Search

The immunoreactivity of cortical and brainstem-type Lewy bodies has been investigates with antibodies to the cyclin-dependent kinase 5 (cdk5), to the extracellular regulated kinase 1 (ERK-1), and to the cdc2p34 kinase and with antibodies specific for phosphorylation epitopes typical of paired helical filament-tau (PHF-tau). Both cortical and brainstem-type Lewy bodies in diffuse Lewy body disease and brainstem-type Lewy bodies in Parkinson's disease were found to be immunoreactive for cdk5 but not for cdc2p34 or ERK-1 or with the PHF-tau antibodies. Double immunolabeling showed that cdk5-positive Lewy bodies were also ubiquitin immunoreactive and that cdk5 antibodies labeled as many Lewy bodies as ubiquitin antibodies in adequately fixed tissue. The cdk5 immunoreactivity of Lewy bodies was abolished by preabsorption of the antibody with a cdk5 peptide. The antibodies to cdk5 labeled a single 33-kd species on Western blots of human brain homogenates, with a similar intensity in control, diffuse Lewy body disease, and Alzheimer's disease, and this cdk5 species was found mainly in the particulate fraction of brain homogenates. This observation suggests that cdk5 might be a protein kinase involved in the phosphorylation of a molecular component of Lewy bodies, for example, neurofilament proteins known to be present in these inclusions.

Mice and rabbits were injected with various forms of a 16 amino acid synthetic peptide representing PSTAIR, the evolutionarily conserved region of the protein kinase p34cdc2, for polyclonal antisera and hybridoma-monoclonal antibody production. Antisera from mice injected with an unconjugated monomeric form of the peptide showed no reaction to the peptide. Of four animals injected with the monomeric form of the peptide conjugated to keyhole limpet hemocyanin via m-maleimidobenzoyl-N-hydroxysulfosuccinimide (MBS), antisera from only one mouse had a very low titer to the peptide, and all four animals produced antibody to the MBS bridge. Both mice injected with an octameric multiple antigen peptide (MAP) of PSTAIR produced antisera reactive to the octameric MAP form of the peptide in ELISA and also to the cdc2 protein expressed in bacteria in an immunoblotting assay. Splenocytes from one mouse injected with the octameric MAP form of the peptide were successfully used for hybridoma-monoclonal antibody production. A monoclonal antibody was produced that reacted with octamer, monomer and cdc2-expressed protein and specifically with the carboxyl terminus of the 16 amino acid peptide.

At the onset of mitosis, eukaryotic cells display an abrupt increase in a Ca2(+)- and cyclic nucleotide-independent histone H1 kinase activity, referred to as growth-associated or M phase-specific H1 kinase. The molecular basis for this activity is generally attributed to a kinase complex that consists of the p34cdc2 protein and cyclin, and exhibits maturation-promoting factor (MPF) activity. In the present study, we show that more than one kinase contributes to M phase-specific H1 kinase activity. When mature Xenopus oocyte extract prepared with ATP gamma S and NaF was fractionated by gel filtration, two prominent peaks of H1 kinase activity were detected, with apparent molecular masses of 600 and 150 kDa. The 150-kDa kinase copurified with the p34cdc2 protein and was immobilized by the suc 1 gene product p13 and anti-cyclin B2, which are specific for the cdc2 kinase complex. However, the 600-kDa kinase did not satisfy any of these criteria, thus identifying it as a novel M phase-specific H1 kinase. Only the 600-kDa kinase was recognized by the mitosis-specific monoclonal antibody, MPM-2, which inhibits Xenopus oocyte maturation and immunodepletes MPF activity. Furthermore, not only did the full activation of this kinase (MPM-2 kinase) coincide with the activation of MPF during the cell cycle, but also MPM-2 kinase-positive fractions obtained by gel filtration accelerated progesterone-induced oocyte maturation. It is, therefore, likely that MPM-2 kinase is a positive regulator in the M phase induction pathway.