Journal: Frontiers in Cell and Developmental Biology

Article Title: Stimulation of c-Jun/AP-1-Activity by the Cell Cycle Inhibitor p57 Kip2

doi: 10.3389/fcell.2021.664609

Figure Lengend Snippet: p57 activates AP-1-dependent promoters by inducing c-Jun transactivation independent of FHL2. (A) Protein levels of FHL2 and p57 in different cell lines. 100 μg of total protein extract were analyzed by immunoblotting for FHL2 and p57 using a mouse monoclonal FHL2- and a rabbit polyclonal p57-specific antibody. CEM, human leukemic; HEL, human erythroleukemic; HL-1, mouse cardiac muscle; WM35, human melanoma; 293, human embryonic kidney; MCF-7, human breast cancer; HRT-18, human colon cancer; U2OS, human osteosarcoma; HeLa, human cervix carcinoma. The unspecific band at 70 kD ( ∗ ) after probing for p57 was used to judge equal loading. (B) Knockdown of FHL2 using four different doxycyclin-inducible shRNAs in stable pools of lentivirally transduced HRT-18 cells. Western blot analysis of extracts obtained from HRT-18 cells. The expression of shRNAs was induced by the addition of doxycycline (1 μg/mL) for 72 h. Cells expressing shRNA targeting luciferase mRNA (shLuc) served as a control. The FHL2-specific antibody revealed efficient (sh215, sh718) and less efficient (sh428, sh598) inhibition of FHL2 expression. GAPDH served as a loading control. (C) Regulation of an artificial AP-1-dependent promotor (schematic representation top panel) by p57. HRT-18.sh215 cells either untreated (– Doxy) or treated with 1 μg/mL doxycycline (+ Doxy) for 72 hours were transfected with 5xTRE-Luc reporter gene construct and 10 ng pUbi-Rluc (expressing renilla luciferase as a transfection control) together with the p57 expression vectors as indicated. A total of 48 h after transfection, cells were harvested and a Dual luciferase assays performed. Relative promoter activities were expressed as firefly luciferase activities normalized for renilla-luciferase activities. Shown are the mean values ± SD from six independent experiments. (D) Gal4-dependent luciferase reporter gene experiments comparing the effect of p57 on c-Jun and c-Fos transactivation. 293 cells with stably integrated Gal4-dependent luciferase reporter gene (293FR) were transfected with DNA-expression plasmids for Gal-DBD, Gal-Jun or Gal-Fos and p57. Schematic representation of the reporter construct is shown in the top panel. In addition, 10 ng pUbi-Rluc expressing renilla luciferase were cotransfected as control. A total of 30 h after transfection, cells were extracted and subjected to Dual luciferase assays. Renilla-normalized values are expressed as fold activation relative to control transfected (–) for Gal-Jun and Gal-Fos which are set to 1. For showing lower fold activations, y-axis is split into two segments (bottom: 0 to 15, top: 100 to 2,250). Shown is the mean ± SD from three independent experiments. (E) Gal4-dependent luciferase reporter gene experiments analyzing effect of p57 on c-Jun transactivation. HRT-18 cells with stably integrated Gal4-dependent luciferase reporter gene (HRT-18FR) were transfected with DNA-expression plasmids for Gal-Jun, p57 and the indicated shRNAs. pUbi-Rluc (expressing renilla luciferase) was cotranfected as a transfection control. shco was used as a control shRNA not targeting any known mRNA. 72 hours after transfection, cells were harvested and subjected to Dual luciferase assays. Renilla-normalized values are expressed as fold activation relative to control transfected (“–”/“shco”) cells which is set to 1. Shown is the mean ± SD from three independent experiments. Level of significance is indicated. ** = Highly significant, n.s. = not significant.

