br Materials and methods br Results In
Materials and methods
Results In order to evaluate changes in the amount of PKC-θ expressed by MEL cells at different stages of the cell cycle, we measured the level of the kinase in cells collected from cultures containing 90% of cells in the interphase or 80% cells synchronized at the metaphase. As shown in Fig. 1A, Western blot analysis reveals that the amount of the 76 kDa PKC-θ immunoreactive band is very similar in both conditions and no degradation fragments, which produce 40–50 kDa immunoreactive bands containing the catalytic domain of this kinase , are detectable. These data indicate that subcellular redistribution of PKC-θ occurring during the MEL SB 525334 is not accompanied by a proteolytic processing or a down-regulation of the kinase. Due to the fact that at present, a rapid purification procedure is not available to obtain a high yield of native PKC-θ, we selected the single step immunoprecipitation method to separate PKC-θ from the other PKC isoenzymes present in MEL cells. PKC-θ immunoprecipitates, obtained using an anti-peptide antibody raised against the caboxy-terminal peptide of the kinase, were analyzed by a Western blot and resulted free of any other PKC isoenzyme previously reported to be expressed by MEL cells (data not shown) . Furthermore, since the protein or peptide substrates of PKC-θ are poorly characterized, we used a chromosomal preparation to evaluate the presence of an intracellular substrate for PKC-θ localized in a cell structure on which the kinase is recruited during mitosis. As shown in Fig. 2, addition of PKC-θ immunoprecipitated from MEL cells to a chromosome preparation in the presence of [γ32P]ATP results in the incorporation of 32P in a protein band having a molecular mass of approximately 66 kDa. It has been reported that phorbol-12-myristate-13-acetate (PMA) and phosphatidylserine (PS) stimulate PKC-θ activity . Addition of these lipid cofactors to the assay mixture enhances the incorporation of 32P into the 66 kDa chromosomal protein 4-fold (cf. lanes 5 and 6). Chromosomes or PKC-θ alone do not show any appreciable phosphorylated band in these conditions. We evaluated the possibility that PKC-θ undergoes changes of catalytic properties following its translocation on the spindle pole during mitosis. The kinase was isolated by immunoprecipitation from MEL cells in the interphase or synchronized at the metaphase and the two PKC-θ preparations were then incubated with chromosomes in the absence or presence of lipid cofactors. As shown in Table 1, both PKC-θ samples exhibit a similar phosphorylating activity when assayed in the presence of PMA and PS. On the contrary, the enzyme isolated from cells in the interphase expresses 15% of its maximal activity in the absence of lipids, whereas the enzyme obtained from metaphasic cells expresses more than 60% of its maximal catalytic activity. This difference indicates that the kinase is more active during the metaphase, i.e. in the form associated with centrosomes and kinetochores. To establish whether the PKC-θ activating agent, present in immunoprecipitates from metaphasic cells, was due to the presence of a protein or a lipid molecule, the immunoprecipitates were at first heated for 3 min at 75°C to obtain a complete inactivation of PKC-θ (data not shown) followed by incubation with trypsin or, alternatively, extraction with ether. Each immunoprecipitate was finally assayed for the presence of the PKC-θ activating factor by addition of chromosomes, as source of a protein substrate, and PKC-θ obtained from interphasic or metaphasic cells. As shown in Table 2, PKC-θ from metaphasic cells phosphorylates the 66 kDa chromosomal substrate with a comparable high efficiency both in the absence or in the presence of the heated immunoprecipitate. On the contrary, PKC-θ from cells in the interphase shows a 5-fold increase in the 66 kDa chromosomal protein phosphorylation when the heated immunoprecipitate is added to the assay mixture. Treatment of the heated immunoprecipitate with trypsin results in the disappearance of the PKC-θ activating effect, whereas, following extraction with ether, the activating factor is recovered in the aqueous phase. These data indicate that, in mitotic MEL cells, PKC-θ associates and co-immunoprecipitates together with a protein factor acting as a stimulating cofactor for the kinase activity. PKC-θ immunoprecipitates from cells in the interphase, on the contrary, do not contain any detectable PKC-θ activating factor (data not shown).