Polyclonal antibodies against rat liver cytosolic casein kinase II (CK-2) cross-react with CK-2 from other tissues and nuclear form (PK-N2) of the enzyme

Casein kinase II (CK-2) is a ubiquitous serine/threonine protein kinase, and is localized to both the cell nucleus and cytoplasm. Despite extensive biochemical similarities in their properties, there is evidence that the two forms of the enzyme exhibit certain distinctions (1). This prompted us to produce antibodies against CK-2, which could be utilized as a possible tool for investigations of the various forms of this enzyme. Specific polyclonal antibodies against the rat liver cytosolic CK-2 were raised in egg yolk of laying hens; the enzyme had repeatedly failed to elicit an immunogenic response in rabbits. The purified polyclonal antibody (egg yolk immunoglobulin, IgY) recognized all three subunits (42, 38, and 28 kDa) of the enzyme in immunoblots. The antibody when bound to a matrix was capable of removing CK-2 from solution, and the bound enzyme could be recovered from the immunoaffinity matrix with 0.1 M diethylamine. The antibody exhibited a high affinity towards CK-2 prepared from cytosol of liver, ventral prostate, and several other rat tissues, but no immunoreactivity was detected towards a number of other protein kinases tested. The subunits of the nuclear form of CK-2 (PK-N2) migrated differently when electrophoresed in parallel in the same gel. However, the antibody did cross-react with the various subunits of PK-N2 suggesting a significant homology in the immunogenic domains in the various subunits of the two forms of the enzyme.     

The homologous operons for P1 and P7 plasmid partition are autoregulated from dissimilar operator sites

The plasmid-partition regions of the P1 and P7 plasmid prophages in Escherichia coli are homologues which each encode two partition proteins, ParA and ParB. The equivalent PI and P7 proteins are closely related. In each case, the proteins are encoded by an operon that is autoregulated by the ParA and ParB proteins in concert. This regulation is species-specific, as the P1 proteins are unable to repress the P7 par operon and vice versa. The homologous ParA proteins are primarily responsible for repression and bind to regions that overlap the operon promoter in both cases. The DNA-binding domain of the P7 auto-repressor lies in the amino-terminal end of the P7 ParA protein. This region includes a helix-turn-helix motif that has a clear counterpart in the P1 ParA sequence. However, despite the common regulatory mechanism and the similarity of the proteins involved in repression, the promoter-operator sequences of these two operons are very different in sequence and organization. The operator is located downstream of the promoter in P1 and upstream of it in P7, and the two regions show little, if any, homology. How these differences may have arisen from a common ancestral form is discussed.     

Alzheimer's disease-like phosphorylation of the microtubule-associated protein tau by glycogen synthase kinase-3 in transfected mammalian cells

Background: Paired helical filaments (PHFs) are a characteristic pathological feature of Alzheimer's disease; their principal component is the microtubule-associated protein tau. The tau in PHFs (PHF-tau) is hyperphosphorylated, but the cellular mechanisms responsible for this hyperphosphorylation have yet to be elucidated. A number of kinases, including mitogen-activated protein (MAP) kinase, glycogen synthase kinase (GSK)-3α, GSK-3β and cyclin-dependent kinase-5, phosphorylate recombinant tau in vitro so that it resembles PHF-tau as judged by its reactivity with a panel of antibodies capable of discriminating between normal tau and PHF-tau, and by a reduced electrophoretic mobility that is characteristic of PHF-tau. To determine whether MAP kinase, GSK-3α and GSK-3β can also induce Alzheimer's disease-like phosphorylation of tau in mammalian cells, we studied the phosphorylation status of tau in primary neuronal cultures and transfected COS cells following changes in the activities of MAP kinase and GSK-3.Results Activating MAP kinase in cultures of primary neurons or transfected COS cells expressing tau isoforms did not increase the level of phosphorylation for any PHF-tau epitope investigated. But elevating GSK-3 activity in the COS cells by co-transfection with GSK-3α or GSK-3β decreased the electrophoretic mobility of tau so that it resembled that of PHF-tau, and induced reactivity with eight PHF-tau-selective monoclonal antibodies.Conclusion Our data indicate that GSK-3α and/or GSK-3β, but not MAP kinase, are good candidates for generating PHF-type phosphorylation of tau in Alzheimer's disease. The involvement of other kinases in the generation of PHFs cannot, however, be eliminated. Our results suggest that aberrant regulation of GSK-3 may be a pathogenic mechanism in Alzheimer's disease.