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

Xiao S, Caglar E, Maldonado P, Das D, Nadeem Z, Chi A, Trinité B, Li X, Saxena A - PLoS ONE (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Biology Department, Brooklyn College, Brooklyn, New York, United States of America; City University of New York, Graduate Center, New York, New York, United States of America.

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.

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NARF6-NCL clones with inducible NCL (WT or 6/S*A) expression.(A) Western blot of a representative clone that expresses 3xFlag-NCL under Tet-off promoter in NARF6 cells (derived from U2OS). (B) Western blot analyses for cells grown without Dx for the indicated period showing inducible NCL-expression (WT or 6/S*A). Both WT and 6/S*A led to a net increase in p53 protein levels and corresponding p21 protein levels -the downstream target of p53. (C) Plots of p53 and p21 protein levels shown in 2B. The quantification was done by NIH Image J software. Values were first corrected for the β-actin levels and then compared to Ctrl (no exogenous NCL, no Dx day 7) cells. The graph is representative of at least three independent experiments.
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pone-0109858-g002: NARF6-NCL clones with inducible NCL (WT or 6/S*A) expression.(A) Western blot of a representative clone that expresses 3xFlag-NCL under Tet-off promoter in NARF6 cells (derived from U2OS). (B) Western blot analyses for cells grown without Dx for the indicated period showing inducible NCL-expression (WT or 6/S*A). Both WT and 6/S*A led to a net increase in p53 protein levels and corresponding p21 protein levels -the downstream target of p53. (C) Plots of p53 and p21 protein levels shown in 2B. The quantification was done by NIH Image J software. Values were first corrected for the β-actin levels and then compared to Ctrl (no exogenous NCL, no Dx day 7) cells. The graph is representative of at least three independent experiments.

Mentions: We created retroviral constructs that express both the Tet activator and a 3xFlag-tagged NCL-WT or NCL-6/S*A from a single DNA molecule. We stably transfected NARF6 cells with these constructs; the NARF6 cells also express p14ARF from an IPTG-inducible promoter [41]. Stable clones were isolated that showed tetracycline (or doxycycline) regulated expression of NCL. Multiple clones were selected: Control cells (Ctrl, with no exogenous NCL expression, vector alone), WT (that express 3xFlag-NCL WT) and 6/S*A (expressing phosphorylation-deficient NCL mutant). Tests of a representative clone demonstrates expression of 3xFlag-NCL only upon doxycycline removal (Figure 2A) that almost completely shuts off when doxycycline is added back in the growth medium. In this study we present data from inducible NCL cells when exogenous NCL expression was induced by removal of doxycycline for a range of 1–28 days.


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

Xiao S, Caglar E, Maldonado P, Das D, Nadeem Z, Chi A, Trinité B, Li X, Saxena A - PLoS ONE (2014)

NARF6-NCL clones with inducible NCL (WT or 6/S*A) expression.(A) Western blot of a representative clone that expresses 3xFlag-NCL under Tet-off promoter in NARF6 cells (derived from U2OS). (B) Western blot analyses for cells grown without Dx for the indicated period showing inducible NCL-expression (WT or 6/S*A). Both WT and 6/S*A led to a net increase in p53 protein levels and corresponding p21 protein levels -the downstream target of p53. (C) Plots of p53 and p21 protein levels shown in 2B. The quantification was done by NIH Image J software. Values were first corrected for the β-actin levels and then compared to Ctrl (no exogenous NCL, no Dx day 7) cells. The graph is representative of at least three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4196967&req=5

pone-0109858-g002: NARF6-NCL clones with inducible NCL (WT or 6/S*A) expression.(A) Western blot of a representative clone that expresses 3xFlag-NCL under Tet-off promoter in NARF6 cells (derived from U2OS). (B) Western blot analyses for cells grown without Dx for the indicated period showing inducible NCL-expression (WT or 6/S*A). Both WT and 6/S*A led to a net increase in p53 protein levels and corresponding p21 protein levels -the downstream target of p53. (C) Plots of p53 and p21 protein levels shown in 2B. The quantification was done by NIH Image J software. Values were first corrected for the β-actin levels and then compared to Ctrl (no exogenous NCL, no Dx day 7) cells. The graph is representative of at least three independent experiments.
Mentions: We created retroviral constructs that express both the Tet activator and a 3xFlag-tagged NCL-WT or NCL-6/S*A from a single DNA molecule. We stably transfected NARF6 cells with these constructs; the NARF6 cells also express p14ARF from an IPTG-inducible promoter [41]. Stable clones were isolated that showed tetracycline (or doxycycline) regulated expression of NCL. Multiple clones were selected: Control cells (Ctrl, with no exogenous NCL expression, vector alone), WT (that express 3xFlag-NCL WT) and 6/S*A (expressing phosphorylation-deficient NCL mutant). Tests of a representative clone demonstrates expression of 3xFlag-NCL only upon doxycycline removal (Figure 2A) that almost completely shuts off when doxycycline is added back in the growth medium. In this study we present data from inducible NCL cells when exogenous NCL expression was induced by removal of doxycycline for a range of 1–28 days.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Biology Department, Brooklyn College, Brooklyn, New York, United States of America; City University of New York, Graduate Center, New York, New York, United States of America.

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.

Show MeSH
Related in: MedlinePlus