Phosphorylation of the Chromatin Binding Domain of KSHV LANA

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.

Author Summary

The Kaposi sarcoma associated herpesvirus (KSHV) is associated with cancers that have an increased incidence in individuals with compromised immune systems. KSHV expresses a protein, LANA, that is needed to maintain KSHV genomes in infected cells and also promotes the growth of KSHV associated tumors. Kinases regulate protein function through phosphorylation. To identify kinases that may affect LANA function, we performed a screen in which 268 human kinases were isolated and tested for the ability to phosphorylate LANA in vitro. We focused on the region of LANA that contains the chromatin binding domain, a motif essential for tethering KSHV genomes to the cell chromatin and maintaining latent infection. We identified serine 10 and threonine 14 as amino acids within the chromatin binding domain whose phosphorylation was important for histone binding. Serine 10 and threonine 14 were targets of the CK1, PIM1, GSK-3 and RSK3 kinases. Treatment with an inhibitor of RSK kinase reduced LANA binding to histones, decreased LANA protein levels and caused a loss of KSHV infected PEL cell viability. Our experiments show that phosphorylation affects LANA function and suggest that KSHV infected cells may be particularly vulnerable to kinase inhibitors.