Signal transduction of constitutively active protein kinase C epsilon.

The protein kinase C (PKC) family is the most prominent target of tumor-promoting phorbol esters. For the PKCepsilon isozyme, different intracellular localizations and oncogenic potential in several but not all experimental systems have been reported. To obtain information about PKCepsilon-signaling, we investigated the effects of constitutively active rat PKCepsilon (PKCepsilonA/E, alanine 159 is replaced by glutamic acid) in HeLa cells in a doxycycline-inducible vector. Upon induction of PKCepsilonA/E expression by doxycycline, the major part of PKCepsilonA/E was localized to the Golgi. This led (i) to phosphorylations of PKCepsilon(S729), Elk-1(S383), PDK1(S241) and Rb(S807/S811), (ii) to elevated expression of receptor of activated C kinase 2 (RACK2) after 12 h, and (iii) increased colony formation in soft agar, increased cell migration and invasion, but not to decreased doubling time. Following induction of PKCepsilonA/E-expression by doxycycline for 24 h and additional short-term treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), PKCepsilonA/E translocated to the plasma membrane and increased phosphorylation of MARCKS(S152/156). Treatment with doxycycline/TPA or TPA alone increased phosphorylations of Elk-1(S383), PDK1(S241), Rb(S807/S811), PKCdelta(T505), p38MAPK(T180/Y182), MEK1/2(S217/S221) and ERK2(T185/T187). MARCKS was not phosphorylated after treatment with TPA alone, demonstrating that in this system it is phosphorylated only by PKCepsilon localized to the plasma membrane but not by PKCalpha or delta, the other TPA-responsive PKC isozymes in HeLa cells. These results demonstrate that PKCepsilon can induce distinctly different signaling from the Golgi and from the plasma membrane.

p73beta is regulated by protein kinase Cdelta catalytic fragment generated in the apoptotic response to DNA damage.

Protein kinase C (PKC) delta is cleaved by caspase-3 to a kinase-active catalytic fragment (PKCdeltaCF) in the apoptotic response of cells to DNA damage. Expression of PKCdeltaCF contributes to the induction of apoptosis by mechanisms that are presently unknown. Here we demonstrate that PKCdeltaCF associates with p73beta, a structural and functional homologue of the p53 tumor suppressor. The results show that PKCdeltaCF phosphorylates the p73beta transactivation and DNA-binding domains. One PKCdeltaCF-phosphorylation site has been mapped to Ser-289 in the p73beta DNA-binding domain. PKCdeltaCF-mediated phosphorylation of p73beta is associated with accumulation of p73beta and induction of p73beta-mediated transactivation. By contrast, PKCdeltaCF-induced activation of p73beta is attenuated by mutating Ser-289 to Ala (S289A). The results also demonstrate that PKCdeltaCF stimulates p73beta-mediated apoptosis and that this response is attenuated with the p73beta(S289A) mutant. These findings demonstrate that cleavage of PKCdelta to PKCdeltaCF induces apoptosis by a mechanism in part dependent on PKCdeltaCF-mediated phosphorylation of the p73beta Ser-289 site.