Hypothesis

Persistent JNK activation during cellular stress leads to phosphorylation of the autophagy receptor p62/SQSTM1, which drives its phase separation into cytoplasmic aggregates. We propose that the duration and kinase context of p62 phosphorylation determine whether these aggregates remain liquid‑like, autophagy‑competent structures or mature into solid, amyloid‑like scaffolds that sustain senescence‑associated secretory phenotype (SASP). Specifically, early JNK‑dependent phosphorylation of p62 at serine‑403 (S403) enhances its LC3‑interacting region (LIR) exposure and promotes liquid‑liquid phase separation (LLPS) that sequesters damaged proteins for autophagic clearance. Continued JNK activity recruits casein kinase 2 (CK2), which phosphorylates additional acidic residues (e.g., S409, S412) on p62, stabilizing β‑sheet interactions and converting the condensates into protease‑resistant, Thioflavin‑T‑positive aggregates. These solid p62 scaffolds then serve as platforms for NLRP3 inflammasome assembly and NF‑κB activation, locking the cell into a chronic inflammatory state.

Testable Predictions

1. Phospho‑switch detection – In senescent fibroblasts, p62 S403 phosphorylation will peak early (6‑12 h after stress) and correlate with increased LC3‑II puncta and high autophagic flux, whereas later time points (24‑48 h) will show accumulation of p62 S409/S412 phosphorylation alongside Thioflavin‑T positivity and reduced LC3 turnover.
2. CK2 inhibition blocks solidification – Treatment with a selective CK2 inhibitor (CX‑4945) during the late phase will prevent S409/S412 phosphorylation, maintain p62 in a liquid state (measured by FRAP recovery >80 %), preserve autophagic flux, and markedly decrease SASP cytokine secretion (IL‑6, IL‑8) without affecting JNK activation levels.
3. Solid p62 scaffolds drive inflammasome activation – Immunoprecipitation of Thioflavin‑T‑positive p62 aggregates from senescent cells will pull down NLRP3 and ASC; CK2 inhibition will reduce this interaction.
4. Genetic validation – Expression of a phospho‑deficient p62 mutant (S403A/S409A/S412A) will fail to form aggregates and will not support SASP induction, whereas a phospho‑mimetic mutant (S403D/S409D/S412D) will form constitutive aggregates that trigger SASP even in the absence of stress.

Experimental Approach

• Induce senescence in human IMR‑90 fibroblasts via etoposide (10 µM, 24 h) or mitochondrial antimycin A.
• Monitor JNK activity (phospho‑JNK) and p62 phospho‑sites using phospho‑specific antibodies by Western blot and immunofluorescence.
• Assess aggregate dynamics with live‑cell FRAP of GFP‑p62 and Thioflavin‑T staining.
• Measure autophagic flux via LC3‑II turnover in presence/absence of bafilomycin A1.
• Quantify SASP cytokines (IL‑6, IL‑8) by ELISA.
• Apply CK2 inhibitor (CX‑4945, 1 µM) or DMSO control at defined intervals.
• Perform co‑immunoprecipitation of p62 with NLRP3/ASC.

Potential Outcomes and Interpretation

If CK2 inhibition prevents the transition to solid p62 aggregates while preserving autophagy and suppressing SASP, the hypothesis is supported, revealing a molecular “phase‑switch” that converts a protective proteostatic response into a chronic inflammatory driver. Conversely, if CK2 inhibition fails to alter aggregate state or SASP, the model would be falsified, suggesting that other kinases or downstream effectors govern aggregate maturation.

Broader Implications

Targeting the JNK‑p62‑CK2 axis could decouple the beneficial sequestration of misfolded proteins from the deleterious inflammation that underlies age‑related pathology, offering a therapeutic strategy to enhance proteostasis without triggering SASP.