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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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Related in: MedlinePlus

Treatment of PEL cells with BRD 7389 decreases cell proliferation and increases PARP cleavage.A. Growth of BC3 PEL and BJAB B cell lines after treatment with the indicated doses of RSK inhibitor. B. Western blot comparing the levels of cleaved PARP in BJAB B cells and BC3 and BCBL1 PEL cells after treatment for 1 day with 0.85 µM RSK inhibitor. C. Western blot comparing the levels of cleaved (*) and uncleaved PARP in BJAB, BC3 and BCBL1 cells after treatment with 0.4 or 1.7 µM RSK inhibitor.
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ppat-1002972-g009: Treatment of PEL cells with BRD 7389 decreases cell proliferation and increases PARP cleavage.A. Growth of BC3 PEL and BJAB B cell lines after treatment with the indicated doses of RSK inhibitor. B. Western blot comparing the levels of cleaved PARP in BJAB B cells and BC3 and BCBL1 PEL cells after treatment for 1 day with 0.85 µM RSK inhibitor. C. Western blot comparing the levels of cleaved (*) and uncleaved PARP in BJAB, BC3 and BCBL1 cells after treatment with 0.4 or 1.7 µM RSK inhibitor.

Mentions: To determine the effect of the loss of LANA protein on PEL cell growth, duplicate cultures of BC3 PEL cells and KSHV negative BJAB cells were treated with RSK inhibitor and each sample was measured in duplicate for cell viability using the CellTiter-Glo assay. RSK inhibition has been reported to reversibly inhibit proliferation of tumor derived cell lines and indeed all three inhibitor concentrations stopped the growth of both BC3 PEL cells and BJAB B cells (Figure 9A). While, the BJAB cultures maintained their original cell numbers, the BC3 cultures showed a decrease in viable cell numbers over time suggesting that drug treatment was both cytostatic and cytotoxic for BC3 cells. The IC50 for BRD 7389 was 0.27 µM for BC3 PEL cells and 1.88 µM for BJAB B cells.


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)

Treatment of PEL cells with BRD 7389 decreases cell proliferation and increases PARP cleavage.A. Growth of BC3 PEL and BJAB B cell lines after treatment with the indicated doses of RSK inhibitor. B. Western blot comparing the levels of cleaved PARP in BJAB B cells and BC3 and BCBL1 PEL cells after treatment for 1 day with 0.85 µM RSK inhibitor. C. Western blot comparing the levels of cleaved (*) and uncleaved PARP in BJAB, BC3 and BCBL1 cells after treatment with 0.4 or 1.7 µM RSK inhibitor.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002972-g009: Treatment of PEL cells with BRD 7389 decreases cell proliferation and increases PARP cleavage.A. Growth of BC3 PEL and BJAB B cell lines after treatment with the indicated doses of RSK inhibitor. B. Western blot comparing the levels of cleaved PARP in BJAB B cells and BC3 and BCBL1 PEL cells after treatment for 1 day with 0.85 µM RSK inhibitor. C. Western blot comparing the levels of cleaved (*) and uncleaved PARP in BJAB, BC3 and BCBL1 cells after treatment with 0.4 or 1.7 µM RSK inhibitor.
Mentions: To determine the effect of the loss of LANA protein on PEL cell growth, duplicate cultures of BC3 PEL cells and KSHV negative BJAB cells were treated with RSK inhibitor and each sample was measured in duplicate for cell viability using the CellTiter-Glo assay. RSK inhibition has been reported to reversibly inhibit proliferation of tumor derived cell lines and indeed all three inhibitor concentrations stopped the growth of both BC3 PEL cells and BJAB B cells (Figure 9A). While, the BJAB cultures maintained their original cell numbers, the BC3 cultures showed a decrease in viable cell numbers over time suggesting that drug treatment was both cytostatic and cytotoxic for BC3 cells. The IC50 for BRD 7389 was 0.27 µM for BC3 PEL cells and 1.88 µM for BJAB B cells.

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