We also explored the possibility
We also explored the possibility that this PKC-θ activating protein could operate on the auto-phosphorylation process of the kinase, a specific event accompanying PKC activation . The heated PKC-θ immunoprecipitate from metaphasic cells was then added to PKC-θ isolated both from interphasic or metaphasic cells in the absence or presence of the classical lipid cofactors PMA and PS. As shown in Fig. 3, in the absence of lipids, the incorporation of 32P into PKC-θ isolated from interphasic cells (76 kDa phosphorylated band, lane 1) is very poor. However, addition of PKC-θ-heated immunoprecipitate increases the extent of auto-phosphorylation of the kinase 5-fold . A similar enhancement of the auto-phosphorylating activity is obtained by addition of the lipid cofactors to the assay mixture. Conversely, auto-phosphorylation of PKC-θ obtained from metaphasic cells occurs at a high extent in the absence of any addition and is not further stimulated by lipid cofactors. The identity between the phosphorylated protein band showing a molecular mass of 76 kDa and the PKC-θ molecule was also confirmed by overlapping of the 32P band and PKC-θ immunoreactive band detectable by a Western blot on the same electrophoretic run (data not shown).
Discussion We recently established that, although many PKC isoenzymes are located in the nucleus of MEL cells, PKC-θ is the only isoform completely recovered on mitotic spindle structures . A specific association of PKC-θ to recognition sites on centrosomes and kinetochores occurs in mitotic cells both of human and murine origin, suggesting that this PKC-θ targetting process is probably involved in triapine progression. This conclusion is also supported by results demonstrating that PKC-θ is absent from the nucleus of ungrowing cells and is also almost completely down-regulated in terminal differentiated erythroid cells . We now provide experimental evidence indicating that in mitotic MEL cells, PKC-θ is present in an active form. In fact, a chromosomal protein with a molecular mass of 66 kDa is phosphorylated by PKC-θ isolated from mitotic cells with a four times higher efficiency, in the absence of lipid cofactor, as compared to the kinase obtained from interphasic cells. This activation of PKC-θ at the metaphase is also detectable as auto-phosphorylation of the kinase molecule. The loss of lipid requirement, shown by PKC-θ from mitotic cells, is not due to accumulation of free catalytic fragments produced by proteolytic events during mitosis, as indicated by the presence of similar levels of the native form of kinase in metaphasic and interphasic cells and also by the absence of low Mr PKC-θ immunoreactive forms in both conditions. A heat-stable protein factor, which co-immunoprecipitates with PKC-θ in MEL cells at the metaphase, has been found to be responsible for the catalytic activation of this kinase and substituting the classical lipid cofactors. The presence of intracellular complexes between PKC isoenzymes and PKC activating proteins has been yet identified and proposed as an alternative mechanism to localize active PKCs in a specific cell site. It has recently been demonstrated that PKC-α binds and is activated by syndecan-4 transmembrane heparan sulfate , whereas PKC-βII and PKC-ϵ bind on different domains and are activated by F-actin , . Another stimulating agent of PKC is the HMG1 protein, known as a DNA binding molecule . This protein stimulates both PKC-α and PKC-β activity increasing their maximal catalytic activity and substituting their requirement for diacylglycerol . A similar effect on cPKC isoenzymes has also been described for stratifin, a member of the 14.3.3 protein family . It has been reported that some PKC isoenzymes operate as modulators of different cell functions following their interaction with specific proteins, such as phosphatidylinositol-4-kinase and phosphatidylinositol-4-phosphate-5-kinase, which bind PKC-μ at specific membrane sites  and the metalloprotease-disintegrin MDC9 involved in the regulated shedding through binding to PKC-δ .