Casein kinase 2 phosphorylates and induces the SALL2 tumor suppressor degradation in cancer cells - Proteomics data
Notice bibliographique
Résumé
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. ******************** Proteomics data: ******************** 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.
Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.
Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,002 | 0,001 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».