ATF4 levels oscillate during the cell cycle. A, lysates from synchronized NIH3T3 cells were immunoblotted with the indicated antibodies. Degree of synchrony was confirmed by flow cytometry. As a control, MG-132 was used to stabilize endogenous ATF4. As, asynchronous; G₁/S, G₁ phase/S-phase; S, S-phase; M, mitosis. B, lysates from NIH3T3 cells synchronized in S-phase (thymidine) and M-phase (nocodazole) were subjected to alkaline phosphatase treatment (+CIP). C, NIH3T3 cells synchronized in early S phase with a double thymidine block were released for the indicated durations. Nuclear extracts were collected and immunoblotted with the indicated antibodies. D, HEK293T cells were transfected with the indicated ATF4 truncation constructs. Lysates were collected, subjected to CIP treatment, and immunoblotted with FLAG antibody. E, schematic of potential proline-directed serines and threonines in murine ATF4. Asterisk, the proline following Ser²⁵⁴ is not conserved across species. F, HEK293T cells were transfected with mutant variants of the 190–272 fragment and full-length ATF4 as indicated and subjected to immunoblot analysis with FLAG antibody. Light, light exposure; dark, dark exposure. G, lysates from 293T cells transfected with full-length WT or triple mutant (3A) ATF4 were subjected to immunoblot analysis with FLAG antibody. WB, Western blot.

PDP events converge to regulate phosphorylation of the β-TrCP phosphodegron. A, schematic of the conserved β-TrCP degron in ATF4 across multiple species. B, 293T cells were transfected with the indicated FLAG-tagged ATF4 constructs. Lysates were subjected to FLAG immunoprecipition followed by treatment with alkaline phosphatase (CIP) where indicated. Samples were then analyzed by Western blot (WB) using the phospho-Ser²¹⁸ antibody. C, the indicated FLAG-tagged ATF4 mutants were expressed in 293T cells, which were treated with MG-132 (30 μm) for 4 h prior to lysis with RIPA buffer. Subsequently, lysates were subjected to FLAG immunoprecipitation and Western blot with phospho-Ser²¹⁸ antibody. D, quantification of C by densitometry. Data are presented as the mean ± S.E. (n = 3). E, 293T cells were transfected with the various ATF4 mutant constructs and GST-tagged β-TrCP2 and treated with MG-132 (30 μm) for 4 h prior to lysis with RIPA buffer. Lysates were subjected to GST pulldown followed by Western blot with FLAG antibody. F, 293T cells were transfected individually or in combination with the indicated plasmids (FLAG-S218E and/or GST-β-TrCP2) as depicted. Following CIP treatment, cell lysates were subjected to GST pulldown followed by Western blot with the indicated antibodies.

Stabilized ATF4 induces an early G₁ cell cycle arrest. A, NIH3T3 cells were transfected with the indicated pCAG-IRES-GFP plasmids for 48 h. Cells were given a 20 μm BrdU pulse for the last hour, fixed with antibodies against GFP and BrdU. Presented is the percentage of GFP and BrdU double positive cells. BrdU incorporation for the control (pCAG) was set to 100% and other samples were normalized to the control. Data are presented as the mean ± S.E. (n = 3). *, p < 0.01; **, p < 0.001 (one-way analysis of variance). B, NIH3T3 cells were transfected as in A. Mitotic cells were analyzed by costaining with antibodies against GFP and phospho-histone H3 antibodies. Data are presented as the mean ± S.E. (n = 3). *, p < 0.01; **, p < 0.001 (one-way analysis of variance). C, FACS analysis of HeLa cells transfected with the indicated plasmids. GFP-positive cells were gated and analyzed for DNA content. Below, quantification of the percent of G₀/G₁ cells from C. Data are presented as the mean ± S.E. (n = 3). *, p < 0.001 (one-way analysis of variance). D, NIH3T3 cells were transfected with the indicated plasmids and stained with Ki-Mcm6 and Ki-67 antibodies. Early G₁ arrest is calculated as the ratio of GFP + Ki-Mcm6 + Ki67, to total GFP + Ki-Mcm6 + cells. Data are presented as the mean ± S.E. (n = 3). *, p < 0.001 (one-way analysis of variance). E, NIH3T3 cells infected with retrovirus encoding the indicated variants of ATF4 were selected by puromycin for 3 days. Cells were replated at 20,000 cells per well, and cell number was counted over the course of 4 days.

