Protein kinase CK2 phosphorylates Hsp105α at Ser509 and modulates its function

The 105 kDa heat-shock protein (Hsp) Hsp105alpha is a mammalian stress protein that belongs to the HSP105/HSP110 family. We have shown previously that Hsp105alpha 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 Hsp105alpha, we first analysed the site of phosphorylation of Hsp105alpha by CK2. Peptide mapping analysis of Hsp105alpha phosphorylated by CK2 and in vitro phosphorylation experiments using various deletion and substitution mutants of Hsp105alpha revealed that Hsp105alpha is phosphorylated at Ser(509) in the beta-sheet domain. Furthermore, Ser(509) in Hsp105alpha was also phosphorylated in mammalian COS-7 cells, although other sites were phosphorylated as well. Next, we examined the effects of phosphorylation of Hsp105alpha on its functions using CK2-phosphorylated Hsp105alpha. Interestingly, Hsp105alpha suppressed 70 kDa heat-shock cognate protein (Hsc70)-mediated protein folding, whereas the phosphorylation of Hsp105alpha at Ser(509) abolished the inhibitory activity of Hsp105alpha in vitro. In accordance with these findings, wild-type Hsp105alpha, 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 Hsp105alpha suppressed the Hsp70-mediated refolding of heat-denatured luciferase in mammalian cells. Thus it was suggested that CK2 phosphorylates Hsp105alpha at Ser(509) and modulates the function of Hsp105alpha. The regulation of Hsp105alpha function by phosphorylation may play an important role in a variety of cellular events.