Article Snippet: Mouse monoclonal FHL2 (F4B2-B11): sc-52667, rabbit polyclonal p57 (C-20): sc-1040, mouse monoclonal p57 (KP39): sc56341, mouse monoclonal anti GAPDH (6C5): sc-32233, c-Jun (G-4): sc-74543, rabbit polyclonal HDAC1 (H-51): sc-7872 and HDAC1 (H-51): sc-7872 were purchased from Santa Cruz, rabbit polyclonal Phospho-c-Jun (Ser73): #9164, rabbit polyclonal Phospho-c-Jun (Ser63) II: #9261 antibodies from Cell Signaling, rabbit polyclonal ANTI-FLAG and mouse monoclonal ANTI-FLAG M2 from Sigma-Aldrich, PSTAIR-motif containing CDKs were detected by using mouse monoclonal anti-PSTAIRE ( ) and mouse monoclonal anti-HA (12CA5 ab16918, Abcam, Cambridge, MA, United States).

Techniques: Western Blot, Knockdown, Expressing, shRNA, Luciferase, Control, Inhibition, Transfection, Construct, Stable Transfection, Activation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Stimulation of c-Jun/AP-1-Activity by the Cell Cycle Inhibitor p57 Kip2

doi: 10.3389/fcell.2021.664609

Figure Lengend Snippet: Strong activation of Gal-Jun activity is specific for p57 and requires both, the N -terminal and C -terminal domains of p57. (A) Gal4-dependent luciferase reporter gene experiments comparing the effect of the Cip/Kip-family members p57, p27, and p21 on c-Jun transactivation. 293FR cells were cotransfected with DNA-expression plasmids for Gal-DBD, Gal-Jun and p57, p27 or p21. pUbi-Rluc (expressing renilla luciferase) served as a transfection control. A total of 30 h after transfection cells were harvested and subjected to Dual luciferase assays. Renilla-normalized values are expressed as fold activation relative to control transfected (Co.) which is set to 1. To represent minor activation, the y-axis is split into two segments (bottom: 0 to 15, top: 100 to 1,500). Shown is the mean ± SD from three independent experiments. (B) Similar experiment to (A) comparing p57 and cyclin/CDK-binding deficient p57 mutant. 293FR cells were cotransfected with DNA-expression plasmids for Gal-DBD, Gal-Jun and p57 or the cyclin/CDK-binding deficient p57-CK- mutant (CK-) and pUbi-Rluc. A total of 30 h after transfection, cells were harvested and subjected to dual luciferase assays. Renilla-normalized values are expressed as fold activation relative to control transfected (Co.) which is set to 1. Shown is the mean ± SD from four independent experiments. (C) Schematic representation of p57 and its N -terminal and C -terminal domains. Key structural or functional regions of human p57 are indicated. Numbers above indicate positions of amino acid flanking distinct regions. Amino acid including the N -terminal (p57-Nt) and C -terminal (p57-Ct) domains are shown in brackets. (D) Experiment as described above (A) analyzing the contribution of p57-domains to the activation of Gal-Jun by p57. 293FR cells were cotransfected with DNA-expression plasmids for Gal-Jun and p57 or the indicated p57 domain mutants. A total of 30 hours after transfection cells were harvested and subjected to Dual luciferase assays. Renilla-normalized values are expressed as fold activation relative to control transfected (Co.) which is set to 1. For showing lower fold activations, y-axis is split into two segments (bottom: 0 to 100, top: 100 to 1,500). Shown is the mean ± SD from three independent experiments.

Article Snippet: Mouse monoclonal FHL2 (F4B2-B11): sc-52667, rabbit polyclonal p57 (C-20): sc-1040, mouse monoclonal p57 (KP39): sc56341, mouse monoclonal anti GAPDH (6C5): sc-32233, c-Jun (G-4): sc-74543, rabbit polyclonal HDAC1 (H-51): sc-7872 and HDAC1 (H-51): sc-7872 were purchased from Santa Cruz, rabbit polyclonal Phospho-c-Jun (Ser73): #9164, rabbit polyclonal Phospho-c-Jun (Ser63) II: #9261 antibodies from Cell Signaling, rabbit polyclonal ANTI-FLAG and mouse monoclonal ANTI-FLAG M2 from Sigma-Aldrich, PSTAIR-motif containing CDKs were detected by using mouse monoclonal anti-PSTAIRE ( ) and mouse monoclonal anti-HA (12CA5 ab16918, Abcam, Cambridge, MA, United States).

