Distinct forms of human CDC2 identified by novel monoclonal antibodies.
Studies on the functional and structural properties of the cdc2 kinase, a key cell-cycle regulator, have been possible thanks to the availability of cdc2-specific immunoreagents. In an attempt to elucidate the biochemical regulation of the cdc2 kinase in more detail, we have raised a series of novel mouse monoclonal antibodies against human recombinant cdc2 protein. The five Mab reported here can be subclassified into two groups according to their interspecies cross-reactivity and distinct immunoprecipitation patterns. Thus, the target epitopes of Mab POH-1, POH-2 and POH-7 (group 1) appear to be limited to a few mammalian species and the fraction of cdc2 immunoprecipitable by these Mab from cellular extracts is considerably enhanced by denaturation. In contrast, the POH-3 and POH-8 (group 2) Mab recognize a denaturation-sensitive epitope on cdc2 which is present in all tested mammalian species. More importantly, each of the two groups of Mab immunoprecipitate forms of cdc2 associated with a characteristic set of cellular proteins, none of which appears to be cyclin A or cyclin B. None of the antibodies precipitated a histone-H1 or casein-kinase activity, although an activity which phosphorylated some of the coprecipitated proteins was coprecipitated with the group 2 Mab. These novel Mab did not interfere with the association of cdc2 with cyclin A in vitro and efficient immunoprecipitation of a panel of cdc2 mutant proteins suggests that the target epitopes may not involve amino acid residues essential for currently known cdc2 functions. The results of the present study provide evidence for the existence of additional forms of the cdc2 protein in exponentially growing human cells, distinct from both the monomeric and the cyclin-bound cdc2 identified so far.
Lukás
J
J
Department of Tumor Biology, Institute of Haematology and Blood Transfusion, Prague, Czechoslovakia.
Draetta
G
G
Bartek
J
J
D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D016203:CDC2 Protein Kinase / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D003429:Cross Reactions; D005260:Female; D006801:Humans; D015151:Immunoblotting; D007527:Isoenzymes / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D011994:Recombinant Proteins / Q000032:analysis / Q000276:immunology / Q000378:metabolism
D000906:Antibodies; D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D011994:Recombinant Proteins; D016203:CDC2 Protein Kinase