DOI: 10.5281/zenodo.10475930
title: Casein kinase 2 phosphorylates and induces the SALL2 tumor suppressor degradation in cancer cells - Proteomics data

description: Spalt-like proteins are Zinc finger transcription factors from C. elegans to vertebrates, with critical roles in development. In vertebrates, four paralogues have been identified (SALL1-4), and SALL2 is the family’s most dissimilar member. SALL2 is required during brain and eye development. It is downregulated in cancer and acts as a tumor suppressor, promoting cell cycle arrest and cell death. Despite its critical functions, information about SALL2 regulation is scarce. Public data indicate that SALL2 is ubiquitinated and phosphorylated in several residues along the protein, but the mechanisms, biological consequences, and enzymes responsible for these modifications remain unknown. Bioinformatic analyses identified several putative phosphorylation sites for Casein Kinase II (CK2) located within a highly conserved C-terminal PEST degradation motif of SALL2. CK2 is a serine/threonine kinase that promotes cell proliferation and survival and is often hyperactivated in cancer. We demonstrated that CK2 phosphorylates SALL2 residues S763, T778, S802, and S806 and promotes SALL2 degradation by the proteasome. Accordingly, pharmacological inhibition of CK2 with Silmitasertib (CX-4945) restored endogenous SALL2 protein levels in SALL2-deficient breast MDA-MB-231, lung H1299, and colon SW480 cancer cells. Silmitasertib induced a methuosis-like phenotype and cell death in SW480 cells. However, the phenotype was significantly attenuated in CRISPr/Cas9-mediated SALL2 knockout SW480 cells. Similarly, Sall2-deficient tumor-derived organoids were more resistant to Silmitasertib-induced cell death, confirming that SALL2 sensitizes cancer cells to CK2 inhibition. We identified a novel CK2-dependent mechanism for SALL2 regulation and provided new insights into the interplay between these two proteins and their role in cell survival and proliferation.

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Proteomics data: 

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VH1: It corresponds to the IP'd protein treated with lambda phosphatase. (antes de fosforilar in vitro hay que remover todos los phosphatos que vienen ya incorporados en la proteina inmunoprecipitada desde las células en cultivo para que se pueda incorporar el fosfato que se añade en la reaccion in vitro. Este es solo un control)VH2: IP'd protein, treated with lambda phosphatase and phosphorylated in vitro without adding any recombinant enzyme (phosphorylated only by Co-IP'd kinases).VH3: IP'd protein, treated with lambda phosphatase and phosphorylated in vitro with recombinant CK2 alpha.VH4: IP'd protein from cells treated with DMSO.VH5: IP'd protein from cells treated with Silmitasertib 30 uM for six hours.

Aim: To identify residues in SALL2 that are phosphorylated in a CK2-dependent manner.Methods: ΔNSALL2-Flag was immunoprecipitated from Flp-In™ T-REx™ U2OS cells transfected withpCMV(NH)/ΔNSALL2-Flag. The immunoprecipitated protein was 1) dephosphorylated for subsequent in vitro phosphorylation (with or without recombinant enzyme) followed by mass spec analyses or 2) directly immunoprecipitated from cells treated with DMSO or 30 μM Silmitasertib for 6 hours followed by mass spectrometry analyses. Samples were subjected to polyacrylamide gel electrophoresis following by gel staining andextraction with the MASSPrep Automated Digestor (Waters/Micromass) from London RegionalProteomics Center at the University of Western Ontario, Canada. After removing the dye,immunoprecipitated ΔNSALL2-Flag was digested with Trypsin in 50 mM NH4HCO3 solution,followed by digestion with Asp-N (Sigma). Peptides obtained from digested samples wereextracted from the gel and sent to London Regional Proteomics Center for Mass Spectrometryanalyses. The samples were subjected to tandem mass spectrometry in the Q-tof Ultima Globalequipment (Micromass), with electrospray ionization (ESI) and fragmentation byactivated/collision-induced dissociation (CAD/CID). The analyzes were performed in PEAKSStudio software in the UniProt database for Homo sapiens, with a 1% FDR (false discoveryrate).

Conclussions: residues S763, T778, S802, and S806 are phosphotylated in a CK2-dependent manner.