The tumor suppressor p53 is a key regulator of apoptosis induced by various cellular stresses. p53 can induce apoptosis by two mechanisms. First, p53 acts as a transcription factor inducing and repressing pro-apoptotic and anti-apoptotic targets genes, respectively. Second, p53 is able to translocate to the mitochondria, where it interacts with BCL-2 family members to induce membrane permeabilization and cytochrome c release. p53 transcriptional activity is regulated by a set of post-translational modifications that have been well documented. However, how these modifications impact the direct mitochondrial pathway of death remain poorly understood. In this study, we focused on the role of serine 392 phosphorylation in the control of p53-dependent apoptosis. We used CRISPR/Cas9 genome editing to substitute serine 392 by a non-phosphorylatable alanine in HCT-116 colon carcinoma cells. The S392A mutant displayed normal transcriptional activity following genotoxic stress, but markedly impaired ability to localize to mitochondria. The decreased mitochondrial localization of the S392A mutant correlated with a lower ability to induce apoptosis. Confirmatory observations were made following enforced expression of the S392A p53 mutant or a phospho-mimetic S392E mutant in H1299 lung carcinoma cells. Our observations support the premise that serine 392 phosphorylation of p53 influences its mitochondrial translocation and transcription-independent apoptotic function.

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Highlights d The HURP protein accumulates on k-fibers inversely proportionally to their length d By setting k-fiber length, centrosomes control HURP accumulation on k-fibers d The relative levels of HURP on k-fibers control plus-end microtubule dynamics d This system allows centrosomes to indirectly control k-fiber plus-end dynamics

Current models infer that the microtubule-based mitotic spindle is built from GDP-tubulin with small GTP caps at microtubule plus-ends, including those that attach to kinetochores, forming the kinetochore-fibres. Here we reveal that kinetochore-fibres additionally contain a dynamic mixed-nucleotide zone that reaches several microns in length. This zone becomes visible in cells expressing fluorescently labelled end-binding proteins, a known marker for GTP-tubulin, and endogenously-labelled HURP - a protein which we show to preferentially bind the GDP microtubule lattice in vitro and in vivo. We find that in mitotic cells HURP accumulates on the kinetochore-proximal region of depolymerising kinetochore-fibres, whilst avoiding recruitment to nascent polymerising K-fibres, giving rise to a growing “HURP-gap”. The absence of end-binding proteins in the HURP-gaps leads us to postulate that they reflect a mixed-nucleotide zone. We generate a minimal quantitative model based on the preferential binding of HURP to GDP-tubulin to show that such a mixed-nucleotide zone is sufficient to recapitulate the observed in vivo dynamics of HURP-gaps.