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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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Half-life analyses of p53 and NCL (WT and 6/S*A) proteins.Inducible NCL-cells grown without Dx for 17d to express either NCL-WT or NCL-6/S*A. Ctrl represents control cells without exogenous NCL expression. Cells were then treated with cycloheximide (CHX, 40 µg/ml) for indicated times. (A) Lysates were prepared and analyzed by Western blotting for p53, FLAG (for NCL expression) and the β-actin loading control. The relative band intensities for NCL and p53 proteins were quantified following normalization with β-actin and are indicated below each blot. (B) Plot of p53-expression levels following CHX treatment corrected for the β-actin levels. The graph is representative of three independent experiments done in duplicates. Half-life of p53 is ∼60 min for NCL-6/S*A, ∼30–40 min for NCL-WT and ∼15–20 min for Ctrl (vector) expressing cells. (C) NCL-6/S*A expression levels are relatively low as compared to NCL-WT under steady state conditions. Half-life of NCL-6/S*A is significantly lower <2 h as against>6 h for NCL-WT suggesting CK2 phosphorylation might regulate NCL protein stability.
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pone-0109858-g003: Half-life analyses of p53 and NCL (WT and 6/S*A) proteins.Inducible NCL-cells grown without Dx for 17d to express either NCL-WT or NCL-6/S*A. Ctrl represents control cells without exogenous NCL expression. Cells were then treated with cycloheximide (CHX, 40 µg/ml) for indicated times. (A) Lysates were prepared and analyzed by Western blotting for p53, FLAG (for NCL expression) and the β-actin loading control. The relative band intensities for NCL and p53 proteins were quantified following normalization with β-actin and are indicated below each blot. (B) Plot of p53-expression levels following CHX treatment corrected for the β-actin levels. The graph is representative of three independent experiments done in duplicates. Half-life of p53 is ∼60 min for NCL-6/S*A, ∼30–40 min for NCL-WT and ∼15–20 min for Ctrl (vector) expressing cells. (C) NCL-6/S*A expression levels are relatively low as compared to NCL-WT under steady state conditions. Half-life of NCL-6/S*A is significantly lower <2 h as against>6 h for NCL-WT suggesting CK2 phosphorylation might regulate NCL protein stability.

Mentions: First, we determined the p53 protein half-life upon expression of NCL phospho-variants by inhibiting protein synthesis by cycloheximide. The p53 protein half-life was clearly increased with NCL-6/S*A expression as compared to WT as indicated by a representative Western blot (Figure 3A). The graph is a representative of three independent experiments, each performed in duplicate (Figure 3B). We further evaluated p53-stability for shorter time period following cycloheximide blocking. As indicated in Figure S7 (upper panel) the p53 half-life is lower in cells expressing WT (∼30–40') as compared to mutant (∼1 h), while control cells have normal half-life of ∼15–20'. Assuming the decrease in p53 protein levels is a pseudo-first order kinetic process, the data presented in Figure 3B were also plotted on a log scale to indicate indeed a higher p53 protein half-life in cells with mutant-NCL expression (Figure S7, lower panel). Interestingly, we also observed lower steady state levels of NCL-6/S*A which had a reduced half-life (<2 h) as compared to WT (>6 h; Figure 3C). The shorter half-life of NCL-6/S*A indicates that CK2 phosphorylation might regulate NCL protein stability as previously suggested by others [47], [48]. The observed fluctuations in the expression pattern for 6/S*A, in part is due to differential stability of nucleolar vs. nucleoplasmic levels of the protein [49].


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)

Half-life analyses of p53 and NCL (WT and 6/S*A) proteins.Inducible NCL-cells grown without Dx for 17d to express either NCL-WT or NCL-6/S*A. Ctrl represents control cells without exogenous NCL expression. Cells were then treated with cycloheximide (CHX, 40 µg/ml) for indicated times. (A) Lysates were prepared and analyzed by Western blotting for p53, FLAG (for NCL expression) and the β-actin loading control. The relative band intensities for NCL and p53 proteins were quantified following normalization with β-actin and are indicated below each blot. (B) Plot of p53-expression levels following CHX treatment corrected for the β-actin levels. The graph is representative of three independent experiments done in duplicates. Half-life of p53 is ∼60 min for NCL-6/S*A, ∼30–40 min for NCL-WT and ∼15–20 min for Ctrl (vector) expressing cells. (C) NCL-6/S*A expression levels are relatively low as compared to NCL-WT under steady state conditions. Half-life of NCL-6/S*A is significantly lower <2 h as against>6 h for NCL-WT suggesting CK2 phosphorylation might regulate NCL protein stability.
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Related In: Results  -  Collection

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

pone-0109858-g003: Half-life analyses of p53 and NCL (WT and 6/S*A) proteins.Inducible NCL-cells grown without Dx for 17d to express either NCL-WT or NCL-6/S*A. Ctrl represents control cells without exogenous NCL expression. Cells were then treated with cycloheximide (CHX, 40 µg/ml) for indicated times. (A) Lysates were prepared and analyzed by Western blotting for p53, FLAG (for NCL expression) and the β-actin loading control. The relative band intensities for NCL and p53 proteins were quantified following normalization with β-actin and are indicated below each blot. (B) Plot of p53-expression levels following CHX treatment corrected for the β-actin levels. The graph is representative of three independent experiments done in duplicates. Half-life of p53 is ∼60 min for NCL-6/S*A, ∼30–40 min for NCL-WT and ∼15–20 min for Ctrl (vector) expressing cells. (C) NCL-6/S*A expression levels are relatively low as compared to NCL-WT under steady state conditions. Half-life of NCL-6/S*A is significantly lower <2 h as against>6 h for NCL-WT suggesting CK2 phosphorylation might regulate NCL protein stability.
Mentions: First, we determined the p53 protein half-life upon expression of NCL phospho-variants by inhibiting protein synthesis by cycloheximide. The p53 protein half-life was clearly increased with NCL-6/S*A expression as compared to WT as indicated by a representative Western blot (Figure 3A). The graph is a representative of three independent experiments, each performed in duplicate (Figure 3B). We further evaluated p53-stability for shorter time period following cycloheximide blocking. As indicated in Figure S7 (upper panel) the p53 half-life is lower in cells expressing WT (∼30–40') as compared to mutant (∼1 h), while control cells have normal half-life of ∼15–20'. Assuming the decrease in p53 protein levels is a pseudo-first order kinetic process, the data presented in Figure 3B were also plotted on a log scale to indicate indeed a higher p53 protein half-life in cells with mutant-NCL expression (Figure S7, lower panel). Interestingly, we also observed lower steady state levels of NCL-6/S*A which had a reduced half-life (<2 h) as compared to WT (>6 h; Figure 3C). The shorter half-life of NCL-6/S*A indicates that CK2 phosphorylation might regulate NCL protein stability as previously suggested by others [47], [48]. The observed fluctuations in the expression pattern for 6/S*A, in part is due to differential stability of nucleolar vs. nucleoplasmic levels of the protein [49].

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