SKAP interacts with Aurora B to guide end-on capture of spindle microtubules via phase separation.

Chromosome segregation in mitosis is orchestrated by the dynamic interactions between the kinetochore and spindle microtubules. Our recent studies show that mitotic motor CENP-E cooperates with SKAP and forms a link between kinetochore core MIS13 complex and spindle microtubule plus-ends to achieve accurate chromosome alignment in mitosis. However, it remains elusive how SKAP regulates kinetochore attachment from lateral association to end-on attachment during metaphase alignment. Here, we identify a novel interaction between Aurora B and SKAP that orchestrates accurate interaction between the kinetochore and dynamic spindle microtubules. Interestingly, SKAP spontaneously phase-separates in vitro via weak, multivalent interactions into droplets with fast internal dynamics. SKAP and Aurora B form heterogeneous coacervates in vitro, which recapitulate the dynamics and behavior of SKAP comets in vivo. Importantly, SKAP interaction with Aurora B via phase separation is essential for accurate chromosome segregation and alignment. Based on those findings, we reason that SKAP-Aurora B interaction via phase separation constitutes a dynamic pool of Aurora B activity during the lateral to end-on conversion of kinetochore-microtubule attachments to achieve faithful cell division.

Acetylation of ezrin regulates membrane-cytoskeleton interaction underlying CCL18-elicited cell migration.

Ezrin, a membrane-cytoskeleton linker protein, plays an essential role in cell polarity establishment, cell migration, and division. Recent studies show that ezrin phosphorylation regulates breast cancer metastasis by promoting cancer cell survivor and promotes intrahepatic metastasis via cell migration. However, it was less characterized whether there are additional post-translational modifications and/or post-translational crosstalks on ezrin underlying context-dependent breast cancer cell migration and invasion. Here we show that ezrin is acetylated by p300/CBP-associated factor (PCAF) in breast cancer cells in response to CCL18 stimulation. Ezrin physically interacts with PCAF and is a cognate substrate of PCAF. The acetylation site of ezrin was mapped by mass spectrometric analyses, and dynamic acetylation of ezrin is essential for CCL18-induced breast cancer cell migration and invasion. Mechanistically, the acetylation reduced the lipid-binding activity of ezrin to ensure a robust and dynamic cycling between the plasma membrane and cytosol in response to CCL18 stimulation. Biochemical analyses show that ezrin acetylation prevents the phosphorylation of Thr567. Using atomic force microscopic measurements, our study revealed that acetylation of ezrin induced its unfolding into a dominant structure, which prevents ezrin phosphorylation at Thr567. Thus, these results present a previously undefined mechanism by which CCL18-elicited crosstalks between the acetylation and phosphorylation on ezrin control breast cancer cell migration and invasion. This suggests that targeting PCAF signaling could be a potential therapeutic strategy for combating hyperactive ezrin-driven cancer progression.

Acetylation of ACAP4 regulates CCL18-elicited breast cancer cell migration and invasion.

Tumor metastasis represents the main causes of cancer-related death. Our recent study showed that chemokine CCL18 secreted from tumor-associated macrophages regulates breast tumor metastasis, but the underlying mechanisms remain less clear. Here, we show that ARF6 GTPase-activating protein ACAP4 regulates CCL18-elicited breast cancer cell migration via the acetyltransferase PCAF-mediated acetylation. CCL18 stimulation elicited breast cancer cell migration and invasion via PCAF-dependent acetylation. ACAP4 physically interacts with PCAF and is a cognate substrate of PCAF during CCL18 stimulation. The acetylation site of ACAP4 by PCAF was mapped to Lys311 by mass spectrometric analyses. Importantly, dynamic acetylation of ACAP4 is essential for CCL18-induced breast cancer cell migration and invasion, as overexpression of the persistent acetylation-mimicking or non-acetylatable ACAP4 mutant blocked CCL18-elicited cell migration and invasion. Mechanistically, the acetylation of ACAP4 at Lys311 reduced the lipid-binding activity of ACAP4 to ensure a robust and dynamic cycling of ARF6-ACAP4 complex with plasma membrane in response to CCL18 stimulation. Thus, these results present a previously undefined mechanism by which CCL18-elicited acetylation of the PH domain controls dynamic interaction between ACAP4 and plasma membrane during breast cancer cell migration and invasion.

Phosphorylation of PP1 Regulator Sds22 by PLK1 Ensures Accurate Chromosome Segregation.

During cell division, accurate chromosome segregation is tightly regulated by Polo-like kinase 1 (PLK1) and opposing activities of Aurora B kinase and protein phosphatase 1 (PP1). However, the regulatory mechanisms underlying the aforementioned hierarchical signaling cascade during mitotic chromosome segregation have remained elusive. Sds22 is a conserved regulator of PP1 activity, but how it regulates PP1 activity in space and time during mitosis remains elusive. Here we show that Sds22 is a novel and cognate substrate of PLK1 in mitosis, and the phosphorylation of Sds22 by PLK1 elicited an inhibition of PP1-mediated dephosphorylation of Aurora B at threonine 232 (Thr232) in a dose-dependent manner. Overexpression of a phosphomimetic mutant of Sds22 causes a dramatic increase in mitotic delay, whereas overexpression of a non-phosphorylatable mutant of Sds22 results in mitotic arrest. Mechanistically, the phosphorylation of Sds22 by PLK1 strengthens the binding of Sds22 to PP1 and inhibits the dephosphorylation of Thr232 of Aurora B to ensure a robust, error-free metaphase-anaphase transition. These findings delineate a conserved signaling hierarchy that orchestrates dynamic protein phosphorylation and dephosphorylation of critical mitotic regulators during chromosome segregation to guard chromosome stability.