p pka  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: OSTM1 is a ubiquitin E3 ligase that suppresses B-cell malignancy by activating the cAMP/PKA/CREB pathway

doi: 10.64898/2026.01.23.701155

Figure Lengend Snippet: (A) Venn diagram showing overlap of B cell-specific CREB1 and CREBBP ChIP-seq targets. All targets identified in each dataset were used to determine overlap, and the top 1,500 common targets were selected for downstream analyses. (B) RNA expression data from the TCGA MDACC B-cell malignancies cohort. Heatmaps showing correlations between OSTM1, CREB1, or PDE3B mRNA levels and the top 1,500 CREB1/CREBBP co-occupied target genes identified in ( A ). (C) Venn diagrams showing overlap among genes significantly correlated with OSTM1, CREB1, or PDE3B within the top 1,500 CREB1/CREBBP targets. As OSTM1 negatively regulates PDE3B, genes positively correlated with OSTM1 and CREB1 expression and negatively correlated with PDE3B expression showed substantial overlap (78.2%). Conversely, genes negatively correlated with OSTM1 and CREB1 expression and positively correlated with PDE3B expression also overlapped significantly (66.8%). (D) CRISPR gene-effect scores from the CCLE database for OSTM1 and PDE3B across BCL lines, showing generally negative effects upon PDE3B silencing and positive effects upon OSTM1 silencing. (E) Pde3b was silenced by sgRNA in sg Ostm1 clone #3 Ba/F3 cells. PDE3B protein levels were determined by IB, and sg Ostm1/Pde3b double knockout (DKO) Ba/F3 cells were selected for subsequent experiments. (F) sgControl, sg Ostm1 , and DKO Ba/F3 cells were cutured in the presense or absence of IL3. Pde3b silencing reversed IL3-independence in sg Ostm1 cells. (G) GFP-expressing sg Ostm1 or DKO Ba/F3 cells were transplanted into nude mice via i.p. injection. Mice were harvested at the endpoint of the sg Ostm1 cohort. Spleens and livers were photographed and weighed. P values were calculated using Student’s t-test. GFP-positive tumor cells were detected only in sg Ostm1 recipients, but not in DKO recipients. (H) qRT-PCR in sgControl and sg Ostm1 Ba/F3 cells showing that Ostm1 silencing reduced expression of PKA/CREB/CREBBP target genes. (I) sgCtrl, sg Ostm1 , and DKO Ba/F3 cells were probed for phospho-PKA substrates and phospho-CREB (Ser133). (J) OSTM1 was silenced using two independent sgRNAs in SU-DHL-5 cells. Left: qPCR validation of OSTM1 knockout using on-target primers. Right: IB showing stablization of PDE3B and downregulation of cAMP/PKA signaling upon OSTM1 deletion. (K) OSTM1 was silenced in ARH-77 cells. IB of two clones shows increased PDE3B protein levles and decreased cAMP/PKA signaling upon OSTM1 silencing. (L) PDE3B-His was stably expressed in SU-DHL-10 cell line, which suppressed cAMP/PKA signaling. (M) OSTM1-Flag or OSTM1Δ31-Flag was stably expressed in OPM2 and RPMI-8226 cells. IB shows that OSTM1Δ31, but not the full-length OSTM1, promoted PDE3B degradation and enhanced cAMP/PKA signaling. (N) IB of whole-spleen lysates from indicated age-matched mice collected at the endpoints of O +/- ;C -/- or DKO cohorts. Phosphorylation levels of CREB and PKA substrates were generally reduced in the O -/- , O +/- C -/- , and DKO mice. (O) Bulk RNA-seq of purified splenic B cells from the indicated genotypes (as in ). Genes up-or down-regulated in DKO versus C -/- mice were intersected with the CREB1/CREBBP ChIP-seq targets identified in (A) . (P) Spleens from two-month-old C -/- and DKO mice were harvested, and 10,000 cells per mouse were analyzed by scRNA-seq. UMAP plots of B-cell subpopulations are shown by genotype. Bar graphs show the relative proportions of B-cell subsets. (Q) Genes down-regulated in DKO vs C -/- B cells, as identified by both bulk RNA-seq of splenic B cells and scRNA-seq of follicular B cells, were intersected with CREB1/CREBBP targets. Eight genes were commonly identified across all 4 datasets.

Article Snippet: Flag-M2 (Millipore Sigma, F1804), Flag (Millipore Sigma, F7425), 6X-His (Thermo Fisher Scientific, MA1-21315), p-AKT Ser-473 (Cell Signaling Technology, 4060), t-AKT (Cell Signaling Technology, 9272), GAPDH (ProteinTech, 10494-1-AP), p-S6 Ser235/236 (Cell Signaling Technology, 4858), t-S6 (Cell Signaling Technology, 2217), p-STAT5 Y694 (Cell Signaling Technology, 9359), t-STAT5 (Cell Signaling Technology, 25656), p-p44/42 MAPK (Erk1/2) Thr202/Tyr204 (Cell Signaling Technology, 4376), t-p44/42 MAPK (Erk1/2) (Cell Signaling Technology, 9102), PDE3B (SMCP3B) (used for IB) (Novus Biologicals, NBP1-43333), PDE3B (used for IP) (Abcam, ab99290), Ubiquitin (P37) (Cell Signaling Technology, 58395), LC3B (Cell Signaling Technology, 2775), K48-linkage specific polyubiquitin (D9D5) (Cell Signaling Technology, 8081), GFP (Santa Cruz, sc-8334), BiP/GRP78 (BD Biosciences, 610978), calnexin (Transduction Labs, C45520-050), CLC7 (Thermo Fisher, A305-381A-T), p-CREB Ser133 (87G3) (Cell Signaling Technology, 9198), t-CREB (48H2) (Cell Signaling Technology, 9197), p-(Ser/Thr) PKA substrates (Cell Signaling Technology, 9621), OSTM1 (Millipore Sigma, ABN1365), goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 680 (Thermo Fisher, A-21057), goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa FluorTM 594 (Thermo Fisher, A11012), goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 488 (Thermo Fisher, A11008), goat anti-rabbit secondary antibody (LI-COR, 926-32211), goat anti-rat secondary antibody for 680 channel (Thermo Fisher, A-21096)

Techniques: ChIP-sequencing, RNA Expression, Expressing, CRISPR, Double Knockout, Injection, Quantitative RT-PCR, Biomarker Discovery, Knock-Out, Clone Assay, Stable Transfection, Phospho-proteomics, RNA Sequencing, Purification