A conserved Hsp70 phosphorylation regulates cell cycle progression after DNA damage

Background

Hsp70 chaperones are highly conserved proteins that play a critical role in maintaining cellular homeostasis. They’re essential in metabolism, DNA repair, and cell cycle control, and their function depends on an ATP-driven cycle, where they switch between an open ATP-bound state and a closed ADP-bound state to bind and release client proteins. Post-translational modifications (PTMs) can fine-tune how Hsp70 works. In fact, studies in model organisms like budding yeast have helped identify hotspots where PTMs regulate this chaperone, providing important clues about how its activity is controlled.

The primary driver for this study was the observation that the Legionella pneumophila kinase LegK4 phosphorylates human cytosolic Hsp70 (Hsc70) at threonine 495 (T495) during infection. Interestingly, this site is similar to T518 in BiP, the ER version of Hsp70, which is known to undergo AMPylation. That modification keeps BiP locked in an open state and changes its activity. This made the authors wonder whether this phosphorylation at Hsc70(T495) could have a similar effect, and if this happened naturally in mammalian cells. To test this, they turned to phosphoproteomics, which showed that the equivalent residue in yeast Hsp70 (Ssa1-T492) is phosphorylated during DNA alkylation damage and when cells are blocked from exiting mitosis. These findings set the stage for asking whether this phosphorylation event plays a role in basic cellular processes, especially how cells respond to DNA damage.

 

Key findings

• Base excision repair leads to a conserved Hsp70 phosphorylation

Hsp70 phosphorylation was specifically induced by DNA alkylating agents like methyl methanesulfonate and reactive oxygen species generated by sodium arsenite, both of which produce substrates for the base excision repair (BER) pathway. The phosphorylation was conserved: it occurred at T495 in human cells and T492 in budding yeast. Inhibiting the BER endonuclease APE1 or chemically blocking abasic site recognition and repair prevented this phosphorylation, indicating it is selectively triggered by repair intermediates generated during BER.

• Phosphomimetic Hsc70 is locked in an open-like conformation

The Hsc70(T495E) mutant locked in an open-like conformation, although this did not impair nucleotide binding. Despite this conformational lock showing reduced ATPase activity, Hsc70(T495E) retained the ability to bind client proteins.

• Requires DNA Damage Response (DDR) Kinases

Pharmacological inhibition of ATM, DNA-PKcs, Chk2, and CK1 reduced or prevented Hsp70 phosphorylation, suggesting that BER-induced double-stranded breaks activate these DDR kinases, which in turn contribute to Hsp70 phosphorylation.

• Hsp70 phosphorylation occurs after M phase onset and regulates G1/S transition

Hsp70 phosphorylation is cell cycle dependent and prevents premature S-phase entry under conditions of BER stress. In human cells, this phosphorylation requires prolonged DNA damage exposure and progression through mitosis. Blocking mitotic entry with a CDK1 inhibitor prevented Hsp70 phosphorylation. In yeast, both phosphomimetic (T492E) and phosphonull (T492A) Hsp70 variants disrupted G1/S progression, leading to growth defects and altered cell cycle dynamics, especially in the absence of the compensatory Ssa2 isoform.

 

Why this new work is important

This preprint provides detailed mechanistic, biochemical, and genetic evidence of a conserved mechanism of cell cycle regulation in response to DNA damage. The paper identifies a novel and conserved Hsp70 phosphorylation event that acts as a reversible molecular brake to prevent premature S-phase entry during BER stress. This is a critical checkpoint mechanism that safeguards genome integrity when DNA repair capacity is overburdened.

The work details the conditions under which this phosphorylation occurs, demonstrating that it is not a general DNA damage response, but specifically answers to BER stress, and the authors elucidate the intricate signaling pathway upstream of Hsp70 phosphorylation. The finding that Hsp70 phosphorylation is cell cycle-dependent and requires prolonged DNA damage exposure and progression through mitosis is highly significant, as it suggests a sophisticated timing mechanism.

Hsp70 chaperones are central to cellular homeostasis and implicated in various diseases such as neurodegenerative disorders and cancer. Thus, adding a new layer of cell cycle-dependent, damage-responsive regulation to Hsp70’s known functions deepens our understanding of its diverse roles and highlights potential novel therapeutic avenues.

Additionally, it is particularly exciting how this work demonstrates the importance of studying host-pathogen interactions in uncovering previously unknown aspects of host biology, as the investigation into Legionella pneumophila‘s modulation of Hsp70 phosphorylation led to the discovery of this important endogenous regulatory mechanism.

 

Future directions and questions for the authors

1. The paper highlights the requirement for DDR kinases for Hsp70 phosphorylation, but it does not identify the direct kinase responsible for this phosphorylation. Is there any evidence pointing to which kinase(s) could be phosphorylating Hsp70?
2. The proposed model suggests that phosphorylated Hsp70 promotes or stabilizes interactions with specific client proteins and accessory machinery to regulate G1/S transition. Which substrates could Hsp70 be engaging to regulate the cell cycle this way?
3. How could the phosphorylation at T495 in the substrate-binding domain affect the nucleotide-binding 82 domain’s ATPase activity?
4. How is Hsp70 phosphorylation reversed? As this is a reversible mechanism for regulating the cell cycle, there should be an active dephosphorylation mechanism for removing the T495/T492 phosphate.
5. A distinct nuclear fraction of phosphorylated Hsp70 is observed after nuclear envelope reformation following mitosis. What role could it be playing there?

Tags: cell cycle, cerevisiae, hsp90, phosphorylation, yeast

doi: https://doi.org/10.1242/prelights.41586

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