The degradation rate of activated RTKs may be controlled by the balance of ubiquitination by E3 ligases (such as c-Cbl) and DUBs such as AMSH 
. ErbB2 is not reduced following indirect activation through stimulation of EGFR, but can be destabilised by CHIP-dependent ubiquitination following dissociation of Hsp90, the target of Geldanamycin 
. Previous studies have determined the influence of ErbB2 on acute EGFR down-regulation following over-expression of ErbB2 
. Here, we have examined the influence of endogenous ErbB2 on endogenous EGFR down-regulation in HeLa cells, for which both receptors are estimated to be expressed at similar levels. In this instance, we clearly show that depletion of ErbB2 does not influence EGFR down-regulation kinetics.
Initially we reasoned that tonic DUB activity may contribute to ErbB2 stability and that we may identify a relevant DUB with a siRNA screen. This predicts that knockdown of a specific DUB would lead to decreased ErbB2 levels due to ubiquitin-dependent degradation. The initial screen using the siGenome pool of 4 oligos per target identified several candidates, but these did not pass the second round of validation i.e.≥two of four On-Target Plus oligos recapitulating the effect.
One DUB, POH1, passed our validation study (4/4 oligos). POH1 is a component of the 19S proteasomal lid complex and has been suggested to couple recycling of ubiquitin to protein degradation 
. Although we see a high degree of loss of ErbB2 following POH1 knock-down, especially when using an antibody directed at the cytosolic domain, when we assay surface associated ErbB2 with an extra-cellular directed antibody we do not observe a corresponding loss of receptor. Furthermore, in these POH1-depleted cells, Western blotting for ErbB2 with an extracellular domain directed antibody reveals a higher molecular weight smear characteristic of ubiquitinated receptor that is susceptible to USP2-cleavage. Thus, we propose that POH1 is an ErbB2 DUB, which may oppose constitutive ubiquitination of the receptor.
We can see the accumulation of ubiquitinated ErbB2 under conditions where proteasome activity is blocked, either by POH1 knockdown or by epoxomicin. Remarkably, the ubiquitinated ErbB2 is not rapidly down-regulated by the lysosomal pathway as happens for example with a truncated EGFR fused to a single linear ubiquitin 
. Our data however fits with the idea of a specific domain within ErbB2 that restricts ligand-dependent degradation independently of ubiquitination status 
Our study also indicates that the proteasomal pathway is unlikely to be a major degradative pathway for ErbB2 under steady state conditions as we do not see any accumulation of ErbB2 upon POH1 knockdown as measured by FACS analysis. Rather we suggest that our results indicate a novel role for POH1 in deubiquitinating ErbB2 that may in fact rescue it from proteasomal and lysosomal degradation. It is also possible that POH1 may de-ubiquitinate EGFR under steady-state conditions, in which case the receptor will be rescued from degradation in the lysosomal pathway. This is consistent with our observation of enhanced EGFR degradation rate following POH1 knock-down () and chimes with reports that both EGFR and TrkA receptor can be deubiquitinated by proteasome associated activity 
. Note that this enhanced turnover of EGFR is unlikely due to alterations in ErbB2 levels, as direct depletion of ErbB2 did not affect the downregulation of EGFR ().
Using doses of proteasome inhibitors in which HeLa cells remain viable, we found that we could recapitulate the apparent loss of ErbB2 and the selectivity for ErbB2 compared with other RTKs (EGFR and Met), once again accumulating a higher molecular weight smear as judged by Western blotting. Marx et al. have also recently observed diminution of ErbB2 levels in breast cancer cell lines following application of the proteasome inhibitor Velcade/Bortezomib 
. However, in their study of cells expressing high levels of ErbB2, they report that proteasome inhibition leads to an intracellular accumulation of remaining ErbB2 consistent with targeting for lysosomal degradation. In contrast, we found that both surface and total levels of ErbB2 are actually little changed in the absence of POH1 activity. We were also unable to rescue or increase ErbB2 levels using a specific inhibitor of lysosomal acidification (concanamycin, data not shown), which is at odds with results obtained with chloroquine in the above study by Marx et al. It is possible that differences in cell lines and disparate expression levels of receptors are responsible for these discrepancies. However this may also suggest that POH1 knockdown does not simply phenocopy proteasome inhibition, a fact that is also indicated by the opposite effects of both treatments on free ubiquitin levels ().
A recent study has indicated a synergistic interaction between an ErbB2 directed monoclonal antibody Herceptin/Trastuzumab and Velcade with respect to cell death of tissue cultured breast cancer cells 
. Further study of this interaction as well as epidemiological data on responsiveness to Velcade and ErbB2 status is clearly warranted.