The high conservation of the complex primary structure of DAPK and the presence of several protein-protein interaction motifs including several ankyrin repeat motifs and a death domain suggested that in addition to substrate binding and activation by calcium/calmodulin, this Ser/Thr kinase might be associated with other cellular proteins. A yeast two-hybrid interaction screen has identified a new protein called DIP-1, which binds to the ankyrin repeat region of DAPK, and co-immunoprecipitation studies have confirmed that DIP-1 and DAPK are associated in cells. Although understanding the structural components and the regulation of DAPK and DIP-1 interactions will be critical to understanding their collaborative function in apoptosis regulation, the similarity between the RING fingers in DIP-1 and those present in IAP-1 and IAP-2 led us to focus on examining the functional properties of the carboxyl-terminal region containing these motifs in DIP-1.
The primary sequence of DIP-1 has several interesting structural motifs including a B-box-type zinc finger, a series of nine ankyrin repeats, and a carboxyl-terminal region that contains three putative RING finger domains with an α
-helical coiled-coil structure that separates RING2 from RING3. Classical RING finger domains are defined by a specific pattern of cysteine and histidine residues that are involved in the binding of zinc, which is important for the folding of the domain and its activities. Many RING domain proteins also have B-box zinc fingers and coiled-coil motifs arranged in a conserved order, and these motifs may function as additional sites of protein interactions (31
). This has led to the suggestion that collectively these motifs function as a molecular scaffold to mediate the organization of large protein signaling complexes, and the identification of DIP-1 as a component of proteins that are associated in a complex with DAPK supports this proposal.
One specific function that has been ascribed to the RING finger domain is its ability to act as an E3 ligase in the ubiquitin proteasome pathway (11
). Consistent with this, we have determined that DIP-1 can autoubiquitinate in vitro
in the presence of ubiquitin-activating E1 and ubiquitin-conjugating E2 enzymes, suggesting that DIP-1 is a member of the “single subunit” class of RING domain E3 ligase (1
). Although we do not know if the autoubiquitination is inter- or intramolecular, the demonstration that DIP-1 is found as a polyubiquitinated protein in vivo
suggests this may serve as mechanism to down-regulate DIP-1 expression.
The determination that DAPK is also found in vivo as a polyubiquitinated protein also suggests that cellular levels of DAPK are regulated by the ubiquitin-proteasome system; the association of DIP-1 with DAPK provided the basis for proposing that DAPK is a target for DIP-1-mediated ubiquitination, and four experimental approaches were utilized to confirm this proposal. First, an in vitro ubiquitination assay using purified RING1–3 as an E3 ligase in the presence of purified E1 and E2 showed that DAPK can be polyubiquitinated by DIP-1. An add-back experiment using purified DIP-1-(492–1006) to supplement DIP-1-immunodepleted cell lysates restored the appearance of polyubiquitinated DAPK. Western blotting to examine the in vivo ubiquitination levels of DAPK in cell lines that overexpress either DIP-1 or RING1–3 showed enhanced ubiquitination of DAPK in these cells compared with the parental HeLa cells. Finally, the endogenous levels of DAPK are diminished significantly in HeLa cell lines overexpressing DIP-1 or RING1–3, suggesting that the endogenous levels of DAPK may be regulated by DIP. Together these findings support the proposal that the apoptosis regulatory protein kinase DAPK is a target for ubiquitination and proteasome degradation by the E3 ligase activity of one of its binding proteins, DIP-1.
Although additional work will be needed to determine whether DIP-1 has other ubiquitination targets, the ability of DIP-1 to deplete cellular levels of DAPK by targeting it for proteasomal degradation suggests a mechanism by which DIP-1 could antagonize the anti-apoptotic effects of DAPK to promote TNF-induced apoptosis. Paralleling our previous findings with DAPK (17
), we also find that DIP-1 enhances caspase-3 and caspase-9 activities while having little effect on TNF-induced caspase-8 activity. These results suggest that both DAPK and DIP-1 act downstream of caspase-8 and before the release of cytochrome c
from the mitochondria. Because previous studies suggest that human DAPK-α
promotes apoptosis or autophagy in variety of cell types (13
), it is still unclear as to why our results consistently show that expression of mouse DAPK-α
has no effect and DAPK-β
is a strong anti-apoptotic factor for TNF-induced apoptosis in several cell lines.
In summary, these studies have identified a novel RING finger protein, which has been named DIP-1 (D
rotein). DIP-1 has intrinsic E3 ligase activity and can self-ubiquitinate in vitro
. In vivo
, DIP-1 can be detected as a polyubiquitinated protein, suggesting that the intracellular levels of DIP-1 are regulated by the ubiquitin proteasome system. The determination that DAPK is an in vitro
as well as in vivo
target for ubiquitination by DIP-1 provides a mechanism by which DAPK activities may be regulated through proteasomal degradation. Finally, we show that expression of DIP-1 can antagonize the anti-apoptotic activity of DAPK to promote a caspase-dependent apoptosis. This result is consistent with our previous determination that DAPK is an important anti-apoptotic survival factor in cells (17