Induced expression of nucleolin phosphorylation-deficient mutant confers dominant-negative effect on cell proliferation

Shu Xiao, Elif Caglar, Priscilla Maldonado, Dibash Das, Zaineb Nadeem, Angela Chi, Benjamin Trinité, Xin Li, Anjana Saxena

Research output: Contribution to journalArticle

Abstract

Nucleolin (NCL) is a major nucleolar phosphoprotein that has pleiotropic effects on cell proliferation and is elevated in a variety of tumors. NCL is highly phosphorylated at the N-terminus by two major kinases: interphase casein kinase 2 (CK2) and mitotic cyclin-dependent kinase 1 (CDK1). Earlier we demonstrated that a NCL-mutant that is partly defective in undergoing phosphorylation by CK2 inhibits chromosomal replication through its interactions with Replication Protein A, mimicking the cellular response to DNA damage. We further delineated that the N-terminus of NCL associates with Hdm2, the most common E3 ubiquitin ligase of p53. We reported that NCL antagonizes Hdm2 to stabilize p53 and stimulates p53 transcriptional activity. Although NCL-phosphorylation by CK2 and ribosomal DNA transcription are closely coordinated during interphase, the role of NCL phosphorylation in regulating cell proliferation remains unexplored. We have therefore engineered unique human cells that specifically induce expression of NCL-wild type (WT) or a phosphorylation-deficient NCL-mutant, 6/S∗A where all the six CK2 consensus serine sites residing in the N-terminus NCL were mutated to alanine. Here we show that this NCL-mutant is defective in undergoing phosphorylation by CK2. We also demonstrate that NCL-phosphorylation by CK2 is required through the S-phase progression in cell cycle and hence proliferation. Induced expression of NCL with mutated CK2 phosphorylation sites stabilizes p53, results in higher expression of Bcl2 (B-cell lymphoma 2) homology 3 (BH3)-only apoptotic markers and causes a dominant-negative effect on cell viability. Our unique cellular system thus provides the first evidential support to delineate phospho-specific functions of NCL on cell proliferation.

Original languageEnglish (US)
Article numbere109858
JournalPLoS One
Volume9
Issue number10
DOIs
StatePublished - Oct 14 2014

Fingerprint

Phosphorylation
Cell proliferation
cell proliferation
phosphorylation
Cell Proliferation
Casein Kinase II
mutants
interphase
Cells
phosphoproteins
ubiquitin-protein ligase
cyclin-dependent kinase
Interphase
non-specific serine/threonine protein kinase
nucleolin
lymphoma
DNA damage
ribosomal DNA
alanine
serine

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Induced expression of nucleolin phosphorylation-deficient mutant confers dominant-negative effect on cell proliferation. / Xiao, Shu; Caglar, Elif; Maldonado, Priscilla; Das, Dibash; Nadeem, Zaineb; Chi, Angela; Trinité, Benjamin; Li, Xin; Saxena, Anjana.

In: PLoS One, Vol. 9, No. 10, e109858, 14.10.2014.

Research output: Contribution to journalArticle

Xiao, S, Caglar, E, Maldonado, P, Das, D, Nadeem, Z, Chi, A, Trinité, B, Li, X & Saxena, A 2014, 'Induced expression of nucleolin phosphorylation-deficient mutant confers dominant-negative effect on cell proliferation', PLoS One, vol. 9, no. 10, e109858. https://doi.org/10.1371/journal.pone.0109858
Xiao, Shu ; Caglar, Elif ; Maldonado, Priscilla ; Das, Dibash ; Nadeem, Zaineb ; Chi, Angela ; Trinité, Benjamin ; Li, Xin ; Saxena, Anjana. / Induced expression of nucleolin phosphorylation-deficient mutant confers dominant-negative effect on cell proliferation. In: PLoS One. 2014 ; Vol. 9, No. 10.
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AU - Xiao, Shu

AU - Caglar, Elif

AU - Maldonado, Priscilla

AU - Das, Dibash

AU - Nadeem, Zaineb

AU - Chi, Angela

AU - Trinité, Benjamin

AU - Li, Xin

AU - Saxena, Anjana

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AB - Nucleolin (NCL) is a major nucleolar phosphoprotein that has pleiotropic effects on cell proliferation and is elevated in a variety of tumors. NCL is highly phosphorylated at the N-terminus by two major kinases: interphase casein kinase 2 (CK2) and mitotic cyclin-dependent kinase 1 (CDK1). Earlier we demonstrated that a NCL-mutant that is partly defective in undergoing phosphorylation by CK2 inhibits chromosomal replication through its interactions with Replication Protein A, mimicking the cellular response to DNA damage. We further delineated that the N-terminus of NCL associates with Hdm2, the most common E3 ubiquitin ligase of p53. We reported that NCL antagonizes Hdm2 to stabilize p53 and stimulates p53 transcriptional activity. Although NCL-phosphorylation by CK2 and ribosomal DNA transcription are closely coordinated during interphase, the role of NCL phosphorylation in regulating cell proliferation remains unexplored. We have therefore engineered unique human cells that specifically induce expression of NCL-wild type (WT) or a phosphorylation-deficient NCL-mutant, 6/S∗A where all the six CK2 consensus serine sites residing in the N-terminus NCL were mutated to alanine. Here we show that this NCL-mutant is defective in undergoing phosphorylation by CK2. We also demonstrate that NCL-phosphorylation by CK2 is required through the S-phase progression in cell cycle and hence proliferation. Induced expression of NCL with mutated CK2 phosphorylation sites stabilizes p53, results in higher expression of Bcl2 (B-cell lymphoma 2) homology 3 (BH3)-only apoptotic markers and causes a dominant-negative effect on cell viability. Our unique cellular system thus provides the first evidential support to delineate phospho-specific functions of NCL on cell proliferation.

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