PKC isoforms interact with and phosphorylate DNMT1

DNA methylation plays a critical role in a large variety of cellular processes by controlling gene transcription via gene silencing. Methylation in most animals occurs at the level of cytosines within the sequence CpG, although low levels of non-CpG methylation have been reported in some species. In mammals, there are two classes of DNA (cytosine-5) methyltransferases, de novo and maintenance methyltransferases. The de novo methyltransferase in mammals has two isoforms, DNMT3a and DNMT3b [1]. The maintenance methyltransferase, DNMT1, is the most prevalent DNA methyltransferase found in cells. DNMT1 has several isoforms, including an oocyte-specific isoform that lacks the first 118 amino acids [2] and a splice variant known as DNMT1b [3]. Maintenance methylation ensures the propagation of tissue-specific methylation patterns established during mammalian development. While the DNMT1 enzymes have a preference for hemimethylated DNA [4], DNMT3a and DNMT3b act on either hemimethylated or unmethylated DNA. Thus, the pattern of mammalian methylation is established and maintained by a set of at least three different DNA methyltransferases.

At present, the signaling cascade by which DNA methylation patterns are imprinted is unclear. Connections between signaling cascades and epigenetic modifications have recently been unraveled by studies showing that the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB) signaling pathway regulates the protein level of DNMT1, protecting it from degradation via the ubiquitin-proteasome pathway [5]. The idea that DNMT1 activity could be regulated at the post-translational level through phosphorylation by a serine/threonine kinase was supported by mass spectrometry studies, which reported phosphorylation sites on the serine and threonine residues located in the N-terminal domain [6–15]. This region of DNMT1 fulfills several regulatory functions by interacting with proteins such as LSH, EZH2, UHRF1, G9a, DMAP1 (DNMT-associated proteins), HDAC2 (a histone deacetylase), HP1β, PCNA, and Rb [16–24]. Recently, Hervouet et al. (2010) [25] have demonstrated that the disruption of DNMT1/PCNA/UHRF1 interactions promote a global DNA hypomethylation in human gliomas. They also found that such interactions were regulated by the phosphorylation status of DNMT1 since phosphorylation of human DNMT1 by Akt and PKC, at the specific residues serine-127/143 and serine-127 respectively, correlated with global hypomethylation [25].

The protein kinase C (PKC) family consists of ubiquitously expressed phospholipid-dependent serine/threonine kinases, which regulate a large number of physiological processes, including cell growth and differentiation. Studies on simple organisms have shown that PKC signaling paradigms are conserved through evolution from yeast to humans. This conservation underscores the importance of this family in cellular signaling and provides novel insight into PKC function in complex mammalian systems. PKC isoenzymes with differential cellular distribution, substrate specificities, and activation responsiveness are divided into three groups: the conventional PKC isoforms, which are activated by calcium, diacylglycerol, and phorbol esters (cPKCs; α, βI, βII and γ); the novel PKCs, which are activated by diacylglycerol but are calcium-insensitive (nPKCs; δ, ε, η/L (mouse/human) and θ); and the atypical PKCs, which are calcium- and diacylglycerol-insensitive (aPKCs; ζ and λ/ι (mouse/human)) [26]. Although each PKC isoform regulates a large number of downstream targets, individual members of the PKC family are, however, regulated in different ways, and an increasing number of studies indicates that they have distinct, and often opposing, roles [27–29]. In fact, it is now well accepted that each of the PKC isoforms is unique in its contribution to specific biological processes [30, 31]. Whether all PKC isoforms can interact with and phosphorylate DNMT1 remains, however, unknown. Here, we have examined the ability of PKC isoforms to phosphorylate the human DNMT1.

In the present report, we have characterized the relation between PKC isoforms and human DNMT1. More specifically, we found that: 1) PKCα, βI, βII, δ, γ, η, ζ and μ preferentially phosphorylate the N-terminal domain of human DNMT1; no such phosphorylation was observed with PKCε; 2) PKCζ and DMNT1 physically interact in vivo in the nucleus of HEK-293 and HeLa cells; 3) PKCζ activity could be detected in DNMT1 immunoprecipitates of endogenous DNMT1; and 4) overexpression of PKCζ and DNMT1 in HEK-293 cells induces a decrease in DNA methylation, consistent with our results showing that phosphorylation of DNMT1 by PKCζ reduces its methyltransferase activity. Overall, these results provide novel insights on the ability of PKC isoforms to play a role in controlling DNA methylation.

