Identification of GTSE1 as a cyclin D1-CDK4 substrate with prognostic significance in cancer

A) Differential proteomic profiling of AMBRA1 KO U2OS cells compared to parental U2OS cells11. Proteins with adjusted p value<0.05 were ranked by the log2 fold change (Log2FC) to quantify expression changes between AMBRA1 KO and parental cells. The top 30 upregulated proteins in this analysis are presented. Statistical significance was assessed by False Discovery Rate (FDR).

B) Subset of proteins from (A) containing a canonical CDK phosphorylation consensus motif [S/T*]PX[K/R]. Annotated list of phosphorylated proteins was generated using PhosphoSitePlus database23.

C) Subset of proteins from (B) whose abundance was reverted to basal levels following treatment of AMBRA1 KO U2OS cells with Palbociclib11 (Log2FC_ambra) with adjusted p-values (adjp_ambra)<0.001 were ranked by log2 fold change (Log2FC) from the original shotgun proteomic analysis (A) to integrate expression changes between AMBRA1 KO and parental cells under Palbociclib-treated versus untreated condition.

D) Transcriptomic analysis of GTSE1 in various cancer types, contrasting tumor (red) and normal tissue (blue) expression levels, using data derived from The Cancer Genome Atlas (TCGA)37. Statistical significance assessed by FDR, p<0.05.

E) Kaplan-Meier curves representing the overall survival analysis based on the 50% upper versus lower expression levels of GTSE1. Survival analysis was conducted across various cancer cohorts, including ACC, KIRP, KIRC, LGG, LUAD, MESO, PAAD, and SKCM (see also Figure supplement 1). The hazard ratio (HR) was calculated to estimate the relative risk, and significance was assessed using the log-rank test with a threshold of p<0.05. Curves were generated using the GEPIA2 platform.38.

F) Survival map of various cancer types according to the indicated gene expression levels (50% upper and lower expression). Color intensity indicates the log of the HR, with red and blue representing poorer and better survival, respectively. Statistically significant differences in survival are denoted by highlighted contour squares, based on the log-rank test. Generated with GEPIA2 platform38, using TCGA data.

Cyclin D1-CDK4 promotes GTSE1 phosphorylation on four serine residues.

A) Immunoblot analysis following transient transfection of the indicated proteins in HEK293T cells detailing their phosphorylation status via differential mobility in phos-tagTM gels in the presence or absence of Cyclin D1-CDK4 co-expression.

B-C) The indicated purified, recombinant proteins were incubated in the presence or absence of recombinant cyclin D1-CDK4 complex. ERK2 was used as a negative control. Post-incubation, differential phosphorylation was analyzed using phos-tagTM gels to detect mobility shifts. Immunoblot analysis was conducted to verify the presence of the recombinant proteins as indicated.

D) HEK293T cells transfected with either the indicated Flag-tagged proteins or an empty vector (EV). Cell lysates were subjected to immunoprecipitation followed by immunoblot analysis. Inputs (5%) represent whole cell extracts before pull down.

E) Schematic representation of candidate CDK phosphorylation sites in GTSE1, as indicated by the PhosphoSitePlus database23.

F) HEK293T cells were transiently transfected with the indicated HA-GTSE1 mutants, in the presence or absence of Cyclin D1-CDK4 co-expression. Changes in protein migration were analyzed by phos-tagTM gels.

G) Immunoblot and phos-tagTM gel analysis displaying cell cycle synchronization effects following 72 hours of serum starvation in parental T98G cells and AMBRA1 KO T98G cells. Cells were collected at various time points post-serum re-supplementation as indicated, followed by immunoblotting with the indicated antibodies.

H) HA-tagged wild type GTSE1 (left) or GTSE1 mutated at positions 91, 261, 454, and 724 (‘Tetra SA’) (right) was subjected to co-expression with various cyclins and CDKs, in the presence or absence of the indicated kinase inhibitors to observe the impact on phosphorylation.

I) Boxplot representation of the log2-transformed Z-scores for bulk phospho-peptide abundance of GTSE1 across a pan-cancer cohort relative to adjacent normal tissue, utilizing data from the Clinical Proteomic Tumor Analysis Consortium (CPTAC)39. Statistical significance was assessed using the Wilcoxon rank-sum test, with a p-value threshold of <0.05.

