Previous studies have established that the catalytic activity of DAPK is regulated by Ca2+
/CaM and by autophosphorylation of Ser308
within its calmodulin-binding domain. It has also been shown that the expression levels of DAPK are modulated by ubiquitin-mediated proteasomal degradation (8
) and that DAPK interacts with HSP90 (14
). Those studies suggest that an intricate network of protein modifications and interactions acts to regulate the activities of this fascinating Ser/Thr protein kinase. In the present study, by identifying novel components of the DAPK signaling complex and defining their roles in modulating the cellular levels of DAPK, we have extended our understanding of how DAPK activities are regulated.
It has been established that in the presence of HSP90 inhibitors like GA or 17-AAG, many client proteins are destabilized and subsequently degraded via the ubiquitin proteasome pathway (29
). In addition, other studies have linked phosphorylation status of proteins to ubiquitination and degradation (26
). Along these lines, we have recently reported that subsequent to its dephosphorylation and activation, DAPK is rapidly degraded through the ubiquitin proteasome pathway (9
). The recent determination that DAPK and HSP90 are associated (14
) prompted us to further investigate the events regulating the cellular levels of DAPK. We show for the first time that GA inhibition of HSP90 results in DAPK degradation by the ubiquitin proteasome pathway and that DAPK degradation is mediated by the activities of two distinct E3 ligases, CHIP and DIP1/Mib1. This conclusion is supported by studies demonstrating that the proteasome inhibitor lactacystin attenuates GA-induced DAPK degradation (). However, we also noted that lactacystin does not completely restore expression of DAPK to control levels, suggesting the possibility that additional proteolytic systems may operate to modulate the cellular levels of DAPK.
The ubiquitin proteasome degradation pathway is an important triage pathway that enforces quality control on protein activities in cells and consists of activating (E1), conjugating (E2), and ligating (E3) enzymes (30
). The E3 ligases are a large group of diverse molecules that function to identify target substrates and mediate the covalent ligation of ubiquitin by direct interaction with the target (31
). We previously identified a novel RING E3 ubiquitin ligase, called DIP1, which was later also identified as mindbomb (Mib1) (32
). Those studies showed that DIP1/Mib1 directly interacts with and ubiquitinates DAPK to promote its proteasomal degradation (8
). In the present study, we have extended those results and determined that siRNA-mediated depletion of DIP1/Mib1 expression rescues DAPK from proteasome-mediated degradation (). In addition, we show that siRNA-mediated depletion of DIP1/Mib1 attenuates GA-induced DAPK degradation, linking DIP1/Mib1-directed ubiquitination and degradation of DAPK to DAPK degradation induced in response to HSP90 inhibitors ().
Previous studies have demonstrated that CHIP, a U-box-containing E3 ligase, binds through its TPR domain to TPR acceptor sites on HSP90 to mediate ubiquitination of at least some HSP90 client proteins (18
). Based on those reports and our finding that DAPK is targeted for proteasome degradation by ubiquitination, we asked whether CHIP could also target DAPK for degradation. The present studies demonstrate that overexpression of CHIP induces the degradation of DAPK and that inhibition of the proteasome with the proteasome inhibitor, lactacystin, attenuates the DAPK degradation caused by CHIP overexpression (). Consistent with this, we also show, using an in vitro
ubiquitination assay, that CHIP increases ubiquitination of DAPK (). Finally, by using siRNA to deplete levels of CHIP, we can rescue DAPK from CHIP-induced degradation. Together, these results suggest that CHIP can also target DAPK for ubiquitination and degradation by the proteasome.
Since CHIP has been previously reported to associate directly with HSP90 in a heterocomplex (18
), we asked whether CHIP, DIP1/Mib1, and DAPK co-associate in a heterocomplex with HSP90. These studies revealed that two distinct DAPK hetero-complexes are present in cells. In one complex, HSP90, CHIP, and DAPK are associated (). In a second complex, however, only DAPK and DIP1/Mib1 are associated (). The finding that two E3 ligases are associated with the DAPK is surprising; however, it may suggest that regulation of DAPK activities by proteasomal degradation is critical and that when DAPK is complexed with HSP90 it can be ubiquitinated by CHIP and when released from HSP90 it can be ubiquitinated by DIP1/Mib1. This heightened surveillance of DAPK activities by HSP90, CHIP, and DIP1/Mib1 may also reflect the central importance of DAPK in regulation of cellular death and survival pathways (1
). Consistent with this suggestion are the recent findings that Cdt-1, a DNA licensing and replication protein, and p27Kip1
, a DNA replication inhibitor, are targeted for degradation by two E3 ubiquitin ligases, DDB1-Cul4 and SCF-Skp2 (34
). One question these results raise that is currently being investigated is whether CHIP and DIP1/Mib1 utilize the same or distinct ubiquitination sites within DAPK and the location of these ubiquitination sites.
In the current report, we also show that treatment of cells with GA results in accumulation of inactive phospho (Ser308)-DAPK, consistent with the low catalytic activity found using immunoprecipitated total DAPK in an in vitro kinase assay (). Together, these data suggest that inhibition of HSP90 activity not only induces DAPK degradation, it also attenuates DAPK catalytic activity by the selective degradation of active DAPK. Unexpectedly, we found that GA also attenuated degradation of DAPK by both E3 ligases ( and ), although HSP90 does not appear to be associated with the DAPK·DIP1/Mib1 complex. This finding suggests that the effect of GA on DIP1/Mib1-mediated degradation of DAPK is not directly related to its association with HSP90. If this is true, then following dephosphorylation and activation, DAPK would be released from the HSP90·CHIP complex, phosphorylate substrates such as myosin II RLC, and then become ubiquitinated through its association with DIP1/Mib1. Prior to its release from HSP90, any misfolded DAPK that is unable to achieve a native conformation could be targeted for degradation by CHIP. Alternatively, it is also possible that a fraction of HSP90 is in fact associated with DIP1/Mib1·DAPK heterocomplexes, but its association is weak or below the level that is detectable by Western blotting.
The finding that DAPK is a target for at least two E3 ubiquitin ligases (DIP1/Mib1 and CHIP), one of which is found complexed with HSP90, suggests that these two heterocomplexes function to scrutinize the folding and activation of DAPK as well as to modulate DAPK activities in cells. The finding that GA inhibition of HSP90 enhances degradation of the active, dephosphorylated DAPK suggests a link between activation and degradation () and is consistent with our previous results showing that tumor necrosis factor treatment results in DAPK dephosphorylation and activation, which is subsequently attenuated by the ubiquitin-mediated proteasomal degradation of DAPK (9
In summary, we find that inhibition of HSP90 results in degradation of active dephosphorylated DAPK via the ubiquitin proteasome pathway. The discovery that GA treatment dramatically increases the expression levels of the inactive form of DAPK, phospho(Ser308)-DAPK, whereas total DAPK decreases them, suggests that HSP90 activity is essential for stabilization of activated DAPK. The finding that DAPK can be isolated from cells in heterocomplexes composed of HSP90 and CHIP or DIP1/Mib1 and that DAPK is targeted for proteasomal degradation through the activities of two distinct E3 ubiquitin ligases (CHIP, a U-box E3 ligase, and DIP1/Mib1, a RING E3 ligase) indicates that heightened surveillance and modulation of DAPK activities is critical to accurate regulation of apoptosis and cellular homeostasis.