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Phosphorylation of the chromatin binding domain of KSHV LANA.

Woodard C, Shamay M, Liao G, Zhu J, Ng AN, Li R, Newman R, Rho HS, Hu J, Wan J, Qian J, Zhu H, Hayward SD - PLoS Pathog. (2012)

Bottom Line: Short-term treatment of transfected cells with inhibitors of these kinases found that only RSK inhibition reduced LANA interaction with endogenous histone H2B.Extended treatment of PEL cell cultures with RSK inhibitor caused a decrease in LANA protein levels associated with p21 induction and a loss of PEL cell viability.The data indicate that RSK phosphorylation affects both LANA accumulation and function.

View Article: PubMed Central - PubMed

Affiliation: High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

ABSTRACT
The Kaposi sarcoma associated herpesvirus (KSHV) latency associated nuclear antigen (LANA) is expressed in all KSHV associated malignancies and is essential for maintenance of KSHV genomes in infected cells. To identify kinases that are potentially capable of modifying LANA, in vitro phosphorylation assays were performed using an Epstein Barr virus plus LANA protein microarray and 268 human kinases purified in active form from yeast. Interestingly, of the Epstein-Barr virus proteins on the array, the EBNA1 protein had the most similar kinase profile to LANA. We focused on nuclear kinases and on the N-terminus of LANA (amino acids 1-329) that contains the LANA chromatin binding domain. Sixty-three nuclear kinases phosphorylated the LANA N-terminus. Twenty-four nuclear kinases phosphorylated a peptide covering the LANA chromatin binding domain (amino acids 3-21). Alanine mutations of serine 10 and threonine 14 abolish or severely diminish chromatin and histone binding by LANA. However, conversion of these residues to the phosphomimetic glutamic acid restored histone binding suggesting that phosphorylation of serine 10 and threonine 14 may modulate LANA function. Serine 10 and threonine 14 were validated as substrates of casein kinase 1, PIM1, GSK-3 and RSK3 kinases. Short-term treatment of transfected cells with inhibitors of these kinases found that only RSK inhibition reduced LANA interaction with endogenous histone H2B. Extended treatment of PEL cell cultures with RSK inhibitor caused a decrease in LANA protein levels associated with p21 induction and a loss of PEL cell viability. The data indicate that RSK phosphorylation affects both LANA accumulation and function.

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LANA protein loss is post-transcriptional and leads to p21 induction.Comparison of LANA protein (A,B) and mRNA levels (C) with p21 mRNA induction (D) in BC3 and BCBL1 PEL cells treated for 1, 2 or 3 days with 1.7 µM BRD 7389. Protein levels were quantified using Image J and RNA was quantified using real time RT-PCR.
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ppat-1002972-g008: LANA protein loss is post-transcriptional and leads to p21 induction.Comparison of LANA protein (A,B) and mRNA levels (C) with p21 mRNA induction (D) in BC3 and BCBL1 PEL cells treated for 1, 2 or 3 days with 1.7 µM BRD 7389. Protein levels were quantified using Image J and RNA was quantified using real time RT-PCR.

Mentions: The effect of a longer exposure to RSK inhibitor was examined firstly in transfected 293 cells. Interestingly, when the cells were treated with 3.4 µM BRD 7389 for 24 hr versus 6 hr, there was not only a further decrease in LANA binding to histone H2B but also a decrease in LANA protein levels (Figure 7A). A comparison of LANA protein levels in cells transfected with wt Flag-LANA or the phosphomimetic Flag-LANA [S10E, S13E, T14E] and treated with RSK inhibitor for 24 hr revealed that the decrease in LANA protein levels that occurred with wt LANA was not seen with the triple phosphomimetic mutant (Figure 7B). This result links LANA stability to phosphorylation of the S10, S13 and T14 residues in the chromatin binding domain and to chromatin binding. Exposure of BC3 and BCBL1 PEL cells to 0.85, 1.7 and 3.4 µM concentrations of BRD 7389 for 48 hr resulted in decreased LANA protein levels in each case (Figure 7C). To determine whether the observed loss of LANA was mediated at the level of protein turnover, LANA protein levels were examined in BCBL1 cells treated with 1.7 µM BRD 7389 for 24 hr with or without the addition of the proteosome inhibitor lactacystin for 6 hr prior to harvesting. LANA levels decreased with BRD 7389 treatment as expected, and were partially restored by proteosome inhibition (Figure 7D, lanes 3 and 4). To strengthen the conclusion that the loss of LANA was mediated at the post-transcriptional level, rather than via transcription, BC3 and BCBL1 PEL cells were treated with 0.85 µM BRD 7389 for 1, 2 or 3 days and harvested cells were examined for LANA protein levels by western blotting (Figures 8A and 8B) and for LANA transcription using RT-PCR (Figure 8C). BC3 and BCBL1 cells differed in the kinetics of LANA protein loss over the 3 day time course. However, at each time point there was a greater decrease in the LANA∶Actin protein ratio than in the LANA∶Actin transcript ratio which remained between 76–100% of that in untreated cells. The data are compatible with RSK inhibition affecting LANA protein stability or turnover.