J) Heatmap illustrating differential abundance in cancer of the three GTSE1 phosphorylation sites found in the current study using CPTAC data39. Color intensity reflects the log2-transformed Z-scores of identified GTSE1 phosphopeptides, indicating relative phosphorylation levels across various tumor cohorts compared to adjacent normal tissues. The complete analysis of all phosphorylation sites can be found in Figure supplement 3D.

GTSE1 protein is stabilized upon its phosphorylation by cyclin D1-CDK4

A) Immunoblot analysis of whole cell extracts from parental HCT-116 cells, AMBRA1 KO HCT-116 cells, and HCT-116 cells expressing either wild-type cyclin D1 or an AMBRA1-insensitive mutant of Cyclin D1 (T286A), in the presence or absence of the CDK4/6 inhibitor palbociclib.

B) Immunoblot analysis of whole cell extracts from HCT-116 cells harboring an endogenous mini-AID domain fused to AMBRA1 N-terminus. Cells were subjected to either incubation with auxin and doxycycline to induce AMBRA1 degradation or transfection with cyclin D1(T286A) in the presence or absence of Palbociclib.

C) Cycloheximide (CHX) chase assay in HCT116 cells with mAID-AMBRA1, treated with or without auxin and doxycycline to induce AMBRA1 degradation. Immunoblot analyses were conducted to assess the stability of GTSE1 and other indicated proteins, with the stable protein SKP1 used as a loading control.

D) Protein stability assessment via CHX chase in U2OS parental cells and AMBRA1 knockout clones (C11 and G11). The protein levels of AMBRA1, GTSE1, and cyclin D1 were analyzed by immunoblotting, with tubulin serving as a loading control.

E) U2OS cells stably transduced with retroviruses expressing the indicated GTSE1 constructs were subjected to cycloheximide CHX chase assays. Subsequent immunoblotting was conducted for the indicated proteins, with tubulin utilized as a loading control.

F) Densitometric quantification of GTSE1 band intensity from (E) and two identical experiments normalized using tubulin. Initial band intensity at time 0 is set as the 100% reference point. Error bars represent SEM (n=3 of biological replicates).

G) Time-lapse microscopy images of U2OS cells stably expressing EGFP-tagged the indicated GTSE1 constructs during a CHX chase. The EGFP signal intensity corresponds to GTSE1 levels, while cells are stained with a far-red cell tracker for cell masking.

H) Quantitative analysis of EGFP fluorescence intensity from time-lapse experiment shown in (G) plus two identical experiments. The initial fluorescence intensity was normalized to 100% at time zero. Data represent the mean fluorescence intensity from the three independent measurements, with error bars indicating the standard error of the mean (SEM).

I) U2OS cells were treated with various inhibitors for 3 hours before harvest: the proteasome inhibitor MG132, the CRL inhibitor MLN4924, and the V-ATPase inhibitor Bafilomycin A1. Immunoblot analysis of the indicated proteins was performed, with tubulin as a loading control. p62 and LC3 serve as autophagy inhibition controls, while p27 is used as control for CRL- and UPS-dependent degradation.

Increased cell proliferation upon cyclin D1-CDK4-mediated phosphorylation and stabilization of GTSE1

A) Bubble plot derived from CPTAC data analysis showing the correlation between the abundance of the bulk phosphopeptides of GTSE1 and the levels of proteins involved in cell proliferation or cell migration across various cancer types. The color intensity of each bubble represents the Spearman correlation coefficient, while the bubble size indicates the negative log10 transformation of the p-values. Bubbles outlined in black indicate statistically significant correlations with q-value < 0.0001, adjusted by Benjamini-Hochberg (BH) method.

B) Growth curve analysis comparing the proliferation of U2OS cells stably expressing GFP-tagged wild-type GTSE1, GTSE1 Tetra SA (phospho-deficient), or GTSE1 Tetra SD (phospho-mimetic). A p-value<0.05 was considered significant (n=3 per cell line)

C) Proliferation analysis using CellTrace™ Far Red dye to assess the division of U2OS cells expressing wild-type GTSE1, Tetra SA, and Tetra SD, alongside a comparison between parental and AMBRA KO U2OS cells. Following staining, the dye dilutes progressively with each cell division, allowing for the distinction of successive generations by the relative decrease in fluorescence intensity. Fluorescence-activated cell sorting was employed to accurately measure the dye dilution and thereby quantify the discrete populations representing each generation of the cell line. The gating strategy was implemented to identify live, single cells positive for GFP, as detailed in Figure supplement 4C. The percentages of cells in each generation, indicative of proliferative capacity, are displayed graphically on the right lower panel.