Journal: The Journal of Biological Chemistry
Article Title: CDC123 is an ATPase that modulates mRNA translation and the integrated stress response by regulating eIF2 complex assembly
doi: 10.1016/j.jbc.2025.111116
Figure Lengend Snippet: CDC123-A109V induces downstream readouts of the ISR independent of increased eIF2α-P . A , schematic representation of the canonical, eIF2α-P-mediated ISR alongside the hypothesis tested in this study. Canonical activation of the ISR occurs in response to various cellular stressors ( top left ). In this pathway, stress-sensing eIF2 kinases phosphorylate eIF2 on its α subunit. eIF2α-P inhibits the activity of the eIF2 GEF, eIF2B, resulting in a decrease in the levels of active eIF2 ( i . e ., eIF2 bound to both GTP and Met-tRNA i Met ). The two outcomes of decreased active eIF2 are a reduction in global protein synthesis and the translational induction of the stress-responsive factor ATF4 through an upstream open reading frame (uORF)-mediated mechanism. We hypothesized that CDC123-A109V ( gray , top right ) would cause a decrease in eIF2 heterotrimer formation, resulting in a downstream reduction in active eIF2 levels and translational readouts of the ISR ( i . e ., reduced global protein synthesis and an induction in ATF4 translation) independent of eIF2α-P. eIF2α, β, and γ subunits are shown in magenta , orange , and cyan , respectively, with GTP indicated by a green circle , and Met-tRNA i Met in black . An ATF4 5′-leader schematic is shown with the two uORFs ( cyan boxes ) and the ATF4 main ORF ( yellow box ; bottom right). B , top panel: Lysates from WT and CDC123-A109V cells placed at 33.8 °C or 37 °C were subjected to co-immunoprecipitation with eIF2α or control IgG antibodies. Immunoblot analyses (WB) were used to detect the eIF2 subunits in the input and immunoprecipitated (IP) samples. The effect of each test condition on eIF2 heterotrimer formation is indicated below the corresponding lane. eIF2α, β, and γ subunits are shown in magenta , orange , and cyan , respectively. Biological replicate 1 is presented in B , while biological replicates 2 and 3 are shown in , A and B , respectively. Bottom panel: Met-tRNA i Met in input and co-immunoprecipitation samples was analyzed by RT-PCR and gel electrophoresis (RT-PCR). Quantification of the WB and RT-PCR images is included in C . Uncropped images corresponding to this panel are located in . C , polysome profiles of lysates from WT and CDC123-A109V cells placed at 33.8 °C or 37 °C. Positions of the 80S monosome peak and polysome peaks are indicated. Biological replicate 1 is presented in Figure 2C, while biological replicates 2 and 3 are shown in , A and B , respectively. Average polysome/monosome ratios with error bars (SD) calculated from the 3 biological replicates is shown in C . D , relative puromycin incorporation with error bars (SD) from 40 individual cells randomly selected from three biological replicates. Statistical comparisons between groups were conducted using a one-way ANOVA followed by a post hoc Tukey’s test. Significance is indicated by ∗ p < 0.05, ∗∗ p < 0.005. Error bars represent SD from three biological replicates. E , protein lysates from WT and CDC123-A109V cells placed at 33.8 °C or 37 °C were subjected to immunoblot analysis for the indicated proteins. Molecular weight markers are shown on the right . ∗nonspecific band detected with the CDC123 antibody. Biological replicate 1 is presented in E , while biological replicates 2 and 3 are shown in , D and E , respectively. Uncropped immunoblots corresponding to this panel are located in . F , average ATF4/β-actin signal with error bars (SD) from the immunoblots shown in panel E and , D and E . Statistical comparisons between groups were conducted using a one-way ANOVA followed by a post hoc Tukey’s test. Significance is indicated by ∗ p < 0.05, ∗∗ p < 0.005 (n = 3 biological replicates). Error bars represent SD. G , Average eIF2α-P/eIF2α signal with error bars (SD) from the immunoblots shown in panel E and , D and E . Statistical comparisons between groups were conducted using a one-way ANOVA followed by a post hoc Tukey’s test. ns = not significant (n = 3 biological replicates). Error bars represent SD. H – I , WT ( H ) and ΔuORF ( I ) P TK -ATF4-Luc constructs were transfected into WT and CDC123-A109V cells placed at 33.8 °C or 37 °C. ATF4-Luc translation was measured via dual-luciferase assay. Reporter constructs contain the WT or ΔuORF 5′-leader of the ATF4 mRNA inserted between a minimal TK promoter and the firefly luciferase CDS. When present, uORFs are represented as blue colored boxes , and the firefly luciferase CDS is represented as a yellow box . In the ΔuORF construct, the uORF start codons have been mutated from AUG to AGG, and the absence of the uORF is denoted by the lack of blue boxes . Statistical comparisons between groups were conducted using a one-way ANOVA followed by a post hoc Tukey’s test. Significance is indicated by ∗∗ p < 0.005, ns = not significant (n = 3 biological replicates). Error bars represent SD. CDS, coding sequence; ISR, integrated stress response; Met-tRNA, methionyl tRNA; TK, thymidine kinase.
Article Snippet: Primary and secondary antibodies used in immunoblots for this study are as follows: eIF2α (Bethyl; Cat #: A300–721A), eIF2β (Bethyl; Cat #: A301–742A), CDC123/C10orf7 (GenScript custom rabbit polyclonal antibody), eIF2γ (GenScript custom rabbit monoclonal antibody), ATF4 (Cell Signaling Technologies; Cat #: 11815S), EIF2S1 (phospho S51) (Cell Signaling Technologies; Cat #: ab32157), β-actin (Sigma-Aldrich; Cat #: A5441), Rabbit IgG HRP Linked F(ab′)2 (Cytiva; Cat #: NA9340), and Mouse IgG HRP Linked F(ab′)2 Fragment (Cytiva; Cat #: NA9310).
Techniques: Activation Assay, Activity Assay, Immunoprecipitation, Control, Western Blot, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Molecular Weight, Construct, Transfection, Luciferase, Sequencing
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