Techniques: Activation Assay, Activity Assay, Luciferase, Expressing, Transfection, Control, Binding Assay, Mutagenesis, Functional Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Stimulation of c-Jun/AP-1-Activity by the Cell Cycle Inhibitor p57 Kip2

doi: 10.3389/fcell.2021.664609

Figure Lengend Snippet: JNK-independent activation of c-Jun by p57. (A) Schematic of human wildtype c-Jun, Gal-Jun and Gal-Jun4A mutant. Functional domains and phosphorylation sites in the transactivation domain (TAD) are indicated. A Gal4-DNA binding domain (DBD) fusion in frame to the aminoterminus of c-Jun or mutant c-Jun4A ranging from amino acids 1–223 is used in reporter gene experiments. (B) Gal4-dependent luciferase reporter gene experiments analyzing the role of the four major phosphorylation sites in the TAD of c-Jun in the activation by p57. 293FR cells were cotransfected with DNA-expression plasmids for Gal-Jun or the TAD phosphosite mutant Gal-Jun4A in absence or presence of p57 or the constitutive active upstream kinase of JNKs (ΔMEKK). pUbi-Rluc (expressing renilla luciferase) served as a transfection control. A total of 30 h after transfection cells were harvested and subjected to dual luciferase assays. Renilla-normalized values are expressed as fold activation relative to control transfected (Co.) Gal-Jun which was set to 1. For showing lower fold activations, y-axis is split into two segments (bottom: 0 to 2.5, top: 50 to 2,000). Shown are the results from three independent experiments. (C) p57 does not induce serine 63 or serine 73 phosphorylation of c-Jun. Immunoblot-analysis investigating the effect of p57 on c-Jun TAD-phosphorylation. 293 cells were transfected with expression vectors for c-Jun and p57 (both HA-tagged) or the constitutive active kinase ΔMEKK. Extracts were prepared 40 h after transfection and subjected to immunoblot-analysis by the LiCor Odyssey system to visualize expression of HA-Jun and Ser 63 and Ser 73 phosphorylation simultaneously. Mixture of Ser 63 and Ser 73 phospho-specific rabbit polyclonal and anti HA mouse monoclonal antibodies were used as first antibodies followed by a mixture of goat derived secondary antibodies coupled with fluorochromes for 680 (anti mouse) or 780 nm (anti rabbit). Three images are shown. Upper: 700 nm channel, phospho-Ser 63/73; middle: 800 nm channel, HA; lower: merged pseudo-colors from channel 700 (red) and 800 (green). Note that molecular weight of HA-Jun is shifted in the presence of the constitutive active kinase ΔMEKK and appears yellow in the merged image.

Article Snippet: Mouse monoclonal FHL2 (F4B2-B11): sc-52667, rabbit polyclonal p57 (C-20): sc-1040, mouse monoclonal p57 (KP39): sc56341, mouse monoclonal anti GAPDH (6C5): sc-32233, c-Jun (G-4): sc-74543, rabbit polyclonal HDAC1 (H-51): sc-7872 and HDAC1 (H-51): sc-7872 were purchased from Santa Cruz, rabbit polyclonal Phospho-c-Jun (Ser73): #9164, rabbit polyclonal Phospho-c-Jun (Ser63) II: #9261 antibodies from Cell Signaling, rabbit polyclonal ANTI-FLAG and mouse monoclonal ANTI-FLAG M2 from Sigma-Aldrich, PSTAIR-motif containing CDKs were detected by using mouse monoclonal anti-PSTAIRE ( ) and mouse monoclonal anti-HA (12CA5 ab16918, Abcam, Cambridge, MA, United States).

