Phosphorylation is a common posttranslational processing event in the synthesis of proteins, and changes in the degree of protein phosphorylation play an important role in modulating protein function. Many cellular processes, including signal transduction, transcription, protein synthesis, and cell growth and differentiation, are regulated by phosphorylation-dependent mechanisms (17
). Therefore, it is not surprising that the protein kinases, a family of enzymes that catalyze the phosphorylation of proteins, comprise one of the largest families of genes in eukaryotes. The protein kinase complement of the human genome (the human “kinome”) is estimated to comprise about 1.7% of all human genes, with a total of 518 genes identified according to a cataloging approach published recently (19
). The presence of such a large number of kinases in the human genome represents a significant challenge to protein kinase studies, suggesting that it would be extremely powerful to develop high-throughput approaches for characterizing kinase-substrate reactions. Although a recent study describes the use of a microarray format for the high-throughput analysis of substrate specificity of yeast protein kinases (36
), the use of such an approach to identify the human kinase(s) responsible for phosphorylation of a biological substrate has not been previously described.
There has been much interest in characterizing the role of protein phosphorylation in mechanisms associated with viral infection, pathogenesis, and persistence (16
). In this regard, it is well established that the hepatitis C virus (HCV) nonstructural 5A (NS5A) protein is a phosphoprotein and that phosphorylation of NS5A is highly conserved among HCV genotypes and other members of the Flaviviridae
). This led to the idea that phosphorylation may play a role in the flavivirus life cycle and inspired our interest in obtaining a better understanding of NS5A phosphorylation, particularly since this protein has been implicated in resistance of HCV to interferon treatment (4
). NS5A is phosphorylated primarily on serine residues both in vitro and in vivo (13
). Although the effects of various kinase inhibitors have implicated a cellular serine/threonine kinase(s) from the CMGC kinase group (25
), it should be noted that this group contains several kinase families, and these studies did not identify a particular kinase in the phosphorylation of NS5A. More recent in vitro kinase assays specifically demonstrated that NS5A can be phosphorylated by casein kinase 2 (CK2) (12
) and protein kinase A (PKA) (12
The interferon-induced double-stranded RNA-activated protein kinase, PKR, is a key component of the antiviral and antiproliferative effects induced by interferon. NS5A forms a complex with PKR, disrupting PKR dimerization, which results in repression of PKR function and inhibition of PKR-mediated eIF-2α phosphorylation. Whether NS5A is a substrate of PKR is yet to be determined. There is no PKR homologue in the yeast kinome, but the yeast kinase GCN2 still has sequence homology with PKR. In this study, we were particularly interested in finding out whether GCN2 is a kinase of NS5A.
In this report, we further investigated the phosphorylation of NS5A using a kinome-scale high-throughput screening approach in order to identify human protein kinases that phosphorylate NS5A. The approach is based on a biochemical genomics strategy, originally described by Martzen et al. (20
), for constructing an array of molecular constructs for expression of all yeast kinases (the yeast kinome) (11
). Our long-term interests are to gain insight into the role of phosphorylation in the ability of NS5A to modulate a multitude of cellular signaling pathways. Although there are already more than 518 known human kinases, more than four times the number of the kinases in yeast, we reasoned that the yeast kinome would represent a good model for beginning a global inspection of NS5A phosphorylation activity, since phosphorylation of NS5A occurs primarily at serine residues (13
) and the major families of serine/threonine kinases are conserved between yeast and humans. The use of the yeast model was based on the idea that the kinase(s) phosphorylating NS5A is very likely to be evolutionarily conserved. This is supported by our previous findings that Ser 2194 is a major phosphorylation site among all HCV genotypes and this site can be phosphorylated by yeast, insect, and mammalian kinases (13
). We screened 119 glutathione S-transferase (GST)-tagged protein kinases purified from Saccharomyces cerevisiae
in a global effort to identify yeast kinases capable of phosphorylating NS5A in vitro. BLAST searches were performed to identify the closest human sequence homologs, and commercially available sources of human protein kinases were used for subsequent in vitro NS5A kinase assays. We demonstrated that several of these human kinases were capable of phosphorylating NS5A in vitro, some of which play a major role in protein translation and antiapoptotic pathways that are regulated by NS5A. Furthermore, the phosphopeptide pattern of NS5A isolated from COS-1 cells suggests that the phosphorylation sites used in vivo were similar to those observed in vitro, allowing us to identify kinases of particular interest for further characterization of NS5A phosphorylation. Finally, rapamycin studies revealed that p70S6 kinase and potentially closely related family members phosphorylate NS5A inside the cell.