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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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Identification of kinases that phosphorylate the LANA chromatin binding domain.A. Phosphorylation of a LANA aa3–21 peptide by the indicated kinases. The peptide was separated from the kinase reaction mixture by electrophoresis and the gel dried and subjected to autoradiography. B. Upper: Amino acid sequence of the wt LANA 3–21 peptide (1) showing the positions of the single and grouped sequence changes in the mutant peptides (2–6). Lower: Examples of phosphorylation of the wild-type and mutant peptides by the indicated kinases. C. GSK-3 phosphorylates S10 after PIM1 priming. Wild-type and S10A mutant peptides were incubated with PIM1 or GSK-3β or with PIM1 plus unlabelled γ-ATP (PIM1 priming) or with GSK-3 β after PIM1 priming followed by inhibition of PIM1 with the PIM1 inhibitor SMI-4a. (−), minus kinase.
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ppat-1002972-g005: Identification of kinases that phosphorylate the LANA chromatin binding domain.A. Phosphorylation of a LANA aa3–21 peptide by the indicated kinases. The peptide was separated from the kinase reaction mixture by electrophoresis and the gel dried and subjected to autoradiography. B. Upper: Amino acid sequence of the wt LANA 3–21 peptide (1) showing the positions of the single and grouped sequence changes in the mutant peptides (2–6). Lower: Examples of phosphorylation of the wild-type and mutant peptides by the indicated kinases. C. GSK-3 phosphorylates S10 after PIM1 priming. Wild-type and S10A mutant peptides were incubated with PIM1 or GSK-3β or with PIM1 plus unlabelled γ-ATP (PIM1 priming) or with GSK-3 β after PIM1 priming followed by inhibition of PIM1 with the PIM1 inhibitor SMI-4a. (−), minus kinase.

Mentions: The possibility that phosphorylation of sites outside of the chromatin binding domain could complicate interpretation of the kinase assays using GST-LANA (1–50) as substrate led us to turn to peptide substrates that covered only the LANA chromatin binding domain. A synthetic peptide representing LANA amino acids 3–21 and covering the S10 and T14 amino acids of interest was used to retest 31 of the nuclear kinases that were positive for phosphorylation of GST-LANA (1–50). In this assay, 24 of these kinases also phosphorylated the amino acid 3–21 LANA peptide (Table 1). Examples of the peptide phosphorylation assay are shown in Figure 5A.


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)

Identification of kinases that phosphorylate the LANA chromatin binding domain.A. Phosphorylation of a LANA aa3–21 peptide by the indicated kinases. The peptide was separated from the kinase reaction mixture by electrophoresis and the gel dried and subjected to autoradiography. B. Upper: Amino acid sequence of the wt LANA 3–21 peptide (1) showing the positions of the single and grouped sequence changes in the mutant peptides (2–6). Lower: Examples of phosphorylation of the wild-type and mutant peptides by the indicated kinases. C. GSK-3 phosphorylates S10 after PIM1 priming. Wild-type and S10A mutant peptides were incubated with PIM1 or GSK-3β or with PIM1 plus unlabelled γ-ATP (PIM1 priming) or with GSK-3 β after PIM1 priming followed by inhibition of PIM1 with the PIM1 inhibitor SMI-4a. (−), minus kinase.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002972-g005: Identification of kinases that phosphorylate the LANA chromatin binding domain.A. Phosphorylation of a LANA aa3–21 peptide by the indicated kinases. The peptide was separated from the kinase reaction mixture by electrophoresis and the gel dried and subjected to autoradiography. B. Upper: Amino acid sequence of the wt LANA 3–21 peptide (1) showing the positions of the single and grouped sequence changes in the mutant peptides (2–6). Lower: Examples of phosphorylation of the wild-type and mutant peptides by the indicated kinases. C. GSK-3 phosphorylates S10 after PIM1 priming. Wild-type and S10A mutant peptides were incubated with PIM1 or GSK-3β or with PIM1 plus unlabelled γ-ATP (PIM1 priming) or with GSK-3 β after PIM1 priming followed by inhibition of PIM1 with the PIM1 inhibitor SMI-4a. (−), minus kinase.
Mentions: The possibility that phosphorylation of sites outside of the chromatin binding domain could complicate interpretation of the kinase assays using GST-LANA (1–50) as substrate led us to turn to peptide substrates that covered only the LANA chromatin binding domain. A synthetic peptide representing LANA amino acids 3–21 and covering the S10 and T14 amino acids of interest was used to retest 31 of the nuclear kinases that were positive for phosphorylation of GST-LANA (1–50). In this assay, 24 of these kinases also phosphorylated the amino acid 3–21 LANA peptide (Table 1). Examples of the peptide phosphorylation assay are shown in Figure 5A.

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