Phosphorylation of the chromatin binding domain of KSHV LANA.

Woodard C, Shamay M, Liao G, Zhu J, Ng AN, Li R, Newman R, Rho HS, Hu J, Wan J, Qian J, Zhu H, Hayward SD - PLoS Pathog. (2012)

LANA protein loss is post-transcriptional and leads to p21 induction.Comparison of LANA protein (A,B) and mRNA levels (C) with p21 mRNA induction (D) in BC3 and BCBL1 PEL cells treated for 1, 2 or 3 days with 1.7 µM BRD 7389. Protein levels were quantified using Image J and RNA was quantified using real time RT-PCR.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475679&req=5

ppat-1002972-g008: LANA protein loss is post-transcriptional and leads to p21 induction.Comparison of LANA protein (A,B) and mRNA levels (C) with p21 mRNA induction (D) in BC3 and BCBL1 PEL cells treated for 1, 2 or 3 days with 1.7 µM BRD 7389. Protein levels were quantified using Image J and RNA was quantified using real time RT-PCR.
Mentions: The effect of a longer exposure to RSK inhibitor was examined firstly in transfected 293 cells. Interestingly, when the cells were treated with 3.4 µM BRD 7389 for 24 hr versus 6 hr, there was not only a further decrease in LANA binding to histone H2B but also a decrease in LANA protein levels (Figure 7A). A comparison of LANA protein levels in cells transfected with wt Flag-LANA or the phosphomimetic Flag-LANA [S10E, S13E, T14E] and treated with RSK inhibitor for 24 hr revealed that the decrease in LANA protein levels that occurred with wt LANA was not seen with the triple phosphomimetic mutant (Figure 7B). This result links LANA stability to phosphorylation of the S10, S13 and T14 residues in the chromatin binding domain and to chromatin binding. Exposure of BC3 and BCBL1 PEL cells to 0.85, 1.7 and 3.4 µM concentrations of BRD 7389 for 48 hr resulted in decreased LANA protein levels in each case (Figure 7C). To determine whether the observed loss of LANA was mediated at the level of protein turnover, LANA protein levels were examined in BCBL1 cells treated with 1.7 µM BRD 7389 for 24 hr with or without the addition of the proteosome inhibitor lactacystin for 6 hr prior to harvesting. LANA levels decreased with BRD 7389 treatment as expected, and were partially restored by proteosome inhibition (Figure 7D, lanes 3 and 4). To strengthen the conclusion that the loss of LANA was mediated at the post-transcriptional level, rather than via transcription, BC3 and BCBL1 PEL cells were treated with 0.85 µM BRD 7389 for 1, 2 or 3 days and harvested cells were examined for LANA protein levels by western blotting (Figures 8A and 8B) and for LANA transcription using RT-PCR (Figure 8C). BC3 and BCBL1 cells differed in the kinetics of LANA protein loss over the 3 day time course. However, at each time point there was a greater decrease in the LANA∶Actin protein ratio than in the LANA∶Actin transcript ratio which remained between 76–100% of that in untreated cells. The data are compatible with RSK inhibition affecting LANA protein stability or turnover.

Bottom Line: Short-term treatment of transfected cells with inhibitors of these kinases found that only RSK inhibition reduced LANA interaction with endogenous histone H2B.Extended treatment of PEL cell cultures with RSK inhibitor caused a decrease in LANA protein levels associated with p21 induction and a loss of PEL cell viability.The data indicate that RSK phosphorylation affects both LANA accumulation and function.

View Article: PubMed Central - PubMed

Affiliation: High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

ABSTRACT
The Kaposi sarcoma associated herpesvirus (KSHV) latency associated nuclear antigen (LANA) is expressed in all KSHV associated malignancies and is essential for maintenance of KSHV genomes in infected cells. To identify kinases that are potentially capable of modifying LANA, in vitro phosphorylation assays were performed using an Epstein Barr virus plus LANA protein microarray and 268 human kinases purified in active form from yeast. Interestingly, of the Epstein-Barr virus proteins on the array, the EBNA1 protein had the most similar kinase profile to LANA. We focused on nuclear kinases and on the N-terminus of LANA (amino acids 1-329) that contains the LANA chromatin binding domain. Sixty-three nuclear kinases phosphorylated the LANA N-terminus. Twenty-four nuclear kinases phosphorylated a peptide covering the LANA chromatin binding domain (amino acids 3-21). Alanine mutations of serine 10 and threonine 14 abolish or severely diminish chromatin and histone binding by LANA. However, conversion of these residues to the phosphomimetic glutamic acid restored histone binding suggesting that phosphorylation of serine 10 and threonine 14 may modulate LANA function. Serine 10 and threonine 14 were validated as substrates of casein kinase 1, PIM1, GSK-3 and RSK3 kinases. Short-term treatment of transfected cells with inhibitors of these kinases found that only RSK inhibition reduced LANA interaction with endogenous histone H2B. Extended treatment of PEL cell cultures with RSK inhibitor caused a decrease in LANA protein levels associated with p21 induction and a loss of PEL cell viability. The data indicate that RSK phosphorylation affects both LANA accumulation and function.

Show MeSH
Related in: MedlinePlus