Stabilization of ATF4 results induces an early G₁ block in neural progenitors and inhibits neurogenesis in vivo. A, embryonic brains (E11.5) were electroporated with pCAG, pCAG-WT, pCAG-S218A, pCAG-5A, fixed 48 h later, and processed for immunohistochemistry. Sections were stained with antibodies against GFP (green) and Tuj1 (red). Nuclei were stained with Hoechst 33258 (blue). A representative image is shown demonstrating that cells remaining in the ventricular zone are negative for Tuj1. Data are presented as the mean ± S.E. (n = 3). *, p < 0.001 (one-way analysis of variance). B, embryonic brains electroporated with the indicated plasmids were analyzed for early G₁ arrest by staining with antibodies against GFP (green), Ki-Mcm6 (red), and Ki-67 (light blue). Shown is the ventricular zone. Yellow arrows, early G₁ phase (GFP + Ki-Mcm6 + Ki-67). White arrowheads, non-early G₁ cells. Data are presented as mean ± S.E. (n = 3). *, p < 0.001 (one-way analysis of variance). C, E11.5 embryonic brains were electroporated with pCAG or pCAG-5A in combination with cyclin D or a vector control. Processed brains were stained as in A, except that Tbr1 antibody was used in place of Tuj1. D, quantification of neurogenesis from brain sections of C stained with Tuj1 antibody in place of the TBR1 antibody. Data are presented as the mean ± S.E. (n = 3). *, p < 0.001 (one-way analysis of variance). E, quantification of cell positioning from C. Data are presented as the mean ± S.E. (n = 3). a, 5A + vector to pCAG + vector, pCAG + cyclin D, 5A + cyclin D, p < 0.001; b, 5A + vector to pCAG + vector, pCAG + cyclin D, 5A + cyclin D, p < 0.001; c, 5A + cyclin D to pCAG + vector and pCAG + cyclin D, p < 0.01; d, 5A + vector, 5A + cyclin D to pCAG + vector, pCAG + cyclin D, p < 0.001; e, 5A + vector to pCAG + vector, pCAG + cyclin D, 5A + cyclin D, p < 0.001. p values were obtained by one-way analysis of variance.

A gradient of phosphorylation targets ATF4 for degradation by the ubiquitin proteasome system. A, HEK293T cells were transfected with the indicated ATF4 constructs and treated with cycloheximide for the indicated durations. ATF4 persistence is plotted as the percentage of signal remaining compared with the vehicle-treated zero time point by densitometry. Results are plotted as the mean of at least three independent experiments. B, plot of percent ATF4 remaining at 100 min from A. Data are presented as the mean ± S.E. (n = 3). p values are denoted on the plot (one-way analysis of variance). The 4A mutants are denoted in the abbreviated form using the last number of each amino acid residue. For example, the 2304 mutant is an abbreviation for T212A/S223A/S230A/S234A. C, HEK293T cells were cotransfected with the indicated ATF4 mutant constructs and GST-tagged β-TrCP2 and subjected to GST pulldowns. Cells were treated with MG-132 (30 μm) prior to lysis to stabilize ATF4. The amount of ATF4 associated with β-TrCP2 was determined by Western blot analysis with FLAG antibody. D, quantification of C by densitometry presented as the mean ± S.E. 1, p < 0.001 WT, 165A, 172A compared with 218A, 3A, 172/3A, 165/172/3A, 5A, and 9A; 2, p < 0.05 3A, 165/3A, 165/172/3A compared with 218A, 5A, and 9A. p values were obtained by one-way analysis of variance. E, HEK293T cells were transfected with the indicated mutant ATF4 constructs. Eighteen hours post-transfection, cells were treated with MG-132 (30 μm) for 4 h prior to lysis with RIPA buffer. Lysates were subjected to FLAG immunoprecipitation and analyzed for the degree of ATF4 ubiquitination by immunoblotting with anti-ubiquitin antibody. WB, Western blot.

