Cyclin E/Cdk2 acts at the G1/S-phase transition to promote the E2F transcriptional program and the initiation of DNA synthesis. To explore further how cyclin E/Cdk2 controls S-phase events, we examined the subcellular localization of the cyclin E/Cdk2 interacting protein p220 NPAT and its regulation by phosphorylation. p220 is localized to discrete nuclear foci. Diploid fibroblasts in Go and G1 contain two p220 foci, whereas S-and G2-phase cells contain primarily four p220 foci. Cells in metaphase and telophase have no detectable focus. p220 foci contain cyclin E and are coincident with Cajal bodies (CBs), subnuclear organelles that associate with histone gene clusters on chromosomes 1 and 6. Interestingly, p220 foci associate with chromosome 6 throughout the cell cycle and with chromosome 1 during S phase. Five cyclin E/Cdk2 phosphorylation sites in p220 were identified. Phospho-specific antibodies against two of these sites react with p220 within CBs in a cell cycle-specific manner. The timing of p220 phosphorylation correlates with the appearance of cyclin E in CBs at the G1/S boundary, and this phosphorylation is maintained until prophase. Expression of p220 activates transcription of the histone H2B promoter. Importantly, mutation of Cdk2 phosphorylation sites to alanine abrogates the ability of p220 to activate the histone H2B promoter. Collectively, these results strongly suggest that p220 NPAT links cyclical cyclin E/Cdk2 kinase activity to replication-dependent histone gene transcription.

GIPC is a PDZ protein located on peripheral endosomes that binds to the juxtamembrane region of the TrkA nerve growth factor (NGF) receptor and has been implicated in NGF signaling. We establish here that endogenous GIPC binds to the C terminus of APPL, a Rab5 binding protein, which is a marker for signaling endosomes. When PC12(615) cells are treated with either NGF or antibody agonists to activate TrkA, GIPC and APPL translocate from the cytoplasm and bind to incoming, endocytic vesicles carrying TrkA concentrated at the tips of the cell processes. GIPC, but not APPL, dissociates from these peripheral endosomes prior to or during their trafficking from the cell periphery to the juxtanuclear region, where they acquire EEA1. GIPC's interaction with APPL is essential for recruitment of GIPC to peripheral endosomes and for TrkA signaling, because a GIPC PDZ domain mutant that cannot bind APPL or APPL knockdown with small interfering RNA inhibits NGF-induced GIPC recruitment, mitogen-activated protein kinase activation, and neurite outgrowth. GIPC is also required for efficient endocytosis and trafficking of TrkA because depletion of GIPC slows down endocytosis and trafficking of TrkA and APPL to the early EEA1 endosomes in the juxtanuclear region. We conclude that GIPC, following its recruitment to TrkA by APPL, plays a key role in TrkA trafficking and signaling from endosomes.

DNA and histone synthesis are both triggered at the beginning of S phase by cyclin/cdk2 activity. Previous studies showed that inhibition of DNA synthesis with hydroxyurea or cytosine arabinoside (AraC) triggers a concerted repression of histone synthesis, indicating that sustained histone synthesis depends on continued DNA synthesis. Here we show that ectopic expression of HIRA, the likely human ortholog of two cell cycleregulated repressors of histone gene transcription in yeast (Hir1p and Hir2p), represses transcription of histones and that this, in turn, triggers a concerted block of DNA synthesis. Thus, in mammalian cells sustained DNA synthesis and histone synthesis are mutually dependent on each other during S phase. Although cyclin/cdk2 activity drives activation of both DNA and histone synthesis at the G 1 /S transition of cycling cells, concerted repression of DNA or histone synthesis in response to inhibition of either one of these is not accompanied by prolonged inhibition of cyclin A/cdk2 or E/cdk2 activity. Therefore, during S phase coupling of DNA and histone synthesis occurs, at least in part, through a mechanism that is independent of cyclin/cdk2 activity. Coupling of DNA and histone synthesis in S phase presumably contributes to the prompt and orderly assembly of newly replicated DNA into chromatin.Progression through the cell cycle is driven by the sequential and periodic activation of cyclin/cdk complexes (23, 27). For example, entry into and progression through S phase is promoted by activation of cyclin E/cdk2 and cyclin A/cdk2, whereas entry into mitosis is triggered by activation of cyclin B/cdc2. Numerous lines of evidence demonstrate that cyclin/ cdk2 activity plays a key role in initiation of S-phase events. Elevation of cyclin/cdk2 activity in G 1 phase causes premature entry into S phase (9,56,57,72), and inhibition of cyclin/cdk2 activity inhibits entry into and progression through S phase (20,52,67,76). Initiation of S phase depends on activation of a number of biosynthetic processes, including DNA synthesis, histone synthesis, and chromatin assembly (58).According to current models distinct S-phase processes, such as DNA synthesis and histone synthesis, are independently activated by cyclin/cdk2 at the start of S phase (17, 30). For example, activation of DNA synthesis depends on phosphorylation of Cdc6 and Cdc45. Increased histone synthesis in S phase is due to regulation at transcriptional and posttranscriptional levels. Histone gene transcription increases threeto fivefold as cells enter S phase, and this depends on phosphorylation of NPAT by cyclin E/cdk2 (30,39,42,50,74). However, posttranscriptional regulation accounts for the majority of the 35-to 50-fold increase in histone synthesis during S phase (42,50). The processing of the immature intronless pre-mRNA to the mature mRNA requires a cleavage within the 3Ј untranslated region (UTR); this occurs more efficiently in S phase (15,19,24,37,65), and the mRNA is also more stable at this time (10,24,25,62). The processing of th...

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...