The dual-specificity phosphatase hYVH1 interacts with Hsp70 and prevents heat-shock-induced cell death

Abstract

hYVH1 [human orthologue of YVH1 (yeast VH1-related phosphatase)] is an atypical dual-specificity phosphatase that is widely conserved throughout evolution. Deletion studies in yeast have suggested a role for this phosphatase in regulating cell growth. However, the role of the human orthologue is unknown. The present study used MS to identify Hsp70 (heat-shock protein 70) as a novel hYVH1-binding partner. The interaction was confirmed using endogenous co-immunoprecipitation experiments and direct binding of purified proteins. Endogenous Hsp70 and hYVH1 proteins were also found to co-localize specifically to the perinuclear region in response to heat stress. Domain deletion studies revealed that the ATPase effector domain of Hsp70 and the zinc-binding domain of hYVH1 are required for the interaction, indicating that this association is not simply a chaperone-substrate complex. Thermal phosphatase assays revealed hYVH1 activity to be unaffected by heat and only marginally affected by non-reducing conditions, in contrast with the archetypical dual-specificity phosphatase VHR (VH1-related protein). In addition, Hsp70 is capable of increasing the phosphatase activity of hYVH1 towards an exogenous substrate under non-reducing conditions. Furthermore, the expression of hYVH1 repressed cell death induced by heat shock, H2O2 and Fas receptor activation but not cisplatin. Co-expression of hYVH1 with Hsp70 further enhanced cell survival. Meanwhile, expression of a catalytically inactive hYVH1 or a hYVH1 variant that is unable to interact with Hsp70 failed to protect cells from the various stress conditions. The results suggest that hYVH1 is a novel cell survival phosphatase that co-operates with Hsp70 to positively affect cell viability in response to cellular insults.

The dual-specificity phosphatase hYVH1 (DUSP12) is a novel modulator of cellular DNA content

“…The ΔCT1 construct terminates right after the phosphatase catalytic domain, the ΔCT2 variant, in addition to the catalytic domain, contains the first zinc coordination site, and the ΔCT3 variant contains an additional hydrophobic motif, which is highly conserved among orthologs. 14 We observed that deletion of the zinc binding region abolished the hYVH1-mediated cell cycle effect as the N-terminal catalytic domain (ΔCT1) essentially had a similar profile to our Flag-empty vector control ( Fig. 3A and B).…”

“…In order to assess the functionality of the hYVH1 phosphorylation sites, non-phosphorylated S/A and phosphomimetic S/E mutations were introduced into Flag-hYVH1, and their effects on hYVH1 localization were analyzed. The phosphorylation mutants for Ser 14 and Thr 252 displayed no detectable difference in subcellular targeting of hYVH1 (data not shown). In contrast, the phosphorylation mutants corresponding to the Ser 335 phosphorylation site exhibited a clear alteration in the nuclear/cytoplasmic distribution of hYVH1.…”

“…It has been demonstrated that nuclear/cytoplasmic shuttling is a common mechanism for regulating key cell cycle proteins, such as p53 and cyclin B. [18][19][20] Human YVH1 has been shown to display a nuclear, perinuclear and cytoplasmic localization pattern in a variety of cell lines, 5,12,14 suggesting hYVH1 can shuttle between the nucleus and the cytoplasm. Furthermore, we were interested in ).…”

“…8 In humans, this domain can function as a redox sensor and is necessary for the cytoprotective function of hYVH1. 14,17 We therefore tested the overexpression of the zinc-binding domain as well hYVH1 remains a poorly understood phosphatase especially regarding how its activities are regulated in the cell.…”

“…Human YVH1 interacts with Hsp70, 14 and overexpression of hYVH1 repressed cell death induced by cellular insults, such as heat shock, hydrogen peroxide exposure and Fas receptor activation. This repression is further enhanced upon co-expression with Hsp70.…”