The results of our present study demonstrate that the regulation of PKCη is unique compared to other PKC isozymes. Although tumor-promoting phorbol esters are potent activators of conventional and novel PKCs [4
], persistent treatment with phorbol esters leads to the downregulation of phorbol ester-sensitive PKCs causing termination of PKC signaling [32
]. Downregulation of PKCs has important implications in regulating long-term cellular responses, such as cell proliferation, differentiation and tumor promotion [2
]. We have shown that in contrast to other PKCs, prolonged treatment with PKC activators led to upregulation of PKCη whereas PKC-specific inhibitors triggered downregulation of PKCη. Furthermore, we made a novel observation that novel PKCs are involved in PKC activator-induced upregulation of PKCη.
It is generally believed that treatment with PKC activators leads to membrane translocation of PKCs followed by dephosphorylation [33
]. The dephosphorylated PKCs are subject to downregulation by proteases [34
]. However, fully phosphorylated PKCα was shown to be downregulated at the plasma membrane via the proteasome-mediated pathway [35
]. In addition, the phosphorylated primed form of PKCε was downregulated by phorbol ester treatment independent of its intrinsic kinase activity [36
]. It has been reported that active conformation of PKCη is necessary for its downregulation in baby hamster kidney (BHK) cells although TPA failed to downregulate PKCη in these cells [37
]. Our results show that prolonged treatment with structurally distinct PKC activators, such as phorbol esters and ILV, caused an upregulation of PKCη in MCF-7 cells (). The upregulation of PKCη by PKC activators was not unique to MCF-7 cells, and was observed in several cell types, including T47D, BT-20 and MCF-10CA1d cells ( and data not shown).
The activity, maturation, stability and localization of PKCs are regulated by phosphorylation and dephosphorylation events [7
]. Our study suggests that PKCη level is regulated by phosphorylation. First, treatment with several PKC activators, such as TPA, PDBu and ILV induced upregulation of PKCη () but this upregulation was not associated with an increase in PKCη mRNA (). Second, PKC-specific inhibitors Gö 6983 () and bisindolylmaleimide (data not shown) led to downregulation of PKCη. Third, upregulation of PKCη by PDBu was associated with an increase in PKCη phosphorylation ().
Phosphorylation of PKCs is regulated by both autophosphorylation and transphosphorylation [3
] and phosphorylation of PKCs at the activation loop is believed to prime them for activation [8
]. Phosphoinositide-dependent kinase-1 (PDK1) has been shown to phosphorylate PKCs, at the activation loop and contributes to the stability of cPKCs and PKCε [32
] PDK1 was also shown to phosphorylate PKCη at the activation loop [30
]. However, knockdown of PDK1 did not prevent PKC activator-induced upregulation of PKCη ().
PKCs can also undergo transphosphorylation by other members of the PKC family [3
]. For example, PKCε rather than PDK1 was shown to phosphorylate PKCδ and PKCε at the activation loop whereas PKCδ induced autophosphorylation as well as transphosphorylation of PKCε at the hydrophobic motif [12
]. We recently reported that depletion of PKCε enhanced PDBu-induced downregulation of PKCδ in HeLa cells [11
]. Since the general PKC inhibitor Gö 6983 but not the conventional PKC inhibitor Gö 6976 induced PKCη downregulation () and atypical PKCs are phorbol ester insensitive, it is likely that PKCη is also regulated by novel PKC isozyme(s). Consistent with this notion, we found that depletion of cPKCα had little effect on PKC activator-induced upregulation of PKCη whereas knockdown of nPKCε attenuated PKCη upregulation ().
The observation that PKCη is the only PKC isozyme upregulated by tumor-promoting phorbol esters suggests that PKCη may play an important role in tumorigenesis. Depending on the cellular context, PKCη may suppress tumorigenesis or promote malignant cell growth. For example, PKCη knockout mice were more susceptible to tumor promotion in two-stage skin carcinogenesis model [39
]. In contrast, PKCη has also been implicated in breast cancer [19
], glioblastoma [21
], Hodgkin's lymphoma [40
], lung cancer [22
] and hepatocellular carcinoma [42
]. This contrasting function of PKCη in different cell types is not unique to PKCη and has been noted with other novel PKCs, such as PKCδ [5
] and PKCε [43
]. PKCη is often overexpressed in breast cancer [19
] and the level of PKCη is upregulated by estradiol in hormone-sensitive breast cancer cells [44
]. Moreover, overexpression of PKCη confers resistance to chemotherapeutic drugs [17
]. Thus, understanding the mechanism of PKCη upregulation has significant implications in cancer therapy.