In a recent study, the use of broad spectrum inhibitors have suggested that phosphorylation of DNMT1 likely involves Akt and PKC [25]. Here, we provide additional evidence that PKC and DNMT1 physically interact and regulate DNA methylation. Overall, our experiments have shown that most PKC isoforms, including PKCα, β, γ, δ, η and ζ, are able to phosphorylate, albeit with different efficiency, the N-terminal region of human DNMT1. In fact, the preferential ability of PKC isoforms to interact with and phosphorylate the region encompassing amino acids 1 to 446 are consistent with previous results showing preferential phosphorylation of Serine127 [25]. Interestingly, PKCε and, to a lesser degree PKCμ, were inefficient in their ability to phosphorylate DNMT1 or its N-terminal domain. Such differential phosphorylation by PKC has often been observed. For example, phosphorylation of Ser1674 of Ca_v1.2 α_1c, but not Ser1928, is PKC isoform specific, as only PKCα, βI, βII, γ, δ and θ, but not PKCε, ζ and η, phosphorylate this site [34]. Although it is currently unclear why PKCε is unable to phosphorylate DNMT1, our observations provide an interesting experimental model to investigate further the functional interaction between PKC isoforms and DNMT1.

PKC participates in a multitude of cellular processes, including differentiation, proliferation, cell cycle progression and tumorigenesis [30, 35]. Increasing evidence has implicated PKC isoforms in nuclear functions, suggesting that they could represent a pathway to communicate to the nucleus signals generated at the plasma membrane [36]. For example, in PC12 cells, PKCζ has been found at the inner nuclear matrix of the nucleus [37], where DNA replication gene expression and protein phosphorylation take place [38]. PKCζ has also been located in the nucleus of rat H9c2 cells during reoxygenation after ischemic hypoxia [39]. Here we provide further evidence of the presence of activated PKCζ in the nucleus of HeLa cells and of HEK-293 cells, indicating that translocation of PKCζ into the nucleus is a common mechanism not restricted to a specific cell type. Our attempts to demonstrate an interaction between endogenous DNMT1 and PKCζ by co-immunoprecipitation were, however, unsuccessful, most likely due to low expression level of DNMT1. Using a more sensitive approach, we were able to show PKCζ-specific activity in immunoprecipitates of endogenous DNMT1, supporting our hypothesis that endogenous DNMT1 and PKCζ could be found in the same complex within the nucleus. This hypothesis is also supported by our data showing that flagged DNMT1 interacts with the endogenous form of PKCζ. Whether nuclear PKCζ stands in close proximity of DNMT1, ready to act in proliferative cells, is not known. This could be, however, a very effective means to rapidly regulate DNMT1 activity when necessary. A similar paradigm has recently been proposed from studies on the regulation of DNMT1 protein stability through the coordinated interaction of an array of DNMT1-associated proteins, such as UHRF1, Tip60 (Tat-interactive protein) and HAUS (herpes virus-associated ubiquitin specific protease) [40–42].

Given its preferential ability to phosphorylate the N-terminal domain of DNMT1, PKCζ may contribute to the formation of multimolecular complexes copying the DNA methylation pattern from a parental to a replicated DNA strand. Several proteins have indeed been reported to interact with DNMT1 via its N-terminal domain, including PCNA, which recruits DNMT1 at the mammalian DNA replication forks [20, 43–45]. Other proteins, such as HDAC and DMAP1 [21] initiate the formation of DNA replication complexes at the replication fork to mediate transcriptional repression. DNMT1 has also been associated with methyl-CpG-binding proteins such as MBD2, MBD3 and MeCP2 to maintain DNA methylation [46, 47]. Histone methyltransferases and HP1 have been recently found to interact with DNMT1, showing a direct connection between the enzymes responsible for DNA methylation and histone methylation [23, 24, 48]. Furthermore, DNMT1 can interact with cell cycle regulating proteins such as Rb and p53 [22, 33, 49]. It is pertinent to note that PKCζ has been shown to interact with and to phosphorylate DNA-bound Sp1, thereby causing the release of the repressor p107 on the Luteinizing Hormone Receptor gene promoter in TSA-treated MCF-7 cells [50]. Because Sp1 interacts with HDAC1/2/mSin3A on the Luteinizing Hormone Receptor gene promoter in both HeLa and MCF-7 cells [51], and HDAC1/2 binds to DNMT1 [22], it is thus possible that PKCζ could interact with DNMT1 on the promoter via the Sp1/repressor complex. Additional studies will be required to test these possibilities.

