A non-canonical function of Plk4 in centriolar satellite integrity and ciliogenesis through PCM1 phosphorylation

Centrioles are the major constituents of the animal centrosome, in which Plk4 kinase serves as a master regulator of the duplication cycle. Many eukaryotes also contain numerous peripheral particles known as centriolar satellites. While centriolar satellites aid centriole assembly and primary cilium formation, it is unknown whether Plk4 plays any regulatory roles in centriolar satellite integrity. Here we show that Plk4 is a critical determinant of centriolar satellite organisation. Plk4 depletion leads to the dispersion of centriolar satellites and perturbed ciliogenesis. Plk4 interacts with the satellite component PCM1, and its kinase activity is required for phosphorylation of the conserved S372. The nonphosphorylatable PCM1 mutant recapitulates phenotypes of Plk4 depletion, while the phosphomimetic mutant partially rescues the dispersed centriolar satellite patterns and ciliogenesis in cells depleted of PCM1. We show that S372 phosphorylation occurs during the G1 phase of the cell cycle and is important for PCM1 dimerisation and interaction with other satellite components. Our findings reveal that Plk4 is required for centriolar satellite function, which may underlie the ciliogenesis defects caused by Plk4 dysfunction

Casein kinase 1γ acts as a molecular switch for cell polarization through phosphorylation of the polarity factor Tea1 in fission yeast

Fission yeast undergoes growth polarity transition from monopolar to bipolar during G2 phase, designated NETO (New End Take Off). It is known that NETO onset involves two prerequisites, the completion of DNA replication and attainment of a certain cell size. However, the molecular mechanism remains unexplored. Here, we show that casein kinase 1γ, Cki3 is a critical determinant of NETO onset. Not only did cki3∆ cells undergo NETO during G1‐ or S‐phase, but they also displayed premature NETO under unperturbed conditions with a smaller cell size, leading to cell integrity defects. Cki3 interacted with the polarity factor Tea1, of which phosphorylation was dependent on Cki3 kinase activity. GFP nanotrap of Tea1 by Cki3 led to Tea1 hyperphosphorylation with monopolar growth, whereas the same entrapment by kinase‐dead Cki3 resulted in converse bipolar growth. Intriguingly, the Tea1 interactor Tea4 was dissociated from Tea1 by Cki3 entrapment. Mass spectrometry identified four phosphoserine residues within Tea1 that were hypophosphorylated in cki3∆ cells. Phosphomimetic Tea1 mutants showed compromised binding to Tea4 and NETO defects, indicating that these serine residues are critical for protein–protein interaction and NETO onset. Our findings provide significant insight into the mechanism by which cell polarization is regulated in a spatiotemporal manner.T.K. was the recipient of a JSPS fellowship (PD) and was partly supported by ‘Strategic Young Researcher Overseas Visits Program for Accelerating Brain Circulation’ from JSPS. This work was supported by Cancer Research UK (T.T. and A.P.S) and the Ministry of Education, Culture, Sports, Science and Technology (D.H.)

Oncogenic RET Kinase domain mutations perturb the autophosphorylation trajectory by enhancing substrate presentation in trans

To decipher the molecular basis for RET kinase activation and oncogenic deregulation, we defined the temporal sequence of RET autophosphorylation by label-free quantitative mass spectrometry. Early autophosphorylation sites map to regions flanking the kinase domain core, while sites within the activation loop only form at later time points. Comparison with oncogenic RET kinase revealed that late autophosphorylation sites become phosphorylated much earlier than wild-type RET, which is due to a combination of an enhanced enzymatic activity, increased ATP affinity, and surprisingly, by providing a better intermolecular substrate. Structural analysis of oncogenic M918T and wild-type RET kinase domains reveal a cis-inhibitory mechanism involving tethering contacts between the glycine-rich loop, activation loop, and αC-helix. Tether mutations only affected substrate presentation but perturbed the autophosphorylation trajectory similar to oncogenic mutations. This study reveals an unappreciated role for oncogenic RET kinase mutations in promoting intermolecular autophosphorylation by enhancing substrate presentation

Casein kinase 1γ acts as a molecular switch for cell polarization through phosphorylation of the polarity factor T

