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Quantitative analysis of a phosphoproteome readily altered by the protein kinase CK2 inhibitor quinalizarin in HEK-293T cells.

Franchin, Cinzia ; Cesaro, Luca ; Salvi, Mauro ; Millioni, Renato ; Iori, Elisabetta ; Cifani, Paolo LU ; James, Peter LU orcid ; Arrigoni, Giorgio and Pinna, Lorenzo (2015) In Biochimica et Biophysica Acta 1854(6). p.609-623
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... (More)
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. (Less)
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organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Biochimica et Biophysica Acta
volume
1854
issue
6
pages
609 - 623
publisher
Elsevier
external identifiers
  • pmid:25278378
  • wos:000353074000010
  • scopus:84939956047
  • pmid:25278378
ISSN
0006-3002
DOI
10.1016/j.bbapap.2014.09.017
language
English
LU publication?
yes
id
e4b59508-48cf-4301-b419-18ec49c79514 (old id 4738436)
date added to LUP
2016-04-01 13:30:40
date last changed
2023-11-12 17:53:10
@article{e4b59508-48cf-4301-b419-18ec49c79514,
  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.}},
  author       = {{Franchin, Cinzia and Cesaro, Luca and Salvi, Mauro and Millioni, Renato and Iori, Elisabetta and Cifani, Paolo and James, Peter and Arrigoni, Giorgio and Pinna, Lorenzo}},
  issn         = {{0006-3002}},
  language     = {{eng}},
  number       = {{6}},
  pages        = {{609--623}},
  publisher    = {{Elsevier}},
  series       = {{Biochimica et Biophysica Acta}},
  title        = {{Quantitative analysis of a phosphoproteome readily altered by the protein kinase CK2 inhibitor quinalizarin in HEK-293T cells.}},
  url          = {{http://dx.doi.org/10.1016/j.bbapap.2014.09.017}},
  doi          = {{10.1016/j.bbapap.2014.09.017}},
  volume       = {{1854}},
  year         = {{2015}},
}