The 105 kDa heat-shock protein (Hsp) Hsp105α is a mammalian stress protein that belongs to the HSP105/HSP110 family. We have shown previously that Hsp105α exists as non-phosphorylated and phosphorylated forms in vivo, and is phosphorylated by protein kinase CK2 (CK2) in vitro. In this study, to elucidate the role of phosphorylation of Hsp105α, we first analysed the site of phosphorylation of Hsp105α by CK2. Peptide mapping analysis of Hsp105α phosphorylated by CK2 and in vitro phosphorylation experiments using various deletion and substitution mutants of Hsp105α revealed that Hsp105α is phosphorylated at Ser509 in the β-sheet domain. Furthermore, Ser509 in Hsp105α was also phosphorylated in mammalian COS-7 cells, although other sites were phosphorylated as well. Next, we examined the effects of phosphorylation of Hsp105α on its functions using CK2-phosphorylated Hsp105α. Interestingly, Hsp105α suppressed 70 kDa heat-shock cognate protein (Hsc70)-mediated protein folding, whereas the phosphorylation of Hsp105α at Ser509 abolished the inhibitory activity of Hsp105α in vitro. In accordance with these findings, wild-type Hsp105α, which was thought to be phosphorylated in vivo, had no effect on Hsp70-mediated refolding of heat-denatured luciferase, whereas a non-phosphorylatable mutant of Hsp105α suppressed the Hsp70-mediated refolding of heat-denatured luciferase in mammalian cells. Thus it was suggested that CK2 phosphorylates Hsp105α at Ser509 and modulates the function of Hsp105α. The regulation of Hsp105α function by phosphorylation may play an important role in a variety of cellular events.
Inducible Expression of Protein Kinase CK2 in Mammalian Cells
