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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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Impairment of LANA binding to H2B occurs upon short-term treatment with RSK inhibitor but not inhibitors of CKI, PIM1 or GSK-3.A. Cells treated with CKI inhibitor (CKI-7). B. Cells treated with GSK-3 inhibitor (LiCl) or PIM1 inhibitor (SMI-4a) individually or in combination. C. Cells treated with RSK inhibitor (BRD 7389). Western blots examine interaction between transfected Flag-LANA and endogenous histone H2B. Quantitation of the band densities provides a measure of relative binding (LANA bound H2B/input H2B) and was obtained using Image J. The ratio in untreated cells was set at 100%.
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ppat-1002972-g006: Impairment of LANA binding to H2B occurs upon short-term treatment with RSK inhibitor but not inhibitors of CKI, PIM1 or GSK-3.A. Cells treated with CKI inhibitor (CKI-7). B. Cells treated with GSK-3 inhibitor (LiCl) or PIM1 inhibitor (SMI-4a) individually or in combination. C. Cells treated with RSK inhibitor (BRD 7389). Western blots examine interaction between transfected Flag-LANA and endogenous histone H2B. Quantitation of the band densities provides a measure of relative binding (LANA bound H2B/input H2B) and was obtained using Image J. The ratio in untreated cells was set at 100%.

Mentions: To examine the effect of kinase inhibition on LANA interaction with histone H2B, 293 cells were transfected with Flag-LANA and treated with the CK1 inhibitor CKI-7, the PIM1 inhibitor SMI-4a, the GSK-3 inhibitor LiCl, the RSK inhibitor BRD 7389 or with DMSO carrier. Short-term (4 hr) treatment with CKI-7 at 30 and 100 µM had no effect on LANA interaction with histone H2B (Figure 6A). Inhibition of GSK-3 or PIM1, individually or in combination, by treatment for 6 hr with LiCl and SMI-4a also had no impact on LANA binding to H2B (Figure 6B). However, treatment with 1.7 or 3.4 µM BRD 7389 for 6 hr decreased LANA interaction with histones in a dose responsive manner while having little effect on LANA protein levels (Figure 6C).


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)

Impairment of LANA binding to H2B occurs upon short-term treatment with RSK inhibitor but not inhibitors of CKI, PIM1 or GSK-3.A. Cells treated with CKI inhibitor (CKI-7). B. Cells treated with GSK-3 inhibitor (LiCl) or PIM1 inhibitor (SMI-4a) individually or in combination. C. Cells treated with RSK inhibitor (BRD 7389). Western blots examine interaction between transfected Flag-LANA and endogenous histone H2B. Quantitation of the band densities provides a measure of relative binding (LANA bound H2B/input H2B) and was obtained using Image J. The ratio in untreated cells was set at 100%.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002972-g006: Impairment of LANA binding to H2B occurs upon short-term treatment with RSK inhibitor but not inhibitors of CKI, PIM1 or GSK-3.A. Cells treated with CKI inhibitor (CKI-7). B. Cells treated with GSK-3 inhibitor (LiCl) or PIM1 inhibitor (SMI-4a) individually or in combination. C. Cells treated with RSK inhibitor (BRD 7389). Western blots examine interaction between transfected Flag-LANA and endogenous histone H2B. Quantitation of the band densities provides a measure of relative binding (LANA bound H2B/input H2B) and was obtained using Image J. The ratio in untreated cells was set at 100%.
Mentions: To examine the effect of kinase inhibition on LANA interaction with histone H2B, 293 cells were transfected with Flag-LANA and treated with the CK1 inhibitor CKI-7, the PIM1 inhibitor SMI-4a, the GSK-3 inhibitor LiCl, the RSK inhibitor BRD 7389 or with DMSO carrier. Short-term (4 hr) treatment with CKI-7 at 30 and 100 µM had no effect on LANA interaction with histone H2B (Figure 6A). Inhibition of GSK-3 or PIM1, individually or in combination, by treatment for 6 hr with LiCl and SMI-4a also had no impact on LANA binding to H2B (Figure 6B). However, treatment with 1.7 or 3.4 µM BRD 7389 for 6 hr decreased LANA interaction with histones in a dose responsive manner while having little effect on LANA protein levels (Figure 6C).

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