Journal: The Journal of Biological Chemistry

Article Title: The mitotic functions of a fission yeast CK1 enzyme are regulated by Cdk1-dependent and auto-phosphorylation

doi: 10.1016/j.jbc.2025.111007

Figure Lengend Snippet: Mitotic hyperphosphorylation of Hhp2 . A , protein lysates of the indicated strains that were either growing asynchronously (asynch) or arrested in mitosis ( nda3-KM311 and mts3-1 ) were subjected to anti-HA immunoprecipitation (IP). One half of each IP was treated with λ-phosphatase (λPPase) and the other with vehicle control, and proteins were immunoblotted with anti-HA (12CA5) antibodies. The position of molecular size standards is indicated to the right of the blots. B , cdc25 - 22 hhp2-HA 3 -TAP cells were synchronized in G2 and released into mitosis. Cell division progression was monitored by determining the percentage of septated cells over time. At times indicated, cell extracts were subject to anti-HA IP. Hhp2-HA 3 -TAP proteins were immunoblotted with anti-HA (12CA5) antibodies. The positions of molecular size standards are indicated. C , cdc25 - 22 cells (15 min after releasing) from ( B ) were subjected to anti-HA IPs as in ( A ). One half of each IP was treated with λPPase and the other with buffer control, and kinase assays were performed using the bead-bound Hhp2 proteins and equivalent amounts of α-casein as substrate. Immunoprecipitated Hhp2 was visualized by immunoblot with anti-HA (12CA5) antibody and phosphorylated casein was detected by autoradiography. The relative protein kinase activities relative to λPPase-untreated Hhp2-WT are indicated. Casein was visualized by Coomassie blue stain.

Article Snippet: One half was incubated with 1.5 μl of λ protein phosphatase (λPPase) (New England Biolabs) in the presence of 10 mM MnCl 2 for 45 min at 30 °C.

Techniques: Immunoprecipitation, Control, Western Blot, Autoradiography, Staining

Journal: The Journal of Biological Chemistry

Article Title: The mitotic functions of a fission yeast CK1 enzyme are regulated by Cdk1-dependent and auto-phosphorylation

doi: 10.1016/j.jbc.2025.111007

Figure Lengend Snippet: Identification of Hhp2 autophosphorylation sites . A , schematic illustration of the Hhp2 N-terminal domain structure and the C-terminal tail sequence. Major autophosphorylation sites identified in vitro are shown as red-shaded characters. KDE: kinase domain extension. B and C , nda3-KM311 cells and nda3-KM311 cells expressing FLAG 3 -tagged Hhp2 proteins were arrested in mitosis. Protein lysates of the indicated strains were subjected to anti-FLAG (M2) IP. One-half of each IP was treated with λPPase and the other with vehicle control, and proteins were immunoblotted with anti-FLAG M2 antibodies. 30 μM Mn 2+ –Phos-tag gel was used for separating phosphorylated species of Hhp2-FLAG more clearly in ( C ). Asterisks indicate non-specific bands, and solid dots and bracket indicate the positions of phosphorylated forms of Hhp2.

Article Snippet: One half was incubated with 1.5 μl of λ protein phosphatase (λPPase) (New England Biolabs) in the presence of 10 mM MnCl 2 for 45 min at 30 °C.

Techniques: Sequencing, In Vitro, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: The mitotic functions of a fission yeast CK1 enzyme are regulated by Cdk1-dependent and auto-phosphorylation

doi: 10.1016/j.jbc.2025.111007

Figure Lengend Snippet: Identification of Cdk1 phosphorylation sites in Hhp2 . A , potential mitotic phosphorylation sites in the C-terminal tail sequence of Hhp2 are indicated. Purple-shaded boxes denote the sites that had been reported as in vivo phosphorylation sites by global phosphoproteomic studies of S . pombe . Red triangles denote the mitotic phosphorylation sites identified in this study. Asterisks denote sites that fit the minimum consensus motif for Cdk1 (S/TP). B , in vitro Cdk1 kinase assays were performed using equal amounts of the indicated recombinant MBP-Hhp2 proteins as substrates. Reactions were resolved using SDS-PAGE and phosphorylated proteins were detected by phosphoimaging. The relative phosphorylation levels of Hhp2 proteins is provided under the lanes. MBP-Hhp2 was visualized by Coomassie blue stain. C , nda3-KM311 cells and nda3-KM311 cells expressing Hhp2-FLAG proteins were arrested in mitosis. Protein lysates of the indicated strains were subjected to anti-FLAG IP. One half of each IP was treated with λPPase and the other with vehicle control. Phos-tag gel was used for the separation of phosphorylated forms of Hhp2-FLAG and proteins were immunoblotted with anti-FLAG M2 antibodies. Single asterisks indicate non-specific bands and the brackets indicate phosphorylated forms of Hhp2. Solid dots in ( C ) indicate the phosphorylated bands of Hhp2-3A that disappeared in the 7A mutant. The double asterisks indicate kinase-activity-dependent phosphoshifted bands. ( D ) Protein lysates of the indicated strains were subject to immunoblotting with anti-FLAG for Hhp2 ( upper panel ) and anti-PSTAIRE for Cdk1 as a loading control ( lower panel ). E , nda3-KM311 hhp2-HA 3 -TAP cdc2-asM17 ( upper panel ) and nda3-KM311 hhp2-FLAG cdc2-asM17 ( lower panel ) cells were arrested in mitosis. They were then treated for 0, 10, or 20 min with 10 μM 1NM-PP1 or 20 min with DMSO. Protein lysates were subjected to anti-HA ( upper panel ) or anti-FLAG IP ( lower panel ). One-half of each IP was treated with λ-PPase and the other with vehicle control. Immunoprecipitated Hhp2 was visualized by immunoblot with anti-HA ( upper panel ) or anti-FLAG M2 ( lower panel ) antibody. Phos-tag gel was used for the separation of phosphorylated forms of Hhp2-FLAG. ( F ) nda3-KM311 cells were subjected to anti-FLAG IPs. One half of each IP was treated with λPPase and the other with vehicle control, and kinase assays were performed using the bead-bound Hhp2 proteins and equivalent amounts of α-casein as substrate. Immunoprecipitated Hhp2 was visualized by immunoblot with anti-FLAG M2 antibody and phosphorylated casein was detected by autoradiography. Casein was visualized by Coomassie blue stain. The relative protein kinase activities against the λ-PPase-untreated Hhp2-WT from two independent experiments are indicated in a bar graph. Black and white dots represent each experimental data set.

Article Snippet: One half was incubated with 1.5 μl of λ protein phosphatase (λPPase) (New England Biolabs) in the presence of 10 mM MnCl 2 for 45 min at 30 °C.

Techniques: Phospho-proteomics, Sequencing, In Vivo, In Vitro, Recombinant, SDS Page, Staining, Expressing, Control, Mutagenesis, Activity Assay, Western Blot, Immunoprecipitation, Autoradiography