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

Journal: bioRxiv

Article Title: Salt-inducible kinase 3 regulates the mammalian circadian clock by destabilizing PER2 protein

doi: 10.1101/124354

Figure Lengend Snippet: a. HEK293T17 cells were transfected with SIK3 and PER2 expressing constructs, and the effect on PER2 expression levels was examined by western blotting analysis. Overexpression of SIK3 (SIK3-OX) in HEK293T17 cells (WT) and constitutively-active SIK3 cells (T163E), but not kinase-deficient SIK3 mutant cells (K37M), significantly reduced PER2 protein levels (n = 3, P <0.001 by Tukey’s test). b. Load volume of cell lysates in western blotting analysis was adjusted for comparison of phosphorylation rate. Adjustment of PER2 levels in SIK3-OX and non-OX controls revealed increased rates of upshifted PER2 protein (upshift/total) in NIH3T3 cells (n = 3, *** P <0.001 vs. controls by Student’s t-test). c. Myc-tagged PER2 co-expressed with SIK3 was purified from HEK293T17 cell lysate and incubated with ÀPPase for 30 min at 30 °C. PER2 up-shift was decreased after λPPase treatment in SIK3-OX cells, which was attenuated by phosphatase inhibitor, Na 3 VO 4 . d. PER2 degradation assay was performed in Sik3-OX cells. Cells were collected at 0, 3, and 6 h after the addition of CHX. PER2 protein levels at the starting point (t=0) were normalized to 1. All data used for quantification are shown in Figure 4-figure supplement 2b (n = 3, * P <0.05 by Student’s t-test). Sik3-OX cells accelerated PER2 degradation. e. NIH3T3 cells were transfected with each shRNAs of Sik3 (SIK3-KD, #1-4) or negative control shRNA. All of SIK3-KD significantly increased PER2 levels (n = 3, P <0.05 by Tukey’s test). Another set of samples is also shown in Figure 3-figure supplement 3a. f. Sik3 -KD in NIH3T3 cells reduced PER2 up-shift. Load volume of cell lysates in western blotting analysis was adjusted for comparison of phosphorylation rate (n = 3 for control and shRNA#2, *** P <0.001 vs. controls by Student’s t-test). g. λPPase treatment with Myc-PER2 purified from cell lysates of control cells or Sik3 -KD cells. PER2 up-shift was decreased after ÀPPase treatment in SIK3-OX cells, which was attenuated by phosphatase inhibitor, Na3VO4. h. PER2 degradation assay in Sik3 -KD cells was performed. PER2 protein levels at the starting point (t = 0) were normalized to 1 (n = 3, * P <0.05 by Student’s t-test).

Article Snippet: NIH3T3 cells were transfected with myc-PER2 and Flag-SIK3 expression vectors or Sik3 -KD vectors, and cultured for 48 h. Immunoprecipitated myc-PER2 were incubated with 400 IU λPPase (Sigma) in Lambda Protein Phosphatase Buffer (Sigma) containing 2 mM MnCl 2 for 30 min at 30°C in the presence of Protein G Sepharose.

Techniques: Transfection, Expressing, Construct, Western Blot, Over Expression, Mutagenesis, Purification, Incubation, Degradation Assay, Negative Control, shRNA

Journal: bioRxiv

Article Title: Salt-inducible kinase 3 regulates the mammalian circadian clock by destabilizing PER2 protein

doi: 10.1101/124354

Figure Lengend Snippet: a. In vitro kinase assays of Myc-PER2 purified from HEK293T17 cell lysate and recombinant SIK3 catalytic domain. Incubation of PER2 protein with SIK3 increased the up-shifted PER2 band. b. Purified Myc-PER2 was treated with λPPase prior to a kinase assay with SIK3. No-phosphorylated PER2 was also phosphorylated by recombinant SIK3. c. Up-shifted band shown in b was confirmed as phosphorylation-form of PER2 by 2nd λPPase assay. Purified Myc-PER2 from cell lysates was incubated with λPPase and then with SIK3 (200 ng). After washing SIK3 away, PER2 was subjected to secondary ÀPPase treatment. d. NIH3T3 and HEK293T/17 cells expressing SIK3 were incubated with CSNK1 inhibitor (IC261) for indicated time periods. CSNK1 inhibitor did not affect SIK3-mediated PER2 degradation in both cell lines. e. NIH3T3 cells were transfected with Sik3 -KD constructs as well as Csnkle -WT or kinase dead form of Csnkle ( Csnkle -KD). Knockdown of Sik3 did not affect CSNK1-mediated phosphorylation and degradation of PER2. f. A schematic representation of a potential role for SIK3 compared with that of CSNK1. Although both SIK3 and CSNK1 mediate PER2 phosphorylation and degradation, their roles and degradation pathways appears to be different. Therefore, it may not be the case that SIK3 acts as a priming kinase for CSNK1, but the two kinases regulate PER2 independently. P represents phosphate.

Article Snippet: NIH3T3 cells were transfected with myc-PER2 and Flag-SIK3 expression vectors or Sik3 -KD vectors, and cultured for 48 h. Immunoprecipitated myc-PER2 were incubated with 400 IU λPPase (Sigma) in Lambda Protein Phosphatase Buffer (Sigma) containing 2 mM MnCl 2 for 30 min at 30°C in the presence of Protein G Sepharose.

Techniques: In Vitro, Purification, Recombinant, Incubation, Kinase Assay, Expressing, Transfection, Construct