Journal: Cancer Research

Article Title: Cyclin-Dependent Kinase–Mediated Phosphorylation Plays a Critical Role in the Oncogenic Functions of PELP1

doi: 10.1158/0008-5472.can-10-0628

Figure Lengend Snippet: Figure 1. PELP1 is a novel substrate of interphase CDKs. A, expression status of PELP1 in the cell cycle was analyzed in IMR-90 cells (left) and NIH3T3 cells (right). Cells synchronized at G0-G1 phase were released into the cell cycle, and lysates from different time intervals were used in Western blot analysis with the 220B2 PELP1 antibody. B, total lysates from MCF7 cells grown in 10% serum were subjected to immunoprecipitation (IP) using the PELP1 antibody (left), and the CDK4 interaction was verified by Western blotting. Middle, T7-tagged PELP1-overexpressing MCF7 cells were treated with 10−8 mol/L estrogen for various periods of time, PELP1 was immunoprecipitated, and the CDK4 interaction was verified by Western blotting. Right, in vitro kinase assays for the CDK4/cyclin D complex using baculovirus-expressed, GST-tagged, full-length PELP1 as a substrate, and phosphorylation was measured by the amount of 32P incorporation. C, left, total lysates from MCF7 cells grown in 10% serum were subjected to immunoprecipitation using the PELP1 antibody and the CDK2 interaction was verified by Western blot analysis. Middle, MCF7 cells were treated with E2 for various periods of time, and the PELP1 interaction with CDK2 was analyzed by using immunoprecipitation. Right, in vitro kinase assays using CDK2/cyclin E (CyE) complex and CDK2/ cyclin A2 (CyA) complex using full-length PELP1 as a substrate.

Article Snippet: The plaslutathione S-transferase (GST)–PELP1 deletions (16), uc (17), and GFP-PELP1 (16) were described previously. sion vectors for p16INK4A, CDK4, CDK2, and cyclin E urchased from Addgene, Inc.

Techniques: Expressing, Western Blot, Immunoprecipitation, In Vitro, Phospho-proteomics

Journal: Cancer Research

Article Title: Cyclin-Dependent Kinase–Mediated Phosphorylation Plays a Critical Role in the Oncogenic Functions of PELP1

doi: 10.1158/0008-5472.can-10-0628

Figure Lengend Snippet: Figure 2. Identification of phosphorylation sites and generation of phospho-specific antibody. A, bacterially expressed GST-PELP1 deletions were used as substrates for an in vitro kinase assay using CDK4/cyclin D1 (left), CDK2/cyclin E (middle), and CDK2/cyclin A2 (right), and PELP1 domains phosphorylated by each kinase complex were identified by using autoradiography. B, identification of CDK phosphorylation sites using site-directed mutagenesis (serine to alanine). Various single MTs in the region of interest were used along with respective PELP1-WT deletion fragments. Loss of 32P incorporation revealed the successful identification of phosphorylation sites. C, Western blot analysis of native, WT-tagged PELP1 and tagged phospho-PELP1-MT with the Ser991 phospho-PELP1 antibody in the presence or absence of the phosphopeptide. D, MCF7 cells were either arrested and released by E2 stimulation (left) or synchronized into the G1-S boundary by double-thymidine block and released into the cell cycle by addition of thymidine-free medium (right), and phosphorylation status of PELP1 was analyzed by using the phospho-Ser991 antibody.

Article Snippet: The plaslutathione S-transferase (GST)–PELP1 deletions (16), uc (17), and GFP-PELP1 (16) were described previously. sion vectors for p16INK4A, CDK4, CDK2, and cyclin E urchased from Addgene, Inc.

Techniques: Phospho-proteomics, In Vitro, Kinase Assay, Autoradiography, Mutagenesis, Western Blot, Blocking Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Combined CDK4/6 mTOR inhibition is synergistic against glioblastoma via multiple mechanisms

