We have shown here that the E3 ligase, WWP1, ubiquitinates HER4, targeting it for degradation through the proteosomal and lysosomal pathways. Although other E3 ligases are known to target EGFR family members for degradation, WWP1 demonstrates specificity within the EGFR family for HER4. This specificity is driven by three PY motifs, or WW-domain binding motifs, which are present in HER4 but are absent in other EGFR members.
By targeting HER4 for degradation, WWP1 expression has a negative impact on the biologic consequences of HER4 activity, such as lactogenic differentiation (as measured by β-casein promoter activity [Fig. ]), or regulation of BRCA1 expression, which we have postulated is important in HER4-dependent growth inhibition (31
) (Fig. ). Increased differentiation and decreased growth of breast cells can occur in response to HER4 Cyt1 activity (13
) and are consistent with the observations that HER4 expression in breast cancers generally correlates with a more favorable prognosis (3
). The results presented here suggest that increased expression of WWP1 may counteract the potential tumor suppressor effects of HER4 in breast cancers (see reference 48
). Interestingly, not all studies of HER4 in breast cancer suggest that HER4 correlates with a favorable prognosis (4
). The reason for this discrepancy is unknown but may relate to alternative HER4 splicing. In fact, some studies have shown altered signaling potential and increased stability of the HER4Cyt2 isoform compared to HER4Cyt1 (50
). Interestingly, the PY2 motif of HER4 is only present in the Cyt1 splice variant. The absence of PY2 in the HER4Cyt2 variant appears to decrease interaction with WWP1, rendering HER4Cyt2 less susceptible to downregulation by WWP1. Decreased interaction between HER4Cyt2 and WWP1 may explain in part the enhanced stability of HER4Cyt2 over HER4Cyt1. Likewise, the different biologic outcomes between Cyt1 and Cyt2 HER4 may also be due to a decreased association of the Cyt2 isoform with YAP as our data show for the first time (Fig. ). The PY3 motif of HER4 (present in both Cyt1 and Cyt2) supported interaction between each of the WW-domain containing proteins tested, including Wwox (Fig. ), but Wwox interacted equally with HER4Cyt1 or HER4Cyt2. Thus, Wwox may play a similar role in the action of both HER4 isoforms. These data underscore the specificity underlying the overlapping but distinct functions of these (and perhaps other) WW domain-containing proteins in HER4 signaling.
WWP1 is a member of Nedd4 family of related E3 ligases, which also includes Nedd4-1, Nedd4-2, WWP1/Tiul1, WWP2, Aip4/Itch, Smurf1, Smurf2, HecW1/NedL1, and HecW2/NedL2 (18
). Among the family members, Nedd4, WWP1, Smurf1, and Smurf2 have been reported to be overexpressed in several human cancers (6
). It is possible that overexpression of these factors may contribute to tumorigenesis through a negative impact on the balance of tumor suppressors or proteins that promote differentiation, such as pTEN or p53, through ubiquitination-directed degradation. This scenario has been proposed for Nedd4, which ubiquitinates the tumor suppressor pTEN, targeting it for degradation (55
). Because pTEN plays a central role in the growth and survival of prostate and other cancer cells, Nedd4 expression levels could influence cellular decisions along the pathway toward malignancy. Similarly, WWP1 and Smurfs target Smads of growth-inhibitory transforming growth factor β (TGF-β) signaling pathway for degradation (5
). While TGF-β has the ability to inhibit growth of some tumor cells in a Smad-dependent manner, TGF-β signaling also promotes motility and survival of tumor cells in a Smad-independent manner, thus contributing to malignancy (32
). The degradation of Smads as directed by WWP1 and Smurfs may tilt the balance of the TGF-β signaling pathway toward Smad-independent mechanisms that promote survival and motility and remain unchecked by growth control (29
). It has also been reported that WWP1 ubiquitinates p53 to prevent its translocation to nucleus and decrease its transcriptional activities (24
Consistent with the observation that WWP1 is often overexpressed in human cancers and may promote tumorigenesis, the gene for WWP1 is located at 8q21, a region frequently amplified in human prostate and breast cancer. WWP1 overexpression promotes prostate and breast cell proliferation and survival (6
), perhaps due to the degradation of one or more of the WWP1 substrates described above or those unidentified. Our report that WWP1 is an E3 ligase for HER4, is a new addition to our understanding on the WWP1's role in cancer, especially in breast cancer. HER4 signaling decreases cellular proliferation of human breast cells and promotes differentiation (13
). Our work indicates that this is due to the action of the Cyt1 isoform (13
), the isoform most likely to be affected by WWP1. In breast cancers, HER4 expression correlates with the presence of estrogen receptor, a more differentiated tumor grade, and longer survival (37
). The studies cited did not have the reagents to distinguish whether HER4 isoform expressed as Cyt1 or Cyt2. However, degradation of the HER4 Cyt1 signal caused by WWP1 overexpression could be another important mechanism of enhanced tumorigenesis.
