HIPK2 Phosphorylates the Microtubule-Severing Enzyme Spastin at S268 for Abscission

Abstract

Abscission is the final step of cell division, mediating the physical separation of the two daughter cells. A key player in this process is the microtubule-severing enzyme spastin that localizes at the midbody where its activity is crucial to cut microtubules and culminate the cytokinesis. Recently, we demonstrated that HIPK2, a multifunctional kinase involved in several cellular pathways, contributes to abscission and prevents tetraploidization. Here, we show that HIPK2 binds and phosphorylates spastin at serine 268. During cytokinesis, the midbody-localized spastin is phosphorylated at S268 in HIPK2-proficient cells. In contrast, no spastin is detectable at the midbody in HIPK2-depleted cells. The non-phosphorylatable spastin-S268A mutant does not localize at the midbody and cannot rescue HIPK2-depleted cells from abscission defects. In contrast, the phosphomimetic spastin-S268D mutant localizes at the midbody and restores successful abscission in the HIPK2-depleted cells. These results show that spastin is a novel target of HIPK2 and that HIPK2-mediated phosphorylation of spastin contributes to its midbody localization for successful abscission.

HIPK2 Is Required for Midbody Remnant Removal Through Autophagy-Mediated Degradation

“…To evaluate the effect of HIPK2 depletion over more than one cell cycle, cells were transfected with stealth siRNAs that have increased stability in cell culture than standard siRNAs. We showed that HIPK2 depletion delays abscission and induces accumulation of aberrant elongated midbodies and cytokinesis failure (Rinaldo et al, 2012;Pisciottani et al, 2019). During these studies, we also observed the appearance of a large fraction of cells with MBRs in the siHIPK2 cells compared to their relative siCtr ( Figures 1A,B, and Supplementary Figure S1).…”

“…In human cells, HIPK2 RNA interference (RNAi) and Spastin RNAi were obtained by using stealth siRNAs as in Pisciottani et al, 2019. Specific prevalidated stealth siRNAs by Life Technologies were used to obtain HIPK2 RNAi in mouse cells and CEP55 RNAi in human cells.…”

“…Expression vectors were transfected by using Lipofectamine LTX and Plus reagent (Life Technologies). For rescue experiments, human HeLa siHIPK2 cells were transfected with low doses of vectors expressing GFP-tagged murine HIPK2-WT (D'Orazi et al, 2002) or its empty vector (peGFP-c2; Clontech) as in Pisciottani et al, 2019. Murine HIPK2 protein shows >97% identity with human HIPK2, fully recapitulates its functions, but is resistant to RNAi when human-specific siRNAs were used.…”

“…HIPK2-mediated phosphorylation of the extrachromosomal histone H2B (ecH2B) at Ser14 is not required for its midbody localization but contributes to the formation of the abscission site and is essential for successful cytokinesis (Rinaldo et al, 2012;Monteonofrio et al, 2019). HIPK2-mediated phosphorylation of the microtubule severing enzyme Spastin at Ser268 is required for its midbody localization to cut microtubules and the subsequent physical separation of the two daughter cells (Pisciottani et al, 2019;Gatti et al, 2020).…”

Chromatin Targeting of HIPK2 Leads to Acetylation-Dependent Chromatin Decondensation

“…Control of HIP kinases involves dynamic changes in their intracellular localization, and also regulation of their protein abundance by multiple levels including regulated synthesis and decay ( D’Orazi et al, 2012 ; Ohnheiser et al, 2015 ; Wong et al, 2020 ). The best studied member of the HIPK family is HIPK2, a kinase that is involved in many different biological processes including the maintenance of basal cardiac functions, cell abscission, type I interferon-mediated antiviral immunity and neuronal survival ( Shang et al, 2018 ; Guo et al, 2019 ; Pisciottani et al, 2019 ). In addition, HIPK2 contributes to developmental and differentiation processes to control adipogenesis ( Sjölund et al, 2014 ), the development of various neuronal subtypes, angiogenesis, vasculogenesis, and hematopoiesis in different contexts ( Blaquiere and Verheyen, 2017 ).…”