title: Quantitative analysis of a phosphoproteome readily altered by the protein kinase CK2 inhibitor quinalizarin in HEK-293T cells.
abstract: CK2 is an extremely pleiotropic Ser/Thr protein kinase, responsible for the generation of a large proportion of the human phosphoproteome and implicated in a wide variety of biological functions. CK2 plays a global role as an anti-apoptotic agent, a property which is believed to partially account for the addiction of many cancer cells to high CK2 levels. To gain information about the CK2 targets whose phosphorylation is primarily implicated in its pro-survival signaling advantage has been taken of quinalizarin (QZ) a cell permeable fairly specific CK2 inhibitor, previously shown to be able to block endogenous CK2 triggering an apoptotic response. HEK-293T cells either treated or not for 3h with 50μM QZ were exploited to perform a quantitative SILAC phosphoproteomic analysis of phosphosites readily responsive to QZ treatment. Our analysis led to the identification of 4883 phosphosites, belonging to 1693 phosphoproteins. 71 phosphosites (belonging to 47 proteins) underwent a 50% or more decreased occupancy upon QZ treatment. Almost 50% of these fulfilled the typical consensus sequence recognized by CK2 (S/T-x-x-E/D/pS) and in several cases were validated as bona fide substrates of CK2 either based on data in the literature or by performing in vitro phosphorylation experiments with purified proteins. The majority of the remaining phosphosites drastically decreased upon QZ treatment display the pS/T-P motif typical of proline directed protein kinases and a web logo extracted from them differentiates from the web logo extracted from all the proline directed phosphosites quantified during our analysis (1151 altogether). A paradoxical outcome of our study was the detection of 116 phosphosites (belonging to 92 proteins altogether) whose occupancy is substantially increased (50% or more), rather than decreased by QZ treatment: 40% of these display the typical motif recognized by proline directed kinases, while about 25% fulfill the CK2 consensus. Collectively taken our data on one side have led to the disclosure of a subset of CK2 targets which are likely to be implicated in the early steps of CK2 signaling counteracting apoptosis, on the other they provide evidence for the existence of side and off-target effects of the CK2 inhibitor quinalizarin, paving the road toward the detection of other kinases susceptible to this compound. This article is part of a Special Issue entitled: Medical Proteomics.
authors.affiliation: Department of Biomedical Sciences, University of Padova, Via U. Bassi 58/B, 35131 Padova, Italy; Proteomics Center of Padova University, Via G. Orus 2/B, 35129 Padova, Italy.
authors.forename: Cinzia
authors.initials: C
authors.lastname: Franchin
authors.affiliation: Department of Biomedical Sciences, University of Padova, Via U. Bassi 58/B, 35131 Padova, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Cesaro
authors.affiliation: Department of Biomedical Sciences, University of Padova, Via U. Bassi 58/B, 35131 Padova, Italy.
authors.forename: Mauro
authors.initials: M
authors.lastname: Salvi
authors.affiliation: Proteomics Center of Padova University, Via G. Orus 2/B, 35129 Padova, Italy; Department of Medicine, University of Padova, Via Giustiniani 2, 35128 Padova, Italy.
authors.forename: Renato
authors.initials: R
authors.lastname: Millioni
authors.affiliation: Department of Medicine, University of Padova, Via Giustiniani 2, 35128 Padova, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Iori
authors.affiliation: Department of Immunotechnology, Lund University, Medicon Village, S-223 81, Lund, Sweden.
authors.forename: Paolo
authors.initials: P
authors.lastname: Cifani
authors.affiliation: Department of Immunotechnology, Lund University, Medicon Village, S-223 81, Lund, Sweden.
authors.forename: Peter
authors.initials: P
authors.lastname: James
authors.affiliation: Department of Biomedical Sciences, University of Padova, Via U. Bassi 58/B, 35131 Padova, Italy; Proteomics Center of Padova University, Via G. Orus 2/B, 35129 Padova, Italy. Electronic address: giorgio.arrigoni@unipd.it.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Arrigoni
authors.affiliation: Department of Biomedical Sciences, University of Padova, Via U. Bassi 58/B, 35131 Padova, Italy. Electronic address: lorenzo.pinna@unipd.it.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Pinna
mesh_terms: D000880:Anthraquinones / Q000494:pharmacology*; D047390:Casein Kinase II / Q000037:antagonists & inhibitors*; D057809:HEK293 Cells; D006801:Humans; D010750:Phosphoproteins / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D020543:Proteome / Q000378:metabolism*
chemical_list: D000880:Anthraquinones; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D020543:Proteome; C543211:1,2,5,8-tetrahydroxy anthraquinone; D047390:Casein Kinase II
keywords: Apoptosis; CK2; Phosphoproteome; Protein kinase inhibitor; Quinalizarin; casein kinase 2