doi: 10.1158/1078-0432.CCR-17-0803

Figure Lengend Snippet: (A) Enrichment analysis showing increased sensitivity of cancer lines with mutations in RB1 to an mTOR inhibitor, sirolimus. (B) Synergy scores of the combination of CDK4/6 and mTOR inhibition in two GIC lines calculated with both the Bliss and the Chou-Talalay methods. (C) 10 and 100 GICs were cultured in 24-well plates over two weeks to compare sphere formation upon treatment with vehicle, palbociclib (1 μM), everolimus (4 μM), and the combination of palbociclib and everolimus (*P < 0.05; **P < 0.001; ***P < 0.0001; one-way analysis of variance (ANOVA) with post-hoc Tukey analysis). (D) The combination of a different mTOR inhibitor temsirolimus (4 μM) and a different CDK4/6 inhibitor ribociclib (4 μM) is also synergistic against GICs (***P < 0.0001; one-way ANOVA with post-hoc Tukey analysis). (E) The combinations of palbociclib (4 μM) with mTOR siRNA and everolimus (5 μM) with CDK4/6 siRNA are also synergistic against GICs (each treatment line received either DMSO or the indicated drug and either control siRNA or a specific siRNA) (**P < 0.001; ***P < 0.0001; one-way ANOVA with post-hoc Tukey analysis). (F) Constitutively-active CDK4 and mTOR plasmids partially rescued from the effects of the combination treatment (each treatment line received either a control plasmid or the indicated active plasmid and either DMSO or the combination treatment) (*P < 0.05; two-tailed t-test). (G and H) Combined CDK4/6 and mTOR inhibition induces significant apoptosis. Shown is an immunoblot using antibodies specific for Bcl-2 and PARP. Caspase-3/7 level is increased with the three days of combined treatment. (*P < 0.05; ***P < 0.0001; one-way ANOVA with post-hoc Tukey analysis).

Article Snippet: Plasmid and siRNA transfection Constitutively-active CDK4 (RC401060, Origene) and mTOR (69010, Addgene) as well as the respective control plasmids were used for rescue experiments.

Techniques: Inhibition, Cell Culture, Control, Plasmid Preparation, Two Tailed Test, Western Blot

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Ribosomal protein RPL22/eL22 regulates the cell cycle by acting as an inhibitor of the CDK4-cyclin D complex.

doi: 10.1080/15384101.2019.1593708

Figure Lengend Snippet: Figure 1. RPL22/eL22 can interact with CDK4 and/or cyclin D1 (a, b) List of selected proteins found to immunoprecipitate with a FLAG-tagged version of CDK4(K35M) from RSL1D1 knockdown-induced senescent cells classified as known interactors of CDK4 (a) and ribosomal proteins interactors (b). (c) HEK-293T cells were transfected with vectors expressing CDK4 and 3xFLAG control, Myc- FLAG tagged RPL22 wild type (RPL22-Myc-FLAG(WT)) or Myc-FLAG tagged RPS14 wild type (RPS14-Myc-FLAG(WT)) and immuno- precipitated with an anti-FLAG antibody. Lysates and immunoprecipitates were immunoblotted for the indicated proteins. (d) HEK- 293T cells were transfected with vectors expressing Myc-cyclin D1 and 3xFLAG control, RPL22-Myc-FLAG(WT) or RPS14-Myc-FLAG (WT) and immunoprecipitated with an anti-FLAG antibody. Lysates and immunoprecipitates were immunoblotted for the indicated proteins. (e) Indirect immunofluorescence (IF) with a specific anti-Myc tag antibody showing RPL22-Myc in IMR90 cells expressing an empty control vector (Vect) or RPL22-Myc at day 12 post-infection (representative images). Data were quantified from 3-independent cell counts up to a total of at least 100 cells in triplicate and are presented as the mean percentage of positive cells for nucleolar localization of RPL22-Myc ± SD. Brightfield images are shown alongside. Scale bar, 10 µm. (f) Immunoblots for the indicated proteins and ethidium bromide detection of 28S rRNA in ribosome purification by sedimentation (Pellet) or its supernatant (Sup) obtained from extracts of IMR90 cells expressing RPL22-Myc or an empty control vector (Vect) at day 7 post-infection. Blots in C, D and F are representative of 3 independent experiments with similar results.

Article Snippet: GST (50 ng) (Cat#: SRP5348, lot: F664-2, SigmaAldrich, St. Louis, MO) or GST tagged human active CDK4-cyclin D1 (50 ng) (Cat#: C0620, lot: SLBK7657V, Sigma-Aldrich) or GST tagged human CDK4 (50 ng) (Cat#: C31-14G, lot: K127-1, Signal Chem, Richmond, BC) or GST tagged human cyclin D1 (50 ng) (Cat#: 009-001-153S, lot: 37136, Rockland antibodies & assays, Limerick, PA) were incubated with human recombinant His-RPL22 (375 ng) (Cat#: NBP2-23419, lot: 12221401, Novus Biologicals, Oakville, ON) in 500 μl of PB buffer (20 mM Hepes, pH 7.5, 130 mM KCl, 5 mM MgCl2, 1 mM DTT, 0.5 mM EDTA, 0.05% NP40) and mixed using a rotating machine at 30°C for 2 hours.

