Interaction between cyclin-dependent kinases and human papillomavirus replication-initiation protein E1 is required for efficient viral replication

Abstract

We have identified the human papillomavirus (HPV) DNA replication initiation protein E1 as a tightbinding substrate of cyclin E͞cyclin-dependent kinase (Cdk) complexes by using expression cloning. E1, a DNA helicase, collaborates with the HPV E2 protein in ori-dependent replication. E1 formed complexes with cyclin E in insect and mammalian cells, independent of Cdks and E2. Additional cyclins, including A-, B-, and F-type (but not D-type), interacted with the E1͞E2 complex, and A-and E-type cyclin kinases were capable of phosphorylating E1 and E2 in vitro. Association with cyclins and efficient phosphorylation of E1 required the presence of a cyclin interaction motif (the RXL motif). E1 lacking the RXL motif displayed defects in E2-dependent HPV ori replication in vivo. Consistent with a role for Cdk-mediated phosphorylation in E1 function, an E1 protein lacking all four candidate Cdk phosphorylation sites still associated with E2 and cyclin E but was impaired in HPV replication in vitro and in vivo. Our data reveal a link between cyclin͞Cdk function and activation of HPV DNA replication through targeting of Cdk complexes to the E1 replicationinitiation protein and suggest a functional role for E1 phosphorylation by Cdks. The use of cyclin-binding RXL motifs is now emerging as a major mechanism by which cyclins are targeted to key substrates.Cell cycle transitions are coordinated in large part through the action of a family of cyclin-dependent kinases (Cdks), enzymes composed of a catalytic Cdk subunit, and a regulatory cyclin subunit (1). Much of what we know concerning Cdk action in the G 1 ͞S transition comes from analysis of the Rb͞E2F pathway, which has led to the identification of Rb, p107, p130, E2F-1, and DP-1 as Cdk substrates (1, 2). Phosphorylation of these proteins typically is linked with formation of tight complexes between the kinase and the substrate, most frequently through a motif in the substrate (the RXL motif) that interacts with the cyclin box. The RXL motif forms the basis for interaction of the p21 family of Cdk inhibitors with cyclins (3-6) and is important for Cdk-mediated phosphorylation of p107 (6, 7), E2F-1 (6, 8, 9), and Rb (10). Cyclin E͞Cdk2 is a key regulator of S-phase initiation. Removal of cyclin E͞Cdk2 activity from Xenopus egg replication systems (reviewed in ref. 11) or mammalian cells (12) blocks S-phase entry, and inXenopus, this blockade can be overcome by addition of cyclin E͞Cdk2. Conversely, ectopic expression of cyclin E can initiate replication independent of Rb inactivation in mammalian cells and in Drosophila (ref. 13, and reviewed in ref. 2). However, targets of this kinase in the preinitiation complex are unknown. We and others (14) have taken advantage of the tight association of cyclins with their substrates to identify cyclin E͞Cdk-binding proteins and substrates via expression cloning techniques. Here we report the identification of human papillomavirus (HPV) replication-initiation protein E1 as a tightbinding substrate of cyclin͞Cdk complexes.Papill...

Cell cycle–regulated phosphorylation of p220NPATby cyclin E/Cdk2 in Cajal bodies promotes histone gene transcription

“…To generate cyclin E/Cdk2-phosphorylated p220, 2 µg of Flag-p220 immobilized on anti-flag agarose was allowed to associate with 1 µg of cyclin E/Cdk2 and washed complexes incubated with 1 mM ATP (50 min at 25°C). (c) Schematic diagram of p220 phosphorylation sites and the cyclin E binding domain inferred by expression cloning (Zhao et al 1998;Ma et al 1999). (d) Altered mobility of p220 in response to cyclin E/Cdk2-mediated phosphorylation requires S775 and S779.…”

“…The link between p220, a cyclin E/Cdk2 interacting protein (Zhao et al 1998;Ma et al 1999), and the transcription of histone 2B and histone 4 genes (Zhao et al 2000) led us to address whether cyclin E/Cdk2 directly regulates this aspect of p220 function. In principle, cyclin E/Cdk2 could function to relay cell cycle positional information to p220, thereby playing a role in controlling the timing of S-phase-specific histone gene transcription.…”

“…We previously identified a C-terminal fragment of NPAT (residues 1054-1397) in a cyclin E/Cdk3 interaction screen (Ma et al 1999). Affinity-purified anti-NPAT antibodies generated against this C-terminal fragment recognize a closely spaced protein doublet of 220 kD in molecular mass in nuclear extracts from HeLa and 293 cells, as determined by either immunoblotting or immunoprecipitation (Fig.…”

“…Viruses were made using Baculogold (Pharmingen). To examine interaction with Cdks, cyclin/Cdk complexes were purified with glutathione-Sepharose beads (Ma et al 1999). Immobilized complexes were then incubated with insect cell extracts with or without Flag-p220.…”

“…In some cases, this interaction involves a motif in the substrate, the RXL motif, and a conserved pocket in the cyclin box (Zhu et al 1995;Adams et al 1996;Russo et al 1996;Schulman et al 1998;Brown et al 1999;Ma et al 1999). We and others have exploited this property of cyclins to identify relevant cyclin E/Cdk2 substrates with the use of expression cloning (Zhao et al 1998;Ma et al 1999). One of these, p220 NPAT , interacts with cyclin E/Cdk2 in extracts from tissue culture cells and accelerates S-phase entry when overexpressed (Zhao et al 1998).…”