Techniques: Activation Assay, Mutagenesis, Functional Assay, Phospho-proteomics, Binding Assay, Luciferase, Expressing, Transfection, Control, Western Blot, Bioprocessing, Derivative Assay, Molecular Weight

Journal: Frontiers in Cell and Developmental Biology

Article Title: Stimulation of c-Jun/AP-1-Activity by the Cell Cycle Inhibitor p57 Kip2

doi: 10.3389/fcell.2021.664609

Figure Lengend Snippet: p57 is in a complex with c-Jun. (A) Co-immunoprecipitation experiment (Co-IP) followed by immunoblotting showing binding of FLAG-tagged p57 to HA-tagged c-Jun. 293 cells were transfected with expression plasmids for HA-Jun and FLAG-p57. HA-tagged c-Jun was precipitated by using anti-HA mouse monoclonal antibodies. Immunoprecipitates (IP: antiHA) and 1% of the total protein extract used for the IP (Input) were subjected to immunoblot analysis. Upper panels show FLAG-p57, lower panels HA-Jun. Molecular weight markers are indicated left. (B) Complex formation of c-Jun with the aminoterminal domain of p57. 293 cells were transfected with expression constructs for FLAG-Jun, HA-p57 and amino- and carboxyterminal domains of p57 (HA-p57-Ct, HA-p57-Nt). FLAG-tagged YFP (FLAG-YFP) was expressed and used as a control. FLAG-tagged c-Jun was precipitated by using anti-FLAG mouse monoclonal antibodies. Immunoprecipitates (IP: antiFLAG) and 1% of the total protein extract used for the IP (Input) were subjected to western-blot analysis. Upper panels show HA-p57 and mutants, lower panels FLAG-Jun/FLAG-YFP. Molecular weight markers are indicated left. Heavy and light chain of the antibodies used in the IPs are indicated ( ∗ ). (C) Endogenous p57 is in a complex with endogenous c-Jun. p57 was immunoprecipitated from extracts of HRT-18 cells using p57-specific rabbit polyclonal antibodies. An IP with rabbit IgG was performed as a control. Immunoprecipitates (IP: p57, IP: rabbit IgG) and 5% of HRT-18 extract used for the IP (Input) were subjected to immunoblot analysis. Upper panel shows p57 and lower panel c-Jun, both detected with mouse monoclonal antibodies. Molecular weight marker is indicated left.

Article Snippet: Mouse monoclonal FHL2 (F4B2-B11): sc-52667, rabbit polyclonal p57 (C-20): sc-1040, mouse monoclonal p57 (KP39): sc56341, mouse monoclonal anti GAPDH (6C5): sc-32233, c-Jun (G-4): sc-74543, rabbit polyclonal HDAC1 (H-51): sc-7872 and HDAC1 (H-51): sc-7872 were purchased from Santa Cruz, rabbit polyclonal Phospho-c-Jun (Ser73): #9164, rabbit polyclonal Phospho-c-Jun (Ser63) II: #9261 antibodies from Cell Signaling, rabbit polyclonal ANTI-FLAG and mouse monoclonal ANTI-FLAG M2 from Sigma-Aldrich, PSTAIR-motif containing CDKs were detected by using mouse monoclonal anti-PSTAIRE ( ) and mouse monoclonal anti-HA (12CA5 ab16918, Abcam, Cambridge, MA, United States).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Binding Assay, Transfection, Expressing, Bioprocessing, Molecular Weight, Construct, Control, Marker

Journal: Frontiers in Cell and Developmental Biology

Article Title: Stimulation of c-Jun/AP-1-Activity by the Cell Cycle Inhibitor p57 Kip2

doi: 10.3389/fcell.2021.664609

Figure Lengend Snippet: p57 binds HDAC1 and HDAC3 via its N -terminal domain and co-migrates in high molecular weight complexes. (A) Co-IP followed by immunoblotting investigating binding of exogenous p57 and domains of p57 to HDAC1. HA-p57, aminoterminal (HA-p57-Nt) and carboxyterminal (HA-p57-Ct) domains were expressed in 293 cells together with FLAG-HDAC1. p57 proteins were immunoprecipitated using anti-HA antibodies and the precipitated p57 proteins were detected by using mouse anti-HA antibodies. Coprecipitated HDAC1 was detected with mouse anti-FLAG antibodies (“IP: antiHA”). One percent of protein extract used for immunoprecipitations was loaded onto the same gel and served as an input control (Input). Size of p57 and its domains are indicated by arrows (left) and molecular weight marker is indicated (right). (B) Similar experiment as in (A) with the exception that binding of FLAG-HDAC3 was analyzed. (C) p57 is a component of high molecular weight complexes and coelutes with HDACs. Crude nuclear extracts of HRT-18 cells were applied to size exclusion chromatography. Fractionation by Superdex 200 10/300 GL was followed by SDS-PAGE and Western blotting, using anti-p57 and anti-HDAC1 and HDAC3 antibodies as indicated. Void volume and the elution of molecular weight markers is indicated below. PSTAIR-reactive CDK proteins were detected using monoclonal anti-PSTAIR antibodies.

