While considerable progress has been made in defining the functional properties of different USPs using in vitro and cell culture systems, their physiological relevance in the context of the whole organism is only poorly understood. Recently, mice deficient in CYLD, a USP that is altered in patients with familial cylindromatosis, were generated. Contrary to results from transfection experiments, which suggested a negative regulatory function of CYLD on NF-κB activation (8
), bone marrow-derived macrophages from wt and CYLD−/−
animals did not differ in innate immune receptor signaling. Analysis of these mice instead demonstrated an essential role for CYLD in T-cell development (47
), underlining the necessity of mouse models to evaluate functional properties of DUBs in vivo.
It was a matter of debate whether and to what extent different DUBs might have redundant functions (2
). UBPy-deficient animals are embryonic lethal, and induced inactivation in adulthood causes fatal liver failure. This clearly shows that lack of UBPy cannot be compensated for by other DUB family members in vivo. Embryonic lethality might at least partially be attributed to the lack of HRS, as HRS- deficient animals also exhibited embryonic lethality caused by ventral-folding defects. However, while HRS-deficient embryos were often as large as wt controls and even had beating hearts by day 9.5 (27
animals were extremely developmentally retarded and exhibited completely disorganized morphology at that time point. This shows that besides the stabilization of HRS, UBPy is essential for embryonic growth. The observation that UBPy-deficient fibroblasts underwent growth arrest further supports an essential role of UBPy in the control of cellular proliferation. MX-Cre-mediated deletion of floxed genes in general leads to the fastest and most efficient deletion in the liver (29
), which most likely explains why the phenotype manifests in that organ, although UBPy is also expressed in other cell types. The strong liver phenotype demonstrates that, besides a role in proliferation, UBPy is also essential in quiescent cells, like hepatocytes. As loss of UBPy in hepatocytes caused apoptosis and cell death while embryonic fibroblasts did not exhibit enhanced apoptosis, functional differences of UBPy in different cell types or in proliferating versus nonproliferating cells apparently exist.
Pathology in the liver can most likely be attributed to the combined effects of disturbed endosomal function, loss of several RTKs, and disturbed liver regeneration caused by the absence of c-met, which has been shown to be essential for this process (6
). Due to the severity of the phenotype in the liver and early death, cell-type-specific inactivation will be necessary to define the physiological relevance in other organs or cell types.
We have shown that in vivo UBPy is essential to maintain proper protein levels of RTKs, like EGFR, c-met, and ERBB3, and thus reduced growth factor stimulation might at least partially contribute to the growth arrest in UBPy−/−
cells. As a common feature of several RTKs, ligand binding induces ubiquitination as a signal to internalize the receptor via endocytosis (15
). Although the detailed mechanisms leading to the reduction or even absence of several RTKs in UBPy-deficient mice need to be defined, the observed cytoplasmic localization of EGFR in UBPy-deficient hepatocytes strongly suggests that, due to the loss of UBPy, RTKs undergo enhanced internalization followed by lysosomal degradation. The results are compatible with a model in which UBPy counteracts ubiquitination of RTKs or components of the endocytic machinery and thus regulates thresholds for the internalization of growth factor receptors.
The observed accumulation of ubiquitinated proteins in UBPy-deficient cells that colocalize with the enlarged endosomes might not only represent ubiquitinated conjugated substrates associated with the endosome, but could also reflect enhanced amounts of ubiquitinated cargo derived from the cell membrane. Such a functional role of UBPy exerted on cell membrane components is compatible with the cellular localization of endogenous UBPy.
As described here, the absence of UBPy caused a severe reduction of HRS. HRS binds directly to ubiquitinated proteins, which are then sorted to clathrin-coated microdomains while nonubiquitinated receptors, like transferrin or LDL receptor, are rapidly recycled to the cell surface (45
). Endosomal enlargement in UBPy-deficient cells most likely can be attributed to the lack of HRS, as HRS-deficient murine cells and drosophila HRS−/−
mutant larvae (27
) were also reported to exhibit this phenotype. While this work was in progress, two reports also described endosomal enlargement when UBPy was knocked down by RNA interference (7
), supporting an essential role of UBPy in endocytic trafficking.
As HRS itself was identified as a protein that is tyrosine phosphorylated upon stimulation with hepatocyte growth factor, platelet-derived growth factor, and epidermal growth factor (26
) and HRS−/−
drosophila pupae have reduced levels of EGFR (34
), it is appealing to see a functional relationship between the lack of RTKs and the lack of HRS in UBPy-deficient cells and animals. However, the knockdown of HRS in cultured cells, as well as the combined inactivation of STAM1 and STAM2, was reported to even inhibit the degradation of ligand-activated RTK degradation (4
). Thus, the observed reduction of RTKs in UBPy−/−
cells is unlikely to be caused by the lack of HRS but rather reflects a function of UBPy independent of the stabilization of the HRS-STAM complex. This is supported by results from Mizuno et al., who described a direct interaction of UBPy and the EGFR resulting in EGFR deubiquitination (38
). The observed accumulation of MVBs shows that in UBPy−/−
cells, endosomes can efficiently mature to MVBs even in the absence of the HRS-STAM complex, a prerequisite of RTK degradation via the lysosomal pathway.
The results suggest that UBPy exerts two independent functions in the RTK degradation pathway, first by directly counteracting RTK internalization and second by stabilizing the HRS-STAM complex. Both activities might act in concert to regulate thresholds for RTK degradation.