Fission yeast undergoes growth polarity transition from monopolar to bipolar during G2 phase, designated NETO (New End Take Off). It is known that NETO onset involves two prerequisites, the completion of DNA replication and attainment of a certain cell size. However, the molecular mechanism remains unexplored. Here, we show that casein kinase 1γ, Cki3 is a critical determinant of NETO onset. Not only did cki3∆ cells undergo NETO during G1‐ or S‐phase, but they also displayed premature NETO under unperturbed conditions with a smaller cell size, leading to cell integrity defects. Cki3 interacted with the polarity factor Tea1, of which phosphorylation was dependent on Cki3 kinase activity. GFP nanotrap of Tea1 by Cki3 led to Tea1 hyperphosphorylation with monopolar growth, whereas the same entrapment by kinase‐dead Cki3 resulted in converse bipolar growth. Intriguingly, the Tea1 interactor Tea4 was dissociated from Tea1 by Cki3 entrapment. Mass spectrometry identified four phosphoserine residues within Tea1 that were hypophosphorylated in cki3∆ cells. Phosphomimetic Tea1 mutants showed compromised binding to Tea4 and NETO defects, indicating that these serine residues are critical for protein–protein interaction and NETO onset. Our findings provide significant insight into the mechanism by which cell polarization is regulated in a spatiotemporal manner

Photosensitized UVA-Induced Cross-Linking between Human DNA Repair and Replication Proteins and DNA Revealed by Proteomic Analysis

Long wavelength ultraviolet radiation (UVA, 320–400 nm) interacts with chromophores present in human cells to induce reactive oxygen species (ROS) that damage both DNA and proteins. ROS levels are amplified, and the damaging effects of UVA are exacerbated if the cells are irradiated in the presence of UVA photosensitizers such as 6-thioguanine (6-TG), a strong UVA chromophore that is extensively incorporated into the DNA of dividing cells, or the fluoroquinolone antibiotic ciprofloxacin. Both DNA-embedded 6-TG and ciprofloxacin combine synergistically with UVA to generate high levels of ROS. Importantly, the extensive protein damage induced by these photosensitizer+UVA combinations inhibits DNA repair. DNA is maintained in intimate contact with the proteins that effect its replication, transcription, and repair, and DNA–protein cross-links (DPCs) are a recognized reaction product of ROS. Cross-linking of DNA metabolizing proteins would compromise these processes by introducing physical blocks and by depleting active proteins. We describe a sensitive and statistically rigorous method to analyze DPCs in cultured human cells. Application of this proteomics-based analysis to cells treated with 6-TG+UVA and ciprofloxacin+UVA identified proteins involved in DNA repair, replication, and gene expression among those most vulnerable to cross-linking under oxidative conditions

Photosensitized UVA-Induced Cross-Linking between Human DNA Repair and Replication Proteins and DNA Revealed by Proteomic Analysis

Long wavelength ultraviolet radiation (UVA, 320–400 nm) interacts with chromophores present in human cells to induce reactive oxygen species (ROS) that damage both DNA and proteins. ROS levels are amplified, and the damaging effects of UVA are exacerbated if the cells are irradiated in the presence of UVA photosensitizers such as 6-thioguanine (6-TG), a strong UVA chromophore that is extensively incorporated into the DNA of dividing cells, or the fluoroquinolone antibiotic ciprofloxacin. Both DNA-embedded 6-TG and ciprofloxacin combine synergistically with UVA to generate high levels of ROS. Importantly, the extensive protein damage induced by these photosensitizer+UVA combinations inhibits DNA repair. DNA is maintained in intimate contact with the proteins that effect its replication, transcription, and repair, and DNA–protein cross-links (DPCs) are a recognized reaction product of ROS. Cross-linking of DNA metabolizing proteins would compromise these processes by introducing physical blocks and by depleting active proteins. We describe a sensitive and statistically rigorous method to analyze DPCs in cultured human cells. Application of this proteomics-based analysis to cells treated with 6-TG+UVA and ciprofloxacin+UVA identified proteins involved in DNA repair, replication, and gene expression among those most vulnerable to cross-linking under oxidative conditions

Developing high strain rate superplasticity in Al-Mg-Sc-Zr alloys using equal-channel angular pressing

The processing of metallic alloys through the procedure of equal-channel angular pressing (ECAP) provides an opportunity for achieving superplastic ductilities at very high strain rates. This paper reports experimental data from an investigation of a series of Al---Mg---Sc---Zr alloys processed by ECAP. The results show the occurrence of high tensile ductilities at testing strain rates above 10?2 s?1. <br/