pubmed.ncbi.nlm.nih.gov

Over Expression of Nucleophosmin and Nucleolin Contributes to the Suboptimal Activation of a G2/M Checkpoint in Ataxia Telangiectasia Fibroblasts - PubMed

Over Expression of Nucleophosmin and Nucleolin Contributes to the Suboptimal Activation of a G2/M Checkpoint in Ataxia Telangiectasia Fibroblasts

Narasimharao Nalabothula et al. Mol Cell Pharmacol. 2010.

Abstract

Ataxia Telangiectasia (AT) cells exhibit suboptimal activation of radiation-induced cell cycle checkpoints despite having a wild type p53 genotype. Reducing or eliminating this delay could restore p53 function and reinstate normal cellular response to genotoxic stress. Here we show that the levels of Nuclephosmin (NPM), NPM phosphorylated at Serine 125, p53, p53 phosphorylated at Serine 15 and Serine 392 and the levels of Nucleolin (NCL) are high in AT fibroblasts compared to normal cells. Transfection of a functional ATM into AT fibroblasts reduced p53, phospo-p53, phospho-NPM and NCL levels to wild type fibroblasts levels. Our data indicate that ATM regulates phospho-NPM and NCL indirectly through the Protein Phosphatase 1 (PP1). Both, NPM and NCL interact with p53 and hinder its phosphorylation at Serine 15 in response to bleomycin. Moreover, NPM and NCL are phosphorylated by several of the same kinases targeting p53 and could potentially compete with p53 for phosphorylation in AT cells. In addition, our data indicate that down regulation of NCL and to a lesser extent NPM increase the number of AT cells arrested in G2/M in response to bleomycin. Together this data indicate that the lack of PP1 activation in AT cells result in increased NPM and NCL protein levels which prevents p53 phosphorylation in response to bleomycin and contributes to a defective G2/M checkpoint.

PubMed Disclaimer

Conflict of interest statement

Conflicts of Interest

No potential conflicts of interest to disclose.

Figures

Figure 1
Figure 1. Basal levels of stress proteins are high in AT cells. Western blots

Normal human fibroblasts (GM 05659) and SV-40 transformed (GM 05849) and non SV-40 transformed (GM 02052) AT fibroblasts cells were grown as described under Material and Methods. Cellular extracts (10 μg) were prepared and analyzed with the indicated antibodies as described in the text.

Figure 2
Figure 2. A functional ATM is required to prevent over-expression of stress-proteins

Western blots. (A) SV-40 transformed (GM 05849) AT fibroblasts stably transfected with a Flag vector (lane 1) or a Flag-ATM vector (lane 2) were used. Cellular extracts (10 μg) from exponentially growing cells were prepared and analyzed with the indicated antibody as described in the text. (B) Same as (A) except that scrambled siRNA (scRNA, lanes 1, 3 and 5) or PP1 siRNA (lanes 2, 4 and 6) were transiently transfected in ATM corrected AT fibroblasts.

Figure 3
Figure 3. Down regulation of NPM and NCL allows p53 phosphorylation in response to bleomycin

(A) Western blots. AT fibroblasts (GM 05849) were treated with 40 nM siNPM or Scrambled RNA (Scramb.) and exposed to 5 mUnits/ ml bleomycin for the indicated period of time. Western blots were performed with the indicated antibodies. Actin was used as a control for loading. Fold induction was determined by densitometry and normalized to actin. (B, D) The cells were transiently transfected with 7.5 μg antisense NPM (As-NPM) vector or empty vector (pCDNA3.1) and exposed to 5 mUnits/ ml bleomycin for the indicated period of time. Actin was used as a control for loading. Fold induction was determined by densitometry and was normalized to actin. (C) Western blots performed as in A with the exception that where indicated, NCL siRNA (40 nM) were used. NCL + NPM siRNA (40 nM total).

