The phosphomimetic Hsc70 T495E mutant adopts an open-like conformation.

a) Crystal structure of bovine Hsc70(residues 1-554; PDB: 1YUW). The nucleotide-binding domain (NBD, residues 1-383) is shown in cyan, the interdomain linker (residues 384-394) in green, and the substrate binding domain (SBD, residues 395-506) in magenta. T495 is highlighted in white and marked with an asterisk. b) J-protein-stimulated ATPase activity of wild-type (WT) and phosphomimetic Hsc70 (T495E) measured by malachite green assay. Data points represent mean + SD of technical triplicates. c) Fluorescence polarization of ATP-FAM binding to WT and T495E Hsc70. Values were normalized to the minimum and maximum polarization; data points represent mean + SD of technical triplicates. d) Partial proteolysis of WT and T495E Hsc70 by trypsin in the presence of ATP or ADP. Digestion products were resolved by SDS-PAGE, and band intensities were quantified (bar graph, right). Statistical significance was determined by two-way ANOVA with Šidák’s multiple comparisons test (adjusted p = 0.0002, n = 4 technical replicates). e) Tau binding to immobilized WT and T495E Hsc70 measured by ELISA. Data points represent mean + SD of technical triplicates.

Base excision repair drives phosphorylation of Hsp70 in human cells.

a) Hsp70 phosphorylation by MMS treatment or LegK4 overexpression. HEK293T cells were transiently transfected overnight with LegK4Δ1-58:GFP or treated with 10 mM MMS for 5 h. Phosphorylation at T495 was detected using a phospho-specific antibody to pHsp70 T495; GAPDH was used as a loading control. Data are representative of n > 3 independent experiments. b) Schematic of the base excision repair (BER) pathway, highlighting steps relevant to MMS-induced DNA damage. c) MPG overexpression increases pHsp70 levels. HEK293T cells were transiently transfected with MPG overnight, treated with MMS, and analyzed by immunoblotting for pHsp70, the loading controls GAPDH and Hsp70, and the DNA damage marker γH2AX. Data are representative of n = 3 independent experiments. d) Inhibition of APE1 reduces MMS-induced pHsc70. Cells were pretreated for 1h with 5µM, 10 µM, or 50 µM APE1 inhibitor (APE1 compound III) before MMS treatment. Hsp70 and pHsp70 were detected by immunoblotting. Data are representative of n = 3 independent experiments. e) Masking of AP sites prevents pHsp70 accumulation. Cells were pretreated with 60 mM methoxyamine (Mx) for 30 min, followed by cotreatment with 30 mM Mx and 10 mM MMS for 5 h. pHsp70, the loading controls GAPDH and Hsp70, and the DNA damage marker γH2AX were detected by immunoblotting. Data are representative of n = 3 independent experiments. f) DNA damage specificity panel for pHsp70 induction. Cells were treated with bleomycin (10 µM, 5 h or 24 h), camptothecin (10 µM, 5 h), hydroxyurea (2 mM, 24 h), MMS (3 mM or 10 mM, 5 h), sodium arsenite (0.5 mM, 5 h), or DMSO vehicle (5 h). Immunoblotting was performed for pHsp70, DDR kinase activation markers (pDNA-PKcs S2056, pChk2 T68, pChk2 S16, pATM S1981), DNA damage marker γH2AX, and loading controls (Hsp70 and Hsp90). Data are representative of n = 3 independent experiments.

