No abstract

The vaccinia-related kinases (VRKs) comprise a branch of the casein kinase family whose members are characterized by homology to the vaccinia virus B1 kinase. The VRK orthologues encoded by Caenorhabditis elegans and Drosophila melanogaster play an essential role in cell division; however, substrates that mediate this role have yet to be elucidated. VRK1 can complement the temperature sensitivity of a vaccinia B1 mutant, implying that VRK1 and B1 have overlapping substrate specificity. Herein, we demonstrate that B1, VRK1, and VRK2 efficiently phosphorylate the extreme N terminus of the BAF protein (Barrier to Autointegration Factor). BAF binds to both DNA and LEM domain-containing proteins of the inner nuclear membrane; in lower eukaryotes, BAF has been shown to play an important role during the reassembly of the nuclear envelope at the end of mitosis. We demonstrate that phosphorylation of ser4 and/or thr2/thr3 abrogates the interaction of BAF with DNA and reduces its interaction with the LEM domain. Coexpression of VRK1 and GFP-BAF greatly diminishes the association of BAF with the nuclear chromatin/matrix and leads to its dispersal throughout the cell. Cumulatively, our data suggest that the VRKs may modulate the association of BAF with nuclear components and hence play a role in maintaining appropriate nuclear architecture.

The proto‐oncogene c‐kit encodes a transmembrane tyrosine protein kinase receptor for an unknown ligand and is allelic with the murine white‐spotting locus (W). Mutations at the W locus affect various aspects of hematopoiesis, the proliferation and migration of primordial germ cells and melanoblasts during development. The original W mutation and W37 are severe lethal mutations when homozygous. In the heterozygous state the W mutation has a weak phenotype while W37 has dominant characteristics. Wv and W41 are weak W mutations with dominant characteristics. We have characterized the molecular basis of these four W mutations and determined their effects on mast cell differentiation by using a fibroblast/mast cell co‐culture assay. We show that W37, Wv and W41 are the result of missense mutations in the kinase domain of the c‐kit coding sequence (W37 E‐‐‐‐K at position 582; Wv T‐‐‐‐M position 660 and W41 V‐‐‐‐M position 831), which affect the c‐kit associated tyrosine kinase to varying degrees. The c‐kit protein products in homozygous mutant mast cells are expressed normally, although the 160 kd cell membrane form of the c‐kitW37 protein displays accelerated turnover characteristics. The W mutation is the result of a 78 amino acid deletion which includes the transmembrane domain of the c‐kit protein. A 125 kd c‐kit protein was detected in homozygous W/W mast cells which lacks kinase activity and is not expressed on the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)

[1992][1993][1994][1995][1996][1997][1998][1999][2000][2001][2002][2003][2004][2005]. This mammalian kinase family comprises three members, VRK1, VRK2, and VRK3. We have annotated the gene structure for the members of this family and have characterized the enzyme activity and subcellular localization for the human and mouse proteins. VRK1 enzymes show robust autophosphorylation activity and will phosphorylate casein; VRK2 enzymes show modest autophosphorylation activity and will also phosphorylate casein. The VRK3 proteins have key amino acid substitutions that disrupt invariant motifs required for catalytic activity, rendering them enzymatically inert. The VRK1 and VRK2 proteins contain COOH-terminal extracatalytic sequences that mediate intracellular localization. VRK1 proteins possess a basic nuclear localization signal and are indeed nuclear; the extreme C termini of the VRK2 proteins are highly hydrophobic, and the proteins are membrane-associated and colocalize with markers of the endoplasmic reticulum. The NH 2 -terminal region of the VRK3s contains a bipartite nuclear localization signal, which directs these proteins to the nucleus. Our findings provide the basis for further studies of the structure and function of this newly discovered family of protein kinases.Post-translational modification of proteins plays a major role in the coordination of cellular events. Reversible phosphorylation of serine, threonine, or tyrosine residues drives the transmission of signals from the cell surface to the nucleus, modulates faithful progression through the cell cycle, and regulates metabolic pathways. Phosphorylation in eukaryotes is largely conducted by a single superfamily of proteins, the protein kinases. To underscore the significant role that kinases serve in the eukaryotic cell, it has been determined that there are 518 kinases encoded in the human genome, comprising ϳ1.7% of the gene complement (1). Members of this family share a common catalytic domain of 250 -300 amino acids that mediates the transfer of phosphate from ATP to a substrate (2, 3). Pathogens that interface with eukaryotic cells also utilize protein kinases to coordinate intrinsic facets of their life cycle and/or to manipulate the host cell. In viruses, for example, modulation of protein phosphorylation may down-regulate the host's response to infection (4 -7), divert the host translation machinery to preferentially translate viral RNAs (8 -12), regulate the replication of the viral genome (13, 14), or facilitate viral assembly (15-17).Vaccinia virus, the prototypic poxvirus, is a complex DNA virus that replicates solely in the cytoplasm of the eukaryotic cell. Among the ϳ200 viral proteins encoded by the genome are two protein kinases, vvB1 1 and vvF10, each of which plays an essential role in the viral life cycle (15-19). The B1 kinase regulates the replication of the viral genome (18), and F10 is required for the onset and progression of virion morphogenesis (16,17). Analysis of the structure and function of these proteins has been facili...

A rapid assay for retroviral reverse transcriptase activity released into the culture medium by infected cells was developed. With the assay, 4,000 clonally infected cell lines could be tested in a few hours. We have adapted the assay for use as a screen for the detection of spontaneous viral mutants. Mutants of Moloney murine leukemia virus have been isolated which (i) produce a thermolabile reverse transcriptase, (ii) are temperature sensitive for release of enzyme activity, or (iii) can only productively infect cells already producing gag-related polypeptides. The assay has also been useful for the isolation of nonproducer cells infected with various replication-defective transforming viruses.