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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 assays on the EBV plus LANA protein array.A. Protein array probed with anti-GST antibody followed by CY5-labeled secondary antibody. Note that the EBNA1 (392–641)-V5-6xHis, 6xHis-Biotin AviTag-LANA (1–329) and control proteins are not expressed as GST-fusions and are not detected with anti-GST antibody. B. Control incubation with [γ32P]-ATP in kinase buffer. C. Incubation with [γ32P]-ATP, kinase buffer plus casein kinase 1, gamma 2 (CSNK1G2). Boxed signal, NME1 kinase control.
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ppat-1002972-g001: Phosphorylation assays on the EBV plus LANA protein array.A. Protein array probed with anti-GST antibody followed by CY5-labeled secondary antibody. Note that the EBNA1 (392–641)-V5-6xHis, 6xHis-Biotin AviTag-LANA (1–329) and control proteins are not expressed as GST-fusions and are not detected with anti-GST antibody. B. Control incubation with [γ32P]-ATP in kinase buffer. C. Incubation with [γ32P]-ATP, kinase buffer plus casein kinase 1, gamma 2 (CSNK1G2). Boxed signal, NME1 kinase control.

Mentions: We have previously described a protein microarray displaying Epstein–Barr virus (EBV) proteins purified as GST-fusions from yeast and printed in duplicate [56]. This array was additionally printed with a series of EBV EBNA1 and KSHV LANA N-terminal and C-terminal polypetides printed either as 6xHis-GST fusions, V5-6xHis fusions or 6xHis-Biotin AviTag fusions. The array also contained control proteins that were used for orientation and normalization. This platform was used to globally identify human kinases that phosphorylate the KSHV LANA protein. Two hundred and sixty-eight human kinases were purified from yeast in active form as assayed by dot blot phosphorylation assays using a mixture of histone H3, myelin basic protein and casein as the substrate. Phosphorylation assays were performed using kinase buffer containing [γ32P]-ATP and individual kinases. As a negative control, two chips per experiment were incubated with kinase buffer containing [γ32P]-ATP but minus the protein kinase. Phosphorylation signals were detected by exposing the arrays to X-ray film. An example of a kinase assay performed on the protein array is shown in Figure 1. In the subsequent analyses performed with GenePix software, paired signals that were 3 standard deviations (SD) above background were considered positive. These assays identified 101 known or predicted nuclear kinases that phosphorylated KSHV LANA (N+C). The EBV EBNA1 protein is the functional homolog of KSHV LANA although the two proteins have no significant amino acid homology. Interestingly, 99 of the kinases that phosphorylated KSHV LANA also phosphorylated EBV EBNA1, a striking degree of overlap (Table S1). KSHV LANA and EBV EBNA1 were also phosphorylated by significantly more kinases than any of the other EBV proteins on the array (Figure 2A). The median number of nuclear kinases phosphorylating the other EBV proteins on the array was 2. The extensive phosphorylation of EBV EBNA1 and KSHV LANA was not due to a disproportionate representation of serine, threonine and tyrosine residues in these proteins relative to the other EBV encoded proteins (Figure 2B). EBNA1 and LANA proteins contain 45 and 120 serine, threonine and tyrosine residues respectively. The median number for EBV encoded proteins is 62.


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 assays on the EBV plus LANA protein array.A. Protein array probed with anti-GST antibody followed by CY5-labeled secondary antibody. Note that the EBNA1 (392–641)-V5-6xHis, 6xHis-Biotin AviTag-LANA (1–329) and control proteins are not expressed as GST-fusions and are not detected with anti-GST antibody. B. Control incubation with [γ32P]-ATP in kinase buffer. C. Incubation with [γ32P]-ATP, kinase buffer plus casein kinase 1, gamma 2 (CSNK1G2). Boxed signal, NME1 kinase control.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002972-g001: Phosphorylation assays on the EBV plus LANA protein array.A. Protein array probed with anti-GST antibody followed by CY5-labeled secondary antibody. Note that the EBNA1 (392–641)-V5-6xHis, 6xHis-Biotin AviTag-LANA (1–329) and control proteins are not expressed as GST-fusions and are not detected with anti-GST antibody. B. Control incubation with [γ32P]-ATP in kinase buffer. C. Incubation with [γ32P]-ATP, kinase buffer plus casein kinase 1, gamma 2 (CSNK1G2). Boxed signal, NME1 kinase control.
Mentions: We have previously described a protein microarray displaying Epstein–Barr virus (EBV) proteins purified as GST-fusions from yeast and printed in duplicate [56]. This array was additionally printed with a series of EBV EBNA1 and KSHV LANA N-terminal and C-terminal polypetides printed either as 6xHis-GST fusions, V5-6xHis fusions or 6xHis-Biotin AviTag fusions. The array also contained control proteins that were used for orientation and normalization. This platform was used to globally identify human kinases that phosphorylate the KSHV LANA protein. Two hundred and sixty-eight human kinases were purified from yeast in active form as assayed by dot blot phosphorylation assays using a mixture of histone H3, myelin basic protein and casein as the substrate. Phosphorylation assays were performed using kinase buffer containing [γ32P]-ATP and individual kinases. As a negative control, two chips per experiment were incubated with kinase buffer containing [γ32P]-ATP but minus the protein kinase. Phosphorylation signals were detected by exposing the arrays to X-ray film. An example of a kinase assay performed on the protein array is shown in Figure 1. In the subsequent analyses performed with GenePix software, paired signals that were 3 standard deviations (SD) above background were considered positive. These assays identified 101 known or predicted nuclear kinases that phosphorylated KSHV LANA (N+C). The EBV EBNA1 protein is the functional homolog of KSHV LANA although the two proteins have no significant amino acid homology. Interestingly, 99 of the kinases that phosphorylated KSHV LANA also phosphorylated EBV EBNA1, a striking degree of overlap (Table S1). KSHV LANA and EBV EBNA1 were also phosphorylated by significantly more kinases than any of the other EBV proteins on the array (Figure 2A). The median number of nuclear kinases phosphorylating the other EBV proteins on the array was 2. The extensive phosphorylation of EBV EBNA1 and KSHV LANA was not due to a disproportionate representation of serine, threonine and tyrosine residues in these proteins relative to the other EBV encoded proteins (Figure 2B). EBNA1 and LANA proteins contain 45 and 120 serine, threonine and tyrosine residues respectively. The median number for EBV encoded proteins is 62.

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