DDR kinase activity is upstream of Hsp70 phosphorylation.

a. DNA-PKcs knockdown reduces pHsp70 levels. Cells were transiently transfected with three independent siRNAs targeting DNA-PKcs or a scramble control for 72 h, followed by treatment with 10 mM MMS for 5 h. pHsp70 and GAPDH (loading control) were detected by immunoblotting. Data are representative of n = 3 independent experiments. b. Pharmacological inhibition of ATM decreases pHsp70 induction. Cells were pretreated for 1 h with ATM inhibitors (10 µM KU-60019 or 200nM AZD1390), then treated with 10 mM MMS for 5 h. ATM and DNA-PKcs autoactivation were monitored by immunoblotting pATM (S1981) and pDNA-PKcs (S2056), respectively. Tubulin and total Hsp70 served as loading controls. Data are representative of n = 3 independent experiments. c. Pharmacological inhibition of ATM, DNA-PKcs, Chk2, and CK1 decrease Hsp70 phosphorylation during MMS treatment. Cells were pretreated for 1h with inhibitors for ATM (200 nM AZD1390), DNA-PKcs (2 µM AZD7648), Chk2 (5 µM CCT241533), CK1(50 µM PF-670462) or with vehicle control (DMSO), then treated with 10mM MMS for 5h. Immunoblotting was performed against pHsp70 and the loading control Hsp70. Data are representative of n = 3 independent experiments. d. Timecourse of MMS-induced DDR activation and pHsp70 phosphorylation. Cells were treated with 10 mM MMS and harvested hourly. ATM and DNA-PKcs activation were detected by pATM (S1981) and pDNA-PKcs (S2056), respectively. Chk2 activation was monitored by pChk2 (T68) and pChk2 (S516). DNA damage was assessed via γH2AX. Hsp70 and Hsp90 were used as loading controls. Data are representative of n = 3 independent experiments

Mitosis precedes Hsp70 phosphorylation.

a. Variable pulse-chase MMS treatment suggests a complex signaling pathway. Cells were treated with 10 mM MMS for 1-5 h, then washed twice with PBS and incubated in MMS-free media for the remainder of the 5 h time period. ATM and DNA-PKcs activation were detected by pATM (S1981) and pDNA-PKcs (S2056), respectively. Chk2 activation was monitored by pChk2 (T68) and pChk2 (S516). DNA damage was assessed by γH2AX. Cell cycle progression was monitored using the S phase markers thymidine kinase (ThyK) and CDT1; mitotic entry marker pCdk1(Y15) (whose dephosphorylation permits M-phase entry), M phase marker phospho-histone H3 (S10) (pH3); and cyclins B and E. GAPDH and Hsp90 served as loading controls. Data are representative of n = 3 independent experiments. b. Early S-phase synchronization by double thymidine block fails to increase pHsp70 accumulation. Cells were treated with 2.5 mM thymidine for 18 h, released into fresh media for 9 h, then retreated with 2.5 mM thymidine for 17 h. Cells were then washed with PBS and released into fresh media with or without 10 mM MMS. Unsynchronized cells were also treated with 10 mM MMS. Cells were harvested at the indicated time points and immunoblotted for pHsp70, cell cycle markers (pCdk1 Y15, pH3, CDT1, ThyK), and loading controls α-tubulin and Hsp90. Data are representative of n = 2 independent experiments. c. G2/M stalling by CDK1 inhibition reduces pHsp70 levels. Cells were pretreated with 10 µM CDK1 inhibitor Ro3306 or DMSO for 17.5 h, washed twice with PBS, then treated again with Ro3306 or DMSO in the presence or absence of 10 mM MMS for 5 h. Immunoblotting was performed for pHsp70, cell cycle markers(pCdk1 Y15, pH3, CDT1, cyclin A), DDR markers (pDNA-PKcs S2056, pChk2 T68, γH2AX), and loading controls Hsp70 and Hsp90. Data are representative of n = 3 independent experiments. d. Subcellular fractionation of pHsp70 during MMS treatment shows nuclear localization. Cells were treated with 10 mM MMS from 1-5 h or left untreated, then fractionated into cytoplasmic and nuclear extracts using the NE-PER kit. Immunoblotting was performed for pHsp70 and total Hsp70 levels; α-tubulin and lamin B1 served as cytoplasmic and nuclear markers, respectively. Data are representative of n = 3 independent experiments. e. Two-hour MMS pulse chase reveals pHsp70 accumulation post-mitosis. Cells were treated with 10 mM MMS for 2 h, washed twice with PBS, and then incubated in fresh media. Samples were harvested hourly, alongside an untreated control. Immunoblotting was performed for pHsp70, DDR markers (pDNA-PKcs S2056, pChk2 T68, pATM S1981, pChk2 S516, γH2AX), cell cycle markers (cyclin A, pCdk1 Y15, pH3, cyclin B, ThyK), and loading controls Hsp70 and Hsp90. Data are representative of n = 3 independent experiments.