Article Snippet: Mouse monoclonal FHL2 (F4B2-B11): sc-52667, rabbit polyclonal p57 (C-20): sc-1040, mouse monoclonal p57 (KP39): sc56341, mouse monoclonal anti GAPDH (6C5): sc-32233, c-Jun (G-4): sc-74543, rabbit polyclonal HDAC1 (H-51): sc-7872 and HDAC1 (H-51): sc-7872 were purchased from Santa Cruz, rabbit polyclonal Phospho-c-Jun (Ser73): #9164, rabbit polyclonal Phospho-c-Jun (Ser63) II: #9261 antibodies from Cell Signaling, rabbit polyclonal ANTI-FLAG and mouse monoclonal ANTI-FLAG M2 from Sigma-Aldrich, PSTAIR-motif containing CDKs were detected by using mouse monoclonal anti-PSTAIRE ( ) and mouse monoclonal anti-HA (12CA5 ab16918, Abcam, Cambridge, MA, United States).

Techniques: High Molecular Weight, Co-Immunoprecipitation Assay, Western Blot, Binding Assay, Immunoprecipitation, Control, Molecular Weight, Marker, Size-exclusion Chromatography, Fractionation, SDS Page

Journal: Frontiers in Cell and Developmental Biology

Article Title: Stimulation of c-Jun/AP-1-Activity by the Cell Cycle Inhibitor p57 Kip2

doi: 10.3389/fcell.2021.664609

Figure Lengend Snippet: p57 might recruit transcription repressors and activators to c-Jun. (A) Gal4-dependent luciferase reporter gene experiments analyzing the effect of p57-domain coexpression on p57-induced c-Jun-activation. Coexpression of p57-Ct may titrate out activators and coexpression of p57-Nt may titrate out repressors of c-Jun activation by p57. 293FR cells were cotransfected with expression plasmids for Gal-Jun, pUbi-Rluc, and p57 wildtype together with vectors expressing p57-Ct or p57-Nt or the related aminoterminal CDK-inhibitory domain of p27 (p27-Nt). A total of 30 h after transfection cells were harvested and subjected to Dual luciferase assays. Renilla-normalized values are expressed as fold activation relative to control transfected (Co.) which is set to 1. Shown is the mean ± SD from three independent experiments. (B) Mechanistic models for p57 in stimulating Gal-Jun transactivation function and a schematic of a putative transcription complex. (C) Model how p57 and FHL2 might act at endogenous promoters at AP-1 sites (TRE). Question mark in (B) and (C) indicates that there is no experimental proof for a direct interaction of p57 with the depicted proteins. For details see text.

Article Snippet: Mouse monoclonal FHL2 (F4B2-B11): sc-52667, rabbit polyclonal p57 (C-20): sc-1040, mouse monoclonal p57 (KP39): sc56341, mouse monoclonal anti GAPDH (6C5): sc-32233, c-Jun (G-4): sc-74543, rabbit polyclonal HDAC1 (H-51): sc-7872 and HDAC1 (H-51): sc-7872 were purchased from Santa Cruz, rabbit polyclonal Phospho-c-Jun (Ser73): #9164, rabbit polyclonal Phospho-c-Jun (Ser63) II: #9261 antibodies from Cell Signaling, rabbit polyclonal ANTI-FLAG and mouse monoclonal ANTI-FLAG M2 from Sigma-Aldrich, PSTAIR-motif containing CDKs were detected by using mouse monoclonal anti-PSTAIRE ( ) and mouse monoclonal anti-HA (12CA5 ab16918, Abcam, Cambridge, MA, United States).

Techniques: Luciferase, Activation Assay, Expressing, Transfection, Control