[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...