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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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Phosphomimetic mutations of LANA S10 and T14 rescue histone binding.A. Western blots showing the effect on binding to histone H2B of individual alanine substitutions across the LANA chromatin binding domain. Flag-LANA was immunoprecipitated from transfected HEK293T cells and the bound endogenous H2B was detected using anti-H2B antibody. B. Western blots showing the effect of individual and grouped phosphomimetic mutations at S10, S13 and T14 on LANA binding to H2B. Flag-LANA or Flag-LANA deleted for the central repeats (dCR) was immunoprecipitated from transfected HEK293T cells and the bound endogenous H2B was detected using anti-H2B antibody.
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ppat-1002972-g003: Phosphomimetic mutations of LANA S10 and T14 rescue histone binding.A. Western blots showing the effect on binding to histone H2B of individual alanine substitutions across the LANA chromatin binding domain. Flag-LANA was immunoprecipitated from transfected HEK293T cells and the bound endogenous H2B was detected using anti-H2B antibody. B. Western blots showing the effect of individual and grouped phosphomimetic mutations at S10, S13 and T14 on LANA binding to H2B. Flag-LANA or Flag-LANA deleted for the central repeats (dCR) was immunoprecipitated from transfected HEK293T cells and the bound endogenous H2B was detected using anti-H2B antibody.

Mentions: Amino acids 5–22 are important for KSHV association with the cell chromatin [41], [42], [57]. Within this domain are three serine residues and two threonine residues that could be modified by phosphorylation. Previous mutagenesis studies found that mutation of S10 to alanine individually or in the context of a triple mutation prevented chromatin binding, as did mutation of S13 to alanine in the context of a triple mutation. Mutation of T14 to alanine was +/− for chromatin binding, mutation of S22 did not affect binding and mutation of T19 was not examined [41], [57]. Chromatin binding correlated with binding to histones H2A and H2B [41]. The S13A mutation has been examined only in the context of a triple alanine mutation. We consequently first examined the effect on binding to endogenous histone H2B of individual alanine mutations of LANA residues 5 through 14, including S13. In immunoprecipitation assays performed on transfected HEK 293T cells the observed loss of binding of Flag-LANA G5A, M6A, L8A, R9A, S10A, G11A, the retention of binding by R7A and the weak +/− binding of T14A recapitulated published results (Figure 3A). As an individual mutation, conversion of serine 13 to alanine did not affect binding to histone H2B. The mutagenesis data implies that, if phosphorylation impacts on LANA's ability to bind histones, the relevant residues would be S10 and T14.


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)

Phosphomimetic mutations of LANA S10 and T14 rescue histone binding.A. Western blots showing the effect on binding to histone H2B of individual alanine substitutions across the LANA chromatin binding domain. Flag-LANA was immunoprecipitated from transfected HEK293T cells and the bound endogenous H2B was detected using anti-H2B antibody. B. Western blots showing the effect of individual and grouped phosphomimetic mutations at S10, S13 and T14 on LANA binding to H2B. Flag-LANA or Flag-LANA deleted for the central repeats (dCR) was immunoprecipitated from transfected HEK293T cells and the bound endogenous H2B was detected using anti-H2B antibody.
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Related In: Results  -  Collection

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

ppat-1002972-g003: Phosphomimetic mutations of LANA S10 and T14 rescue histone binding.A. Western blots showing the effect on binding to histone H2B of individual alanine substitutions across the LANA chromatin binding domain. Flag-LANA was immunoprecipitated from transfected HEK293T cells and the bound endogenous H2B was detected using anti-H2B antibody. B. Western blots showing the effect of individual and grouped phosphomimetic mutations at S10, S13 and T14 on LANA binding to H2B. Flag-LANA or Flag-LANA deleted for the central repeats (dCR) was immunoprecipitated from transfected HEK293T cells and the bound endogenous H2B was detected using anti-H2B antibody.
Mentions: Amino acids 5–22 are important for KSHV association with the cell chromatin [41], [42], [57]. Within this domain are three serine residues and two threonine residues that could be modified by phosphorylation. Previous mutagenesis studies found that mutation of S10 to alanine individually or in the context of a triple mutation prevented chromatin binding, as did mutation of S13 to alanine in the context of a triple mutation. Mutation of T14 to alanine was +/− for chromatin binding, mutation of S22 did not affect binding and mutation of T19 was not examined [41], [57]. Chromatin binding correlated with binding to histones H2A and H2B [41]. The S13A mutation has been examined only in the context of a triple alanine mutation. We consequently first examined the effect on binding to endogenous histone H2B of individual alanine mutations of LANA residues 5 through 14, including S13. In immunoprecipitation assays performed on transfected HEK 293T cells the observed loss of binding of Flag-LANA G5A, M6A, L8A, R9A, S10A, G11A, the retention of binding by R7A and the weak +/− binding of T14A recapitulated published results (Figure 3A). As an individual mutation, conversion of serine 13 to alanine did not affect binding to histone H2B. The mutagenesis data implies that, if phosphorylation impacts on LANA's ability to bind histones, the relevant residues would be S10 and T14.

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