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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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Phosphorylation of GST-LANA(1–50).A. Diagram of the GST-LANA aa 1–50 wild-type (1) and mutant constructions (2–5) showing the amino acid changes at positions 10, 13 and 14 in the chromatin binding domain. B. Coomassie brilliant blue staining of the purified GST-LANA(1–50) wild-type (1) and mutant (2–5) proteins used in the phosphorylation assays. C. Examples of in vitro phosphorylation assays in which GST-LANA(1–50) wild-type and mutant proteins were incubated with CSNK1G2 or MAPK14/p38a kinases. (−), minus peptide.
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ppat-1002972-g004: Phosphorylation of GST-LANA(1–50).A. Diagram of the GST-LANA aa 1–50 wild-type (1) and mutant constructions (2–5) showing the amino acid changes at positions 10, 13 and 14 in the chromatin binding domain. B. Coomassie brilliant blue staining of the purified GST-LANA(1–50) wild-type (1) and mutant (2–5) proteins used in the phosphorylation assays. C. Examples of in vitro phosphorylation assays in which GST-LANA(1–50) wild-type and mutant proteins were incubated with CSNK1G2 or MAPK14/p38a kinases. (−), minus peptide.

Mentions: The LANA N-terminus contains the chromatin binding domain and we therefore chose to focus on this region of LANA and to limit subsequent experiments to the 63 kinases that phosphorylated both the 6xHis-GST and 6xHis-Biotin AviTag fusions of the N-terminus of LANA (aa 1–329) and are known or predicted to function in the cell nucleus. LANA has a nuclear localization and this subset of kinases is therefore more likely to be biologically relevant (Table 1). Included in this list are the PIM1 and GSK-3 kinases that are known to phosphorylate LANA [51], [53], [54]. We generated GST-fusion proteins of the N-terminal 50 amino acids of LANA along with derivatives in which serine or threonine residues within the chromatin binding domain (S10, S13, T14) were mutated to alanine individually or as a triple mutation (Figure 4A). The purified GST-fusion proteins used in the kinase assays are shown in Figure 4B. In vitro kinase assays were performed with 42 of the kinases that phosphorylated LANA(1–329) on the protein array and 31 of these kinases phosphorylated the N-terminal 50 amino acids of LANA (Table 1). Examples of the kinase assay using these GST-LANA (1–50) proteins as substrates are shown in Figure 4C. MAPK14/p38α (mitogen activated protein kinase 14) phosphorylated GST-LANA(1–50), each of the S10A, S13A and T14A mutants and the S10A, S13A, T14A triple mutant. MAPK14 either phosphorylates LANA 1–50 outside of the chromatin binding domain or alternatively, if S10, S13 or T14 are sites, then MAPK4 must also be capable of phosphorylating the N-terminus outside of this domain. CSNK1G2 (casein kinase 1, gamma 2 isoform) phosphorylated GST-LANA(1–50) and the S13A and T14A mutants but not the S10A or S10A,S13A,T14A mutants. This suggests that S10 within the chromatin binding domain is phosphorylated by casein kinase 1.


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)

Phosphorylation of GST-LANA(1–50).A. Diagram of the GST-LANA aa 1–50 wild-type (1) and mutant constructions (2–5) showing the amino acid changes at positions 10, 13 and 14 in the chromatin binding domain. B. Coomassie brilliant blue staining of the purified GST-LANA(1–50) wild-type (1) and mutant (2–5) proteins used in the phosphorylation assays. C. Examples of in vitro phosphorylation assays in which GST-LANA(1–50) wild-type and mutant proteins were incubated with CSNK1G2 or MAPK14/p38a kinases. (−), minus peptide.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002972-g004: Phosphorylation of GST-LANA(1–50).A. Diagram of the GST-LANA aa 1–50 wild-type (1) and mutant constructions (2–5) showing the amino acid changes at positions 10, 13 and 14 in the chromatin binding domain. B. Coomassie brilliant blue staining of the purified GST-LANA(1–50) wild-type (1) and mutant (2–5) proteins used in the phosphorylation assays. C. Examples of in vitro phosphorylation assays in which GST-LANA(1–50) wild-type and mutant proteins were incubated with CSNK1G2 or MAPK14/p38a kinases. (−), minus peptide.
Mentions: The LANA N-terminus contains the chromatin binding domain and we therefore chose to focus on this region of LANA and to limit subsequent experiments to the 63 kinases that phosphorylated both the 6xHis-GST and 6xHis-Biotin AviTag fusions of the N-terminus of LANA (aa 1–329) and are known or predicted to function in the cell nucleus. LANA has a nuclear localization and this subset of kinases is therefore more likely to be biologically relevant (Table 1). Included in this list are the PIM1 and GSK-3 kinases that are known to phosphorylate LANA [51], [53], [54]. We generated GST-fusion proteins of the N-terminal 50 amino acids of LANA along with derivatives in which serine or threonine residues within the chromatin binding domain (S10, S13, T14) were mutated to alanine individually or as a triple mutation (Figure 4A). The purified GST-fusion proteins used in the kinase assays are shown in Figure 4B. In vitro kinase assays were performed with 42 of the kinases that phosphorylated LANA(1–329) on the protein array and 31 of these kinases phosphorylated the N-terminal 50 amino acids of LANA (Table 1). Examples of the kinase assay using these GST-LANA (1–50) proteins as substrates are shown in Figure 4C. MAPK14/p38α (mitogen activated protein kinase 14) phosphorylated GST-LANA(1–50), each of the S10A, S13A and T14A mutants and the S10A, S13A, T14A triple mutant. MAPK14 either phosphorylates LANA 1–50 outside of the chromatin binding domain or alternatively, if S10, S13 or T14 are sites, then MAPK4 must also be capable of phosphorylating the N-terminus outside of this domain. CSNK1G2 (casein kinase 1, gamma 2 isoform) phosphorylated GST-LANA(1–50) and the S13A and T14A mutants but not the S10A or S10A,S13A,T14A mutants. This suggests that S10 within the chromatin binding domain is phosphorylated by casein kinase 1.

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