A chimeric pre-ubiquitinated EGF receptor is constitutively endocytosed in a clathrin-dependent, but kinase-independent manner.

The roles of EGF receptor (EGFR) kinase activity and ubiquitination in EGFR endocytosis have been controversial. The adaptor protein and ubiquitin ligase Cbl has reportedly been required. Consistently, we now report that siRNA-mediated knock-down of c-Cbl and Cbl-b significantly slowed clathrin-dependent internalization of activated wild-type (wt) EGFR by inhibiting recruitment of the EGFR to clathrin-coated pits. However, a chimeric protein consisting of wt-EGFR, a C-terminal linker and four linearly connected ubiquitins was found to interact with Eps15 and epsin 1 and to be constitutively endocytosed in a clathrin-dependent manner. Interestingly, endocytosis of this fusion protein did not require binding of EGF. Nor was kinase activity required, and the fusion protein was endocytosed in the presence of an EGFR kinase inhibitor, which efficiently counteracted tyrosine phosphorylation. This demonstrates that ubiquitination over-rides the requirement for kinase activity in recruitment of the EGFR to clathrin-coated pits.

The oncoprotein ErbB3 is endocytosed in the absence of added ligand in a clathrin-dependent manner.

The oncoprotein ErbB3 is overexpressed in several human cancers, for example in pancreatic adenocarcinoma and in ovarian cancers, and ErbB3-containing heterodimers have been demonstrated to be potent signaling units in carcinogenesis. This especially applies to ErbB2-ErbB3 and epidermal growth factor receptor (EGFR)-ErbB3 heterodimers providing anti-apoptotic signaling. Relatively little is understood about the signaling of EGFR-ErbB3 heterodimers and especially about mechanisms involved in downregulation of ErbB3 from the plasma membrane. This is in contrast to EGFR homodimers, for which trafficking has been extensively characterized. In the present study, we have investigated mechanisms involved in endocytosis of ErbB3 in porcine aortic endothelial cells stably expressing either ErbB3 only or stably expressing ErbB3 and EGFR. Our data show that ErbB3 is endocytosed in the absence of added ligand, independently of its tyrosine phosphorylation state and in a clathrin-dependent manner. Functional EGFR-ErbB3 heterodimers were observed to be formed, and dimerization with ErbB3 was observed to negatively affect endocytosis of the EGFR.

Expression of epidermal growth factor receptor or ErbB3 facilitates geldanamycin-induced down-regulation of ErbB2.

Overexpression of the epidermal growth factor receptor (EGFR), ErbB2, and ErbB3 promotes growth and antiapoptotic signaling. Overexpression of ErbB2 in breast cancer is associated with poor clinical outcome, and ways of down-regulating ErbB2 are important as therapeutic approaches. In contrast to EGFR, ErbB2 has been shown to be endocytosis deficient. However, down-regulation of ErbB2 can be induced by incubation of cells with geldanamycin and geldanamycin derivatives, counteracting the stabilizing function of heat shock protein 90 on ErbB2. In the present study, we have made use of stably transfected isogenic cell lines expressing ErbB2 only or ErbB2 together with EGFR and/or ErbB3. We now show that whereas ErbB2 can be down-regulated by incubation with geldanamycin in cells expressing ErbB2 only, the rate of geldanamycin-induced down-regulation increases significantly when the cells additionally express EGFR and/or ErbB3. This increase does, however, not correlate with activation/phosphorylation of ErbB2. The potential of heterodimer formation in ErbB2-positive breast cancer cells could thus turn out to be prognostically predictive with respect to outcome of treatment with geldanamycin derivatives.

The inhibitory effect of ErbB2 on epidermal growth factor-induced formation of clathrin-coated pits correlates with retention of epidermal growth factor receptor-ErbB2 oligomeric complexes at the plasma membrane.