Techniques: Knockdown, Transfection, Expressing, Control, Immunoprecipitation, Immunofluorescence, Plasmid Preparation, Infection, Western Blot, Purification, Sedimentation

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Ribosomal protein RPL22/eL22 regulates the cell cycle by acting as an inhibitor of the CDK4-cyclin D complex.

doi: 10.1080/15384101.2019.1593708

Figure Lengend Snippet: Figure 4. RPL22/eL22 directly binds and inhibits CDK4-cyclin D1 complex. (a) Immunoblots of the indicated proteins after in vitro kinase assay containing ATP and GST-CDK4 and GST-cyclin D1, with or without GST-RB (773–928), palbociclib and HIS-RPL22. RB (pS795): RB phosphorylated on serine 795. (b) In vitro GST pull-down of recombinant GST, GST-cyclin D1 and/or GST-CDK4 and recombinant HIS-RPL22 using glutathione beads. Lysate and pull-down were immunoblotted for the indicated proteins. (c) Model showing interactions between RPL22 and cyclin D1, CDK4 or CDK4-cyclin D1. (d) Model showing how nucleolar stress can lead to activation of p53 and RB tumor suppressor pathways. Blots in A and B are representative of 3 independent experiments with similar results.

Article Snippet: GST (50 ng) (Cat#: SRP5348, lot: F664-2, SigmaAldrich, St. Louis, MO) or GST tagged human active CDK4-cyclin D1 (50 ng) (Cat#: C0620, lot: SLBK7657V, Sigma-Aldrich) or GST tagged human CDK4 (50 ng) (Cat#: C31-14G, lot: K127-1, Signal Chem, Richmond, BC) or GST tagged human cyclin D1 (50 ng) (Cat#: 009-001-153S, lot: 37136, Rockland antibodies & assays, Limerick, PA) were incubated with human recombinant His-RPL22 (375 ng) (Cat#: NBP2-23419, lot: 12221401, Novus Biologicals, Oakville, ON) in 500 μl of PB buffer (20 mM Hepes, pH 7.5, 130 mM KCl, 5 mM MgCl2, 1 mM DTT, 0.5 mM EDTA, 0.05% NP40) and mixed using a rotating machine at 30°C for 2 hours.

Techniques: Western Blot, In Vitro, Kinase Assay, Recombinant, Activation Assay

Journal: iScience

Article Title: RPRM negatively regulates ATM levels through its nuclear translocation on irradiation mediated by CDK4/6 and IPO11

doi: 10.1016/j.isci.2022.105115

Figure Lengend Snippet: RPRM translocates from the cytoplasm to the nucleus upon irradiation, which involves CDK4/6 (A) Representative immunofluorescence images showing RPRM translocation from the cytoplasm to the nucleus in H460-RPRM cells shortly after 10 Gy X-irradiation and quantification of the nuclear/cytoplasmic HA-RPRM fluorescence intensity ratios. Scale bar, 10 μm. (B) Immunoblotting on the nuclear and cytoplasmic RPRM of H460-RPRM cells 30 min after 10 Gy X-irradiation confirmed the nuclear translocation of RPRM upon IR. (C) RPRM nuclear import was enhanced with the increase of irradiation dose. (D) Immunoblotting on the nuclear and cytoplasmic RPRM confirmed that the nuclear translocation of RPRM was enhanced by palbociclib (Palb, 50 μM) pre-treatment. (E and F) Co-IP result showing an interaction between RPRM and CDK4/6 in H460-RPRM cells. (G) Inhibition of CDK4/6 promoted ATM nuclear export regulated by RPRM. Representative immunofluorescence images showing the effect of CDK4/6 inhibition by Palb pre-treatment on ATM cytoplasmic translocation in H460-RPRM cells upon 2 Gy X-irradiation and quantification of the ratios of cytoplasmic/nuclear ATM fluorescence intensity. Scale bar, 10 μm. (H) Immunoblotting results confirmed that CDK4/6 inhibitor Palb reduced ATM levels in H460-NC/RPRM cells upon 2 Gy X-irradiation. (I) The effect of CDK4/6 inhibition by Palb pre-treatment on the micronucleus formation of H460-NC/RPRM cells after 2 Gy X-irradiation. (J) The effect of CDK4/6 inhibition by Palb pre-treatment on the plating efficiency of H460-NC/RPRM cells upon 4 Gy X-irradiation. Data shown represent the means (±SEM) of three biological replicates, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; significance was determined by unpaired two-sample t test.

Article Snippet: Purified recombinant CDK4 , Origene , TP301156.

