ERK-dependent phosphorylation of the linker and substrate-binding domain of HSP70 increases folding activity and cell proliferation

Abstract

The enhanced productive folding of translated polypeptides by heat shock protein 70 (HSP70) is often required for the survival of cancer cells. Although the folding activity of HSP70 is considered a significant determinant of the progression of cancer cells, it is still unknown how this activity could be regulated. Here, we report that the phosphorylation of HSP70 facilitates its folding activity, enhancing cell proliferation. Mass spectrometry identified the serine residues at positions 385 and 400 in the linker and substrate-binding domains of HSP70, respectively, as sites of phosphorylation mediated by EGF signaling, and this result was further confirmed by site-directed mutagenesis. ERK is known to be a specific kinase. The phosphorylation of the two sites induces the extended conformation of HSP70 via the regulation of the binding of the linker to the nucleotide- and substrate-binding domains, augmenting the binding affinity of HSP70 to substrates and enhancing its folding activity; this ultimately results in pro-proliferative effects. Cell lines harboring activated ERK showed increased phosphorylation of HSP70, and a positive correlation between the phosphorylation of HSP70 and the activity of ERK was observed. Thus, this study demonstrated that the ERK-dependent phosphorylation of HSP70 facilitated its folding activity and cellular proliferative function.

Post-translational modifications of Hsp70 family proteins: Expanding the chaperone code

“…Whereas it has been known for several years that chaperones can bind and regulate MAP kinase function (66), a recent study suggests that mitogen-activated protein kinase pathway activity can in turn regulate chaperone function (67). Treatment of cells with epidermal growth factor triggers Hsp70 phosphorylation on Ser-385 and Ser-400.…”

“…These sites are particularly interesting as they reside on and adjacent to the flexible linker that con-nects the NBD and SBD of Hsp70. Phosphorylation of Hsp70 at these two sites leads to an extended conformation of the protein, which in turn increases the binding affinity of the clients (67). Mutation of these two sites results in cells that have a reduced viability and growth rate.…”

Genome-Wide Identification and Expression Analysis of Heat Shock Protein 70 (HSP70) Gene Family in Pumpkin (Cucurbita moschata) Rootstock under Drought Stress Suggested the Potential Role of these Chaperones in Stress Tolerance

“…These modifications and their effects on the function of chaperones are also considered as "chaperone code" [12]. As an example, phosphorylation of HSP70 at SBD and NBD regions improves the binding affinity of this chaperone to its targets [13]. SBD and NBD are two functionally crucial regions for PTM, since the mutation in these sites represses the ability of HSP70 to repair misfolded proteins [14].…”

NEK9, a novel effector of IL-6/STAT3, regulates metastasis of gastric cancer by targeting ARHGEF2 phosphorylation

“…The phosphorylation of the human ARHGEF2 recombinant protein (P01) (H00009181-P01, Abnova, Tai Wan, China) by active NEK9 protein (ab125614, Abcam) was monitored using a universal kinase activity kit (R&D Systems). The amount of phosphorylated ARHGEF2 was determined as described previously 34 . All experiments were repeated independently in triplicate.…”