Initial reports of m80HER4
production from full-length HER4 described the rapid ubiquitination and degradation of m80HER4
. The observation was made that m80 was far more susceptible to proteosomal degradation than was full-length HER4 (54
). This is similar to our findings that demonstrate proteosomal/lysosomal degradation of m80 HER4, but unknown mechanisms of full-length HER4 degradation (Fig. ). Most recently, Omerovic et al. screened a brain cDNA phage library with HER4 peptides and found that Itch could interact with HER4 and then demonstrated that Itch could ubiquitinate and target HER4 for degradation (36
). It is very likely that both WWP1 and Itch regulate HER4 protein levels via ubiquitination. Functional redundancy seems to be common among members of the Nedd4 E3 ligase family. For example, Smurf1 and Smurf2 can target Smad1, Smad2, and Smad5 for degradation, and WWP1 targets Smad2 for degradation (5
). Smurf1, Smurf2, and WWP1 each target RUNX2 for ubiquitin-mediated degradation (20
). Consistent with the idea of functional redundancy between WWP1 and Itch, it has been described (unpublished data referred to in reference 5
) that combined loss of both WWP1 and Itch in genetically engineered mice resulted in postnatal lethality within 72 h of birth due to lung hemorrhage, but that single loss of either was compatible with fetal development. The fact that WWP1 knockdown by siRNA in MCF7 cells increases endogenous HER4 suggests that, at least in the MCF-7 breast cell, WWP1 may be the relevant E3 ligase and that the Itch and WWP1 may have tissue or cell-type-specific functions.
Our data do not fully elucidate the “downregulation” mechanism involved in HER4 signaling. Our previous results show that ligand-dependent HER4 signaling in cells can be prolonged (33
). The data presented here indicate that WWP1 binds to HER4 and is most effective in degrading the membrane-associated species HER4 and m80HER4
. WWP1 binds to s80HER4
but does not appear to promote degradation at the same rate as the membrane-associated forms. Previous data suggest that HER4 Cyt1 growth inhibition and differentiation requires the action of the intracellular s80HER4
derived from m80HER4
). Perhaps one major action of WWP1 is to act at the membrane to prevent the m80HER4
conversion to s80HER4
by causing its rapid degradation through a largely proteosome-dependent process. The elimination of the C2-membrane association domain of WWP1 (Fig. ) reverses the membrane-soluble HER4 specificity of WWP1. The overexpression of soluble WWP1 (without C2) and soluble 80-kDa HER4 Cyt1 shows that under these conditions the WWP1-s80HER4
complex can access the degradation machinery efficiently. Under the “normal” circumstance in which WWP1 is membrane associated via its C2 domain, it appears that a more efficient degradation of m80HER4
is achieved, presumably through a membrane complex. What triggers WWP1 activity toward m80, preventing its prolonged signaling (and subsequent stochastic, γ-secretase-dependent release of the potent differentiation and growth inhibitory fragment s80HER4
), is unknown. The mechanism of the WWP1's effect on full-length HER4 is at least in these experiments less well defined. However, the fact that s80HER4
expression stimulates WWP1 RNA accumulation does suggest that a cellular feedback mechanism exists whereby s80 action would result in more WWP1 (presumably sent to the membrane) to downmodulate the signal.
In summary, we have demonstrated that WWP1 binds, ubiquitinates, and promotes the degradation of HER4, but not other members of the EGFR family. Given that HER4 is the unique member of the EGFR family that decreases growth and promotes differentiation of breast cells, it will be important to understand the relationship between HER4 and WWP1, as well as other WW domain-containing E3 ligases, in breast cancer. It is conceivable that inhibition of WWP1 activity could result in the stabilization of HER4 and its cleavage products, resulting in decreased growth and differentiation of breast cancer cells.