Techniques: Irradiation, Immunofluorescence, Translocation Assay, Fluorescence, Western Blot, Co-Immunoprecipitation Assay, Inhibition

Journal: iScience

Article Title: RPRM negatively regulates ATM levels through its nuclear translocation on irradiation mediated by CDK4/6 and IPO11

doi: 10.1016/j.isci.2022.105115

Figure Lengend Snippet: CDK4/6 phosphorylate RPRM at serine 98, but the unphosphorylation status of RPRM is critical for its stabilization and nuclear translocation (A and B) Mass spectrometry analysis of the phosphorylation site of RPRM corresponding to CDK4 (A) and CDK6 (B). (C) Time-course analysis of the changes in the RPRM levels of H460-RPRM cells after treated with different inhibitors. (D) Diagrams of full-length RPRM protein and different mutants. (E) Representative immunofluorescence images of the RPRM −/− MEFs exogenously expressing full-length RPRM, RPRM-S98A and ΔRPRM (79–109) at 30 min after 20 Gy X-irradiation. Scale bars, 10 μm. (F) Co-IP assay confirmed an interaction between ATM and both RPRM-S98A and ΔRPRM (79–109) in H460 cells. (G and H) Change in the ATM levels of H460-NC/RPRM/S98A cells at different times after 2 Gy X-irradiation. (I) Change in the ATM expression of H460-NC/RPRM/S998A cells after cisplatin (CDDP, 15 μM) treatment for 24 h. (J) Relative plating efficiency of H460-NC/RPRM/S98A cells irradiated with 4 Gy X-rays. (K) Exogenous expression of both RPRM and RPRM-S98A increased micronucleus formation in H460 cells upon 2 Gy X-rays. (L) Relative plating efficiency of H460-NC/RPRM/S98A cells treated with 15 μM CDDP. (M) Micronucleus formation of RPRM −/− MEFs expressing full-length RPRM, RPRM-S98A and ΔRPRM (79–109) after exposed to 5 Gy X-irradiation. Data shown represent the means (±SEM) of three biological replicates, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; Significance was determined by unpaired two-sample t test.

Article Snippet: Purified recombinant CDK4 , Origene , TP301156.

Techniques: Translocation Assay, Mass Spectrometry, Phospho-proteomics, Immunofluorescence, Expressing, Irradiation, Co-Immunoprecipitation Assay

Journal: iScience

Article Title: RPRM negatively regulates ATM levels through its nuclear translocation on irradiation mediated by CDK4/6 and IPO11

doi: 10.1016/j.isci.2022.105115

Figure Lengend Snippet:

Article Snippet: Purified recombinant CDK4 , Origene , TP301156.

Techniques: Control, Virus, shRNA, Recombinant, Purification, Sequencing, Dominant Negative Mutation, Mutagenesis, Software

Journal: Cancer Research

Article Title: Contrasting Behavior of the p18INK4c and p16INK4a Tumor Suppressors in Both Replicative and Oncogene-Induced Senescence

doi: 10.1158/0008-5472.can-11-2552

Figure Lengend Snippet: Figure 6. Senescence-dependent and -independent downregulation of p18INK4c. A, HDFs (Hs68) treated with the CDK4/6 inhibitor PD0332991 were harvested at the indicated intervals, and levels of INK4c, B-MYB, and CDC6 RNA were assessed by qRT-PCR and normalized to the untreated control (Con). B, HDFs (TIG3) treated with and without PD0332991 for 14 days were stained for SA-b-galactosidase activity and with 40,6—diamidino-2-phenylindole (DAPI) to visualize SAHFs. C, lysates from the cells described in B were immunoblotted for p18INK4c, menin, and E2F1 with MEK as a loading control. D, HDFs (BF and Leiden) were infected with a retrovirus encoding H-RASG12V or empty vector, and the relative levels of INK4c and INK4a RNA were assessed by qRT-PCR and normalized to the vector-only control. E, lysates from the cells described in D were immunoblotted for p18INK4c and p16INK4a. F, lysates from Leiden cells infected with a retrovirus encoding H-RASG12V or empty vector were immunoblotted for menin, E2F1, p18INK4c, and RAS. MEK, MAP/ERK kinase.

Article Snippet: Primary antibodies were as follows: CDK4 (sc-601), CDK6 (sc-177), E2F1 (sc-22820), p18INK4c (sc-1208), mouse p16Ink4a (sc-1207), b-tubulin (sc9104), p53 (DO-1, sc-126), cyclin E (sc-247), cyclin A (sc-601), and p21CIP1 (sc-397) were from Santa Cruz; MEK1/2 (#9122) was from Cell Signalling; menin (AB2605), HRP-conjugated anti-GAPDH (AB9482) and a monoclonal antibody (mAb) against CDK6 (AB77674) were from Abcam; CDK4 (DCS31) and p18INK4c (DCS118) mAbs were from Novus; the cyclin D1 mAb (DCS6) was from BD Pharmingen; a pan-Ras mAb (OP41) was from Oncogene Research; and the mAb against p16INK4a (JC8) was provided by LRI Cell Services using cells originally obtained from J. Koh and E. Harlow.

Techniques: Quantitative RT-PCR, Control, Staining, Activity Assay, Infection, Plasmid Preparation