Figure 4
Figure 4. NPM is an in vitro substrate for ATM and SMG1ATX

(A) In vitro kinase assay on recombinant NPM (300 ng) with Flag-ATM (lane 1) or its kinase dead (KD, lane 2) mutant immunoprecipitated from RKO cells. (B) Primary sequence of residues surrounding human NPM Serine 125. Acidic residues are underlined.

Figure 5
Figure 5. Nucleolin Serine 145 is an in vitro substrate for ATM, ATR and SMG-1ATX

(A) In vitro kinase assay performed as in Fig.4 with the indicated kinase and N-terminal GST-NCL recombinant proteins (lanes 2-3). GST-NPM fragment 1-180 (lanes 1) was used as a positive control. (B) Three point mutations were analyzed as in A): GST-NCL 101-151 S145A: Serine 145 was replaced by Alanine (lane 4), GST-NCL 152-183 S153A: Serine 153 was replaced with Alanine (lane 6), GST-NCL 184-219 S184A: Serine 184 was replaced with Alanine (lane 8).

Figure 6
Figure 6. Overexpression of NCL and NPM contributes to the suboptimal G2/M checkpoint in AT cells

The non SV-40 transformed (GM 02052) AT fibroblasts were transfected with the indicated siRNA (40 nM) for two consecutive days and then treated with bleomycin (4 μg/ml) for 3h. The cells were then either harvested (0h) or maintained in cultured for an additional 24 h and analyzed by FACS analysis.

Figure 7
Figure 7. Schematic summary and proposed model

(A) In normal cells, ATM can phosphorylate PP1 Inhibitory factor 2 (I-2) and activate PP1. PP1 represses NPM Ser125 phosphorylation and NCL levels. Low levels of NPM and NCL allow p53 to respond to stress by activating cellular checkpoints. PP2A can also dephosphorylate p53 in an ATM dependent manner. (B) In AT cells, the absence of a functional ATM prevents the activation of PP1 and results in high NPM Ser125, NCL and p53 Ser15 phosphorylation levels probably through the remaining active kinases such as ATR and SMG1ATX. The high expression levels of NPM and NCL could compromise p53 activation through binding to either p53 amino (NPM) or carboxy (NCL) terminal end or through competition for the remaining active kinases. (C) Down regulation of NPM and NCL levels in AT cells reduces p53 steric hindrance and decreases the pool of competing substrates for the remaining active kinases which allow p53 phosphorylation and activation of a G2 checkpoint. PP1: protein phosphatase 1; PP2a; Protein Phosphatase 2A; NPM: Nucleophosmin; NCL: Nucleolin. Phosphorylated Serines are indicated; red indicates low level of phosphorylation, green indicates hyper-phosphorylation and yellow indicates p53 inducible levels.

Similar articles

Cited by

References

    1. Kastan MB, Zhan Q, el-Deiry WS, Carrier F, Jacks T, Walsh WV, Plunkett BS, Vogelstein B, Fornace AJ., Jr A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell. 1992;71:587–97. - PubMed
    1. Canman CE, Wolff AC, Chen CY, Fornace AJ, Jr, Kastan MB. The p53-dependent G1 cell cycle checkpoint pathway and ataxia- telangiectasia. Cancer Res. 1994;54:5054–8. - PubMed
    1. Wright JA, Keegan KS, Herendeen DR, Bentley NJ, Carr AM, Hoekstra MF, Concannon P. Protein kinase mutants of human ATR increase sensitivity to UV and ionizing radiation and abrogate cell cycle checkpoint control. Proc Natl Acad Sci U S A. 1998;95:7445–50. - PMC - PubMed
    1. Gatei M, Shkedy D, Khanna KK, Uziel T, Shiloh Y, Pandita TK, Lavin MF, Rotman G. Ataxia-telangiectasia: chronic activation of damage-responsive functions is reduced by alpha-lipoic acid. Oncogene. 2001;20:289–94. - PubMed
    1. Guo CY, Brautigan DL, Larner JM. Ionizing radiation activates nuclear protein phosphatase-1 by ATM-dependent dephosphorylation. J Biol Chem. 2002;277:41756–61. - PubMed

LinkOut - more resources