Ssa1 T492 phosphorylation mutations cause cell cycle defects in S. cerevisiae.

a. Growth curves of S. cerevisiae Ssa1 mutants show delayed growth. Indicated yeast mutants were grown to mid-log phase, diluted to the same starting concentration, and monitored overnight at 30 °C in a plate reader. Data represent the average of technical triplicates. Data are representative of n = 3 independent experiments. b. Half-times (t₁/₂) of both Ssa1 mutants in the ssa2Δ background, and of the phosphomimetic mutant in the SSA2 background, are significantly increased. Sigmoidal fits were applied to growth curves to determine t1/2 values. The data represent three technical replicates with two biological replicates per strain. Bars represent mean + SD of six replicates (n = 6; 2 biological replicates x 3 technical replicates) Statistical significance was determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparison test (** p = 0.0015; **** p < 0.0001). c. Cell cycle distribution analysis reveals G1 stalling of Ssa1 phosphomutants. Yeast were grown to mid-log phase, adjusted to the same concentration, and immediately fixed. Cells were stained with Sytox Green and analyzed by flow cytometry to determine DNA content. Histograms display DNA content (X-axis) with 1N corresponding to G1 phase, 2N to G2/M, and intermediate values to S phase. Left: WT SSA2 background; right: Δssa2 background. Data are representative of n = 4 technical replicates with 2 biological replicates per strain. d. Ssa1 phosphomutants display increased MMS sensitivity in a spot test assay. Yeast were grown to mid-log phase, adjusted to 2e7 cells/mL, serially diluted 1:10, and spotted (5µL) onto YPAD plates with or without 0.0095% MMS. Plates were incubated at 30 °C and imaged after 3 days. Data are representative of n = 3 independent experiments. e. Ssa1 phosphomutants exhibit perturbed G1/S stalling during MMS recovery. Yeast were grown to mid-log phase, treated with 0.05% MMS for 3 h, washed, and resuspended in fresh media for recovery. Samples were collected at the indicated times points and analyzed by staining and flow cytometry as described as in (c). Data are representative of n = 2 technical replicates with 2 biological replicates per strain.

Cartoon model of the causes and consequences of Hsp70 phosphorylation.

a. Non-helix distorting DNA lesions in M phase lead to Hsp70 phosphorylation through APE1-dependent processing and DDR kinase signaling. Treatment with MMS or sodium arsenite generates lesions that are repaired by BER. In M-phase cells, APE1 processing of these lesions creates DSBs, activating DDR kinases and resulting in phosphorylation of Hsp70 at T495. This phosphorylation persists into G1 and stalls cell cycle progression. b. Proposed mechanism of G1 arrest by phosphorylated Hsp70 (pHsp70). WT (upper panel): (1,2) Phosphorylation promotes stable interactions between Hsp70, its client, and an accessory protein (e.g. pro-degradation machinery); (3) subsequent dephosphorylation permits client release and degradation, enabling S phase entry. Phosphomimetic (middle panel): (1,2) Phosphomimetic Hsp70 promotes client and accessory protein engagement. Inability to dephosphorylate impairs substrate release, blocking S phase entry. Phosphonull (lower panel):(1) client sampling occurs, but phosphorylation-dependent stabilization is absent; (2) eventual association allows (3) client release and degradation, permitting S phase entry, albeit in a poorly regulated manner.