphorylation. Phosphorylation of hsp30C gave rise to the formation of smaller multimeric hsp30C complexes and severely compromised its ability to prevent the heat-induced aggregation of citrate synthase and luciferase. Additionally, we demonstrate that this loss of chaperone activity was due to an attenuated binding of phosphorylated hsp30C with target proteins. Data from these studies indicate that phosphorylation of hsp30C affects multiple aspects of small hsp molecular chaperone activity. Taken together, hsp30C acts as a molecular chaperone and displays similar characteristics of other chaperones from the small hsp family. Hsp30 forms part of the p38 stress activated signaling pathway in Xenopus and responds negatively to phosphorylation by MAPKAP kinase-2. We suggest that the phosphorylation induced attenuation of chaperone activity mediates the recovery of cells from heat stress.