Phosphorylation is one of the most common post-translational modifications occurring in animal cells. The previous observations that human DNMT1 was phosphorylated in vivo were indicative that at some point, DNMT1 was interacting with yet unidentified serine/threonine kinases. The results from previous mass spectrometry studies suggested that several phosphorylation sites were targeted depending on the activation status of the cell and/or the cell type [7–15], while Ser154 and Ser714 were shown to be the major phosphorylation sites in HEK-293 cells [8, 12], Ser127, Ser143 and Ser714 in Jurkat cells [13] and Ser143 in lung cancer cells [15]. Although it is unclear at present whether distinct phosphorylation sites are targeted by PKC isoforms in different cell types, it is likely that Ser127 is preferentially targeted [25]. Examination of the phosphorylation profile of human DNMT1 reveals, however, the presence of several alternative phosphosites for PKC isoforms, including some located in the C-terminal regions of DNMT1. Future investigations will be necessary to identify the specific phosphorylation sites in different cell types and different states.

We found that the overexpression of PKCζ along with DNMT1 in HEK-293 cells led to a decrease in DNA methylation and that phosphorylation of DNMT1 by PKCζ reduced its methyltransferase activity in vitro. Our preliminary data indicate that these changes in the methylation status may not, however, be sufficient to induce or modulate gene expression. For example, no significant changes in Egr1 mRNA expression were observed (data not shown). This may not be surprising because DNA hypomethylation of the promoter does not always result in increased gene expression. Moreover, in cancer cells, although gene-specific hypomethylation occurs, much of the effect of global DNA hypomethylation are thought to occur through the activation of the normally dormant transposons and endogenous retroviruses present in the human genome [52]. The fact that overexpression of PKCζ alone was not sufficient to trigger genome hypomethylation may be explained, in part, by the presence of excess of PKCζ as compared to endogenous DNMT1. Unbound PKCζ might also activate signaling pathways critical for cell proliferation, differentiation and survival, such as the ERK/MAPK pathway, thereby providing a counterbalance to the negative regulation of DNMT1. It is well known that PKCζ can activate extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway in different cell types [39, 53, 54]. Moreover, it has been shown that inhibition of ERK/MAPK pathway lead to a decrease in DNA methylation in colon cancer cells [55].

Our data support the idea that PKC-DNMT1 interaction is important in controlling DNA methylation, possibly by regulating DNMT1 interaction with other proteins, such as UHFR1, as recently suggested [25]. This possibility is also supported by data showing that activation of PKC with phorbol ester in mouse hippocampus tissues induced a rapid demethylation of the reelin promoter [56]. To date, it was believed that such a role was essentially mediated through the ability of PKC to down-regulate the DNMT expression at the mRNA level [56]. Moreover, Sun et al., [5] have also shown that treatment of HeLa cells with a specific inhibitor of PI3K, which activates PKC, DNMT1 protein level and genomic content of methylated cytosines were decreased in a time-dependent manner without affecting the DNMT1 mRNA level. Whether phosphorylation of DNMT1 on specific residues was involved in maintaining the functional integrity of the enzyme is in fact a real possibility because mutations of one of the major phosphorylation sites of murine DNMT1, Ser515 (previously referred to as Ser514 by Glickman et al., 1997) [6], has been shown to significantly reduce the in vitro enzymatic activity of recombinant DNMT1 [57]. Alternatively, phosphorylation of DNMT1 could affect its structural integrity, thereby reducing its DNA-binding activity, as shown by Sugiyama et al. via in vitro phosphorylation of murine DNMT1 by CK1δ [58]. It would thus be very interesting to determine, for instance, whether phosphorylation of DNMT1 modulates its ability to bind specific endogenous DNA sequences, thereby contributing to the overall genome hypomethylation. Ideally, however, such experiments will require antibodies that recognize specific PKCζ-mediated phosphorylated residues on human DNMT1. Future investigations will be needed to address this issue.

This study is the first to identify PKC specific isoforms involved in the phosphorylation of DNMT1. Indeed, all PKC isoforms except PKCε, which was very inefficient, preferentially phosphorylated the N-terminal domain (amino acids 1 to 446) of DNMT1. Functional implications of DNMT1 phosphorylation by PKC isoforms have been highlighted by experiments using PKCζ as a model, which suggested possible roles in the control of DNA methylation patterns of the genome, and possibly in the control of gene expression. Based on the importance of PKC signaling in a multitude of biological processes and of a tight regulation of DNA methylation in normal cells, these findings may provide a novel strategy for cancer therapy.