CK1 regulates ATF4 stability. A, cold in vitro kinase assay with purified GST-ATF4 and CK1, and CK2 kinases. Samples were analyzed by immunoblotting with the Ser²¹⁸ phospho-specific antibody. B, FLAG-tagged ATF4 or the corresponding D216A mutant were transfected into 293T cells. Following FLAG immunoprecipitation with cell lysates, immunoprecipitates were subjected to Western blot with the phospho-Ser²¹⁸ antibody. C, FLAG-tagged ATF4 and myc-tagged CK1 α, δ, or ϵ were transfected into 293T cells and cells were treated with MG-132 for 4 h prior to lysis. Following FLAG immunoprecipitation, immunoprecipitates were subjected to Western blot (WB) with the indicated antibodies. IgG heavy chain overlaps with myc-tagged CK1δ and -ϵ. D, FLAG-tagged ATF4 and myc-tagged CK1 were transfected into 293T cells as in C. Phosphorylation of the Ser²¹⁸ residue was analyzed by FLAG immunoprecipitation followed by Western blot with the S218P antibody. E, stability of ATF4 in cells pretreated with 50 μm IC261 was analyzed as described in the legend to Fig. 2A. The exposure was adjusted to achieve comparable starting ATF4 levels for both samples. F, 293T cells were co-transfected with FLAG-tagged ATF4 and dominant-negative CK1 constructs and treated with MG-132 for 4 h prior to lysis. Lysates were subjected to FLAG immunoprecipitation followed by Western blot (WB) with ubiquitin and S218P antibodies. The asterisk represents monoubiquitinated ATF4. CHX, cycloheximide.

ATF4 degradation is required for proper cell positioning in the developing embryonic brain. A, in situ hybridization of wild type and ATF4 knock-out mouse brains from E12.5 and E16.5. B, ATF4 was immunoprecipitated from RIPA brain lysates from the indicated ages and subjected to immunoprecipitation and immunoblot analysis with ATF4 antibody. Input lysates were probed with antibodies against p35, CDK5, and actin. C, in utero electroporation scheme. D, RT-PCR analysis of ATF4 and actin brain mRNA from the indicated ages. E, brains were electroporated with pCAG, pCAG-WT, pCAG-S218A, pCAG-5A, or pCAG-9A (all proline-directed sites mutated) at E11.5 and fixed 2 days later at E13.5. Brain sections (12 μm) were prepared and stained with an anti-GFP antibody (green). Nuclei were stained with Hoechst 33258 (blue). F, quantification of cell positioning from D. Positioning of GFP-positive cells were scored as the percentage of cells in the VZ, IZ, or CP. Data are presented as the mean ± S.E. (n = 3). Ventricular zone: a, GFP to WT, S218A, 5A, and 9A p < 0.001; b, WT to S218A, 5A, and 9A, p < 0.001. Intermediate zone: c, GFP to S218A and 5A, p < 0.01, GFP to 9A, p < 0.001; d, WT to 5A, p < 0.01, WT to S218A, 9A, p < 0.001. Ventricular zone: e, GFP to WT, S218A, 5A, and 9A, p < 0.001; f, WT to S218A, 5A, and 9A, p < 0.001. Intermediate zone + cortical plate: g, GFP to WT, S218A, 5A, and 9A, p < 0.001; h, WT to S218A, 5A, and 9A, p < 0.001. p values were obtained by one-way analysis of variance.