The protein kinase pUL97 of human cytomegalovirus plays important but incompletely defined roles in viral replication. Concerning the early phase of infection, it is postulated that pUL97 possesses regulatory functions in gene expression and/or DNA synthesis. Here we report that pUL97 interacts with an essential component of the replication complex, the DNA polymerase processivity factor pUL44. Interaction was determined by yeast two-hybrid and coimmunoprecipitation analyses and was mapped to the pUL97 region 366-459. In vitro kinase assays demonstrated that pUL44, coimmunoprecipitated either from transfected or from infected cells, is phosphorylated by pUL97 (but not by a catalytically inactive pUL97-mutant). In infected fibroblasts, immunofluorescence analysis revealed that pUL97 and pUL44 accumulate in the nucleus and are both incorporated into viral replication centers. The treatment with inhibitors of DNA synthesis or pUL97 kinase activity largely prevented colocalization. Thus, pUL97 may be indirectly involved in viral genome replication by modifying the replication cofactor pUL44.
Herpesviral protein kinases of the UL97 subfamily are expressed by all known herpesviruses but the degree of functional conservation is unclear. A selection of representative members was investigated by a comparative structural and functional analysis. The coding sequences of human cytomegalovirus (HCMV) pUL97, rat CMV pR97, Epstein-Barr virus BGLF4, and herpes simplex virus UL13 showed a low degree of amino acid identity. A computational approach employing fold recognition techniques revealed structural similarity to the cellular kinase Cdk2 with a high level of conservation of the functionally important residues in ATP binding sites and the catalytic centers. Analyses of in vitro activities of these herpesviral protein kinases, including measurements of phosphorylation of cellular substrates, trans-complementation experiments with a UL97-deleted HCMV mutant, and sensitivity profiles toward protein kinase inhibitors, demonstrated marked similarities between pUL97 and pR97 and to a lesser extent between pUL97 and BGLF4 or UL13. Thus, the structure-activity analysis of pUL97-like herpesviral protein kinases indicates a partial but not a full conservation of their functional properties among the herpesviruses.
Mutations of
inversin
cause type II nephronophthisis, an infantile autosomal recessive disease characterized by cystic kidney disease and developmental defects. Inversin regulates Wnt signaling and is required for convergent extension movements during early embryogenesis. We now show that Inversin is essential for
Xenopus
pronephros formation, involving two distinct and opposing forms of cell movements. Knockdown of Inversin abrogated both proximal pronephros extension and distal tubule differentiation, phenotypes similar to that of
Xenopus
deficient in Frizzled-8. Exogenous Inversin rescued the pronephric defects caused by lack of Frizzled-8, indicating that Inversin acts downstream of Frizzled-8 in pronephros morphogenesis. Depletion of Inversin prevents the recruitment of Dishevelled in response to Frizzled-8 and impeded the accumulation of Dishevelled at the apical membrane of tubular epithelial cells in vivo. Thus, defective tubule morphogenesis seems to contribute to the renal pathology observed in patients with nephronophthisis type II.
Planar cell polarity signaling controls a variety of polarized cell behaviors. In multiciliated Xenopus epidermal cells, recruitment of Dishevelled (Dvl) to the basal body and its localization to the center of the ciliary rootlet are required to correctly position the motile cilia. We now report that the anaphase-promoting complex (APC/C) recognizes a D-box motif of Dvl and ubiquitylates Dvl on a highly conserved lysine residue. Inhibition of APC/C function by knockdown of the ANAPC2 subunit disrupts the polarity of motile cilia and alters the directionality of the fluid movement along the epidermis of the Xenopus embryo. Our results suggest that the APC/C activity enables cilia to correctly polarize in Xenopus epidermal cells.cilium ͉ Dishevelled ͉ planar cell polarity ͉ polycystic kidney disease ͉ ANAPC2
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