By constructing stably transfected cells harboring the same amount of epidermal growth factor (EGF) receptor (EGFR), but with increasing overexpression of ErbB2, we have demonstrated that ErbB2 efficiently inhibits internalization of ligand-bound EGFR. Apparently, ErbB2 inhibits internalization of EGF-bound EGFR by constitutively driving EGFR-ErbB2 hetero/oligomerization. We have demonstrated that ErbB2 does not inhibit phosphorylation or ubiquitination of the EGFR. Our data further indicate that the endocytosis deficiency of ErbB2 and of EGFR-ErbB2 heterodimers/oligomers cannot be explained by anchoring of ErbB2 to PDZ-containing proteins such as Erbin. Instead, we demonstrate that in contrast to EGFR homodimers, which are capable of inducing new clathrin-coated pits in serum-starved cells upon incubation with EGF, clathrin-coated pits are not induced upon activation of EGFR-ErbB2 heterodimers/oligomers.

Nucleosome binding by the bromodomain and PHD finger of the transcriptional cofactor p300.

The PHD finger and the bromodomain are small protein domains that occur in many proteins associated with phenomena related to chromatin. The bromodomain has been shown to bind acetylated lysine residues on histone tails. Lysine acetylation is one of several histone modifications that have been proposed to form the basis for a mechanism for recording epigenetically stable marks in chromatin, known as the histone code. The bromodomain is therefore thought to read a part of the histone code. Since PHD fingers often occur in proteins next to bromodomains, we have tested the hypothesis that the PHD finger can also interact with nucleosomes. Using two different in vitro assays, we found that the bromodomain/PHD finger region of the transcriptional cofactor p300 can bind to nucleosomes that have a high degree of histone acetylation. In a nucleosome retention assay, both domains were required for binding. Replacement of the p300 PHD finger with other PHD fingers resulted in loss of nucleosome binding. In an electrophoretic mobility shift assay, each domain alone showed, however, nucleosome-binding activity. The binding of the isolated PHD finger to nucleosomes was independent of the histone acetylation levels. Our data are consistent with a model where the two domains cooperate in nucleosome binding. In this model, both the bromodomain and the PHD finger contact the nucleosome while simultaneously interacting with each other.

TRAF6 mediates ubiquitination of KIF23/MKLP1 and is required for midbody ring degradation by selective autophagy.

Upon completion of cytokinesis, the midbody ring is transported asymmetrically into one of the two daughter cells where it becomes a midbody ring derivative that is degraded by autophagy. In this study we showed that the ubiquitin-binding autophagy receptor SQSTM1/p62 and the interacting adaptor protein WDFY3/ALFY form a complex with the ubiquitin E3 ligase TRAF6 and that these proteins, as well as NBR1, are important for efficient clearance of midbody ring derivatives by autophagy. The number of ubiquitinated midbody ring derivatives decreases in TRAF6-depleted cells and we showed that TRAF6 mediates ubiquitination of the midbody ring localized protein KIF23/MKLP1. We conclude that TRAF6-mediated ubiquitination of the midbody ring is important for its subsequent recognition by ubiquitin-binding autophagy receptors and degradation by selective autophagy.

The Cbl-interacting protein TULA inhibits dynamin-dependent endocytosis.

The Cbl- and ubiquitin-interacting protein T-cell ubiquitin ligand (TULA) has been demonstrated to inhibit endocytosis and downregulation of ligand-activated EGF receptor (EGFR) by impairing Cbl-induced ubiquitination. We presently report that TULA additionally inhibited clathrin-dependent endocytosis in general, as both uptake of transferrin (Tf) and low-density lipoprotein (LDL) was inhibited. Additionally, endocytosis of the raft proteins CD59 and major histocompatibility complex class I (MHC-I), which we demonstrate were mainly endocytosed clathrin-independently, but dynamin-dependently, was blocked in cells overexpressing TULA. By contrast, the uptake of ricin, which is mainly endocytosed clathrin- and dynamin-independently, was not affected by overexpressed TULA. Consistently, TULA and dynamin co-immunoprecipitated and colocalized intracellularly, and upon overexpression of dynamin the TULA-mediated inhibitory effect on endocytosis of Tf, LDL, CD59 and MHC-I was counteracted. Overexpressed dynamin did not restore ubiquitination of the EGFR, and consistently dynamin did not rescue endocytosis of the EGFR in cells overexpressing TULA. We conclude that TULA inhibits both clathrin-dependent and clathrin-independent endocytic pathways by functionally sequestering dynamin via the SH3 domain of TULA binding proline-rich sequences in dynamin.