Protein microarrays and high throughput techniques for kinase expression and purification have previously been used to characterize the yeast kinome and analyze yeast phosphorylation networks 
. We now describe the use of these technologies to examine phosphorylation of the KSHV LANA protein by human kinases. The ability to screen LANA with 268 different human kinases provided a unique opportunity to examine the potential role of phosphorylation of the chromatin binding domain on LANA function and to identify those kinases able to perform this function.
In vitro kinase screens have limitations. Not all of the kinases that phosphorylate the substrate in vitro will have the appropriate intracellular localization, expression pattern or activating signals to perform this function in the cellular environment while kinases that require specialized reaction conditions or partners such as priming kinases for maximum activity will tend to be under represented. It should be noted that substrates purified from yeast exhibit a limited degree of phosphorylation that can partially support the priming activity required by kinases such as GSK-3. To ensure that we focused on the most biologically relevant LANA partners, kinases that phosphorylated LANA in the initial microarray screen were subjected to database searches for their intracellular localization and only those with a known or predicted nuclear activity were included in subsequent assays. An interesting observation from the initial microarray assay was that the number of kinases phosphorylating LANA and its functional homolog in latency viral DNA replication in EBV, the EBNA1 protein, was an order of magnitude greater than the number detected for most other EBV proteins on the array. This suggests that LANA and EBNA1 functions may be particularly sensitive to regulation by phosphorylation. The extensive overlap in phosphorylating kinases for these two proteins also suggests that they may be subject to regulation by common signaling pathways.
Within the LANA chromatin binding domain, three of the amino acids that had been implicated as critical for histone and chromatin binding in mutagenesis studies were S10, S13 and T14. We tested S13 as a single mutation, S13A, and did not find any impact on LANA binding to histones. This placed the emphasis on S10 and T14 as residues potentially subject to post-translational modification. The ability of phosphomimetic S10E and T14E mutations to restore LANA binding to histone H2B is suggestive of a role for phosphorylation in the regulation of LANA chromatin binding function. Two of the kinases that we identified as phosphorylating these residues, PIM and RSK have been implicated in KSHV lytic replication. Overexpression of PIM1 and PIM3 led to reactivation of KSHV from latency and the role of these kinases was linked to phosphorylation of LANA at serines 205 and 206, an event that abolished LANA mediated repression of RTA 
. Treatment of PEL cells with the RSK inhibitor BI-D1870 or treatment with inhibitors of ERK signaling also inhibited KSHV lytic replication 
. The latency and lytic cycles of herpesviruses are usually thought to have opposing requirements. Proteins or pathways that favor latency generally have an inhibitory effect on lytic replication and vice versa. However, a requirement for the activity of certain kinases in both aspects of the KSHV life-cycle need not be contradictory. For example, ERK signaling is also known to be required for establishment of a KSHV infection 
. ERK signaling, which activates RSK, can have directly opposing effects depending on the duration and strength of the signal and cross-talk with other signaling pathways 
We saw no effect on LANA interaction with histone H2B after a short treatment with CK1, PIM1 or GSK-3 inhibitors. However, a short treatment with the RSK inhibitor BRD 7389 decreased LANA binding to histone H2B. All of the RSK family members, RSK1, 2, 3 and 4 are inhibited by BRD 7389. Interestingly, a longer BRD 7389 treatment resulted in a decrease in LANA protein levels in LANA transfected cells and in PEL cells. Treatment of cancer cell lines with RSK inhibitors results in growth inhibition 
. We observed this same response upon treatment of the BJAB B cell lymphoma cell line and PEL cells. The treated BC3 PEL cells not only stopped growing but also decreased in viable cell numbers suggesting that inhibition of RSK was having an additional impact on these cells. PEL cells are generally wild-type for p53 (BCBL1 cells are heterozygous 
) and BJAB B cells are p53 mutant. One of the functions of LANA is to block p53 mediated apoptosis through interaction with the p53-MdM2 complex 
and through inactivation of GSK-3 
and interaction with angiogenin 
. In RSK inhibitor treated PEL cells, p21 induction by activated p53 occurred later in the time course than induction of caspase cleavage suggesting that the loss of viability of treated PEL cells likely occurred by a p53 independent mechanism.
Overall, our data provide evidence that phosphorylation affects LANA interaction with chromatin and that inhibition of the RSK kinases that phosphorylate the chromatin binding domain has an additional effect on LANA protein levels. The dual outcomes of reduced histone binding and loss of LANA protein stability imply that chromatin binding protects LANA from degradation and contributes to the long half-life demonstrated by LANA in latently infected cells. ERK signaling positively regulates transcription of LANA upon KSHV infection of endothelial cells 
but the loss of LANA in inhibitor treated cells was mediated at the level of protein turnover rather than transcriptionally. The Ras/Raf/MEK/ERK signaling pathway that leads to RSK activation regulates cell growth, proliferation and survival. The sensitivity of PEL cells to treatment with inhibitors of this pathway may represent a vulnerability that can be exploited to limit the growth of latently KSHV infected cells.