Annie Angers

Reciprocal regulation of the ubiquitin ligase Itch and the epidermal growth factor receptor signaling

EGF-mediated stimulation of the EGF receptor activates a plethora of signaling cascades followed by receptor down regulation. Preventing down regulation leads to increased mitogenic signaling and potentially, cancer. Cbl and Endophilin are two key proteins required for EGF receptor down regulation and both become ubiquitylated and subject to proteasome-mediated degradation following EGF activation, providing a negative feedback loop for EGF receptor down regulation. The mechanism of this pathway is unknown. Here, we demonstrate that treatment of cells with EGF leads to JNK-dependent phosphorylation of the ubiquitin ligase Itch, stimulating Itch ligase activity. EGF-stimulated JNK activation causes an increased interaction between Itch and the de-ubiquitylating enzyme FAM, limiting the influence of Itch auto-ubiquitylation on its own degradation. Finally, JNK activation stimulates the association of Itch with its substrates. These effects combine to cause increased ubiquitylation of Itch substrates including Endophilin and Cbl, resulting in the proteasome-dependent down regulation of these key trafficking proteins. Thus, Itch is a key regulatory locus for EGF receptor degradation.

SIMPLE/LITAF Expression Induces the Translocation of the Ubiquitin Ligase Itch towards the Lysosomal Compartments

LITAF is a small cellular protein with an unknown function. The C-terminus of LITAF contains a highly conserved domain termed the SIMPLE-like domain (SLD), while the N-terminus contains two PPXY motifs that mediate protein-protein interactions with WW-domain containing proteins. LITAF also harbors two endosome/lysosome targeting sequences at its C-terminus, but there has been conflicting reports regarding its intracellular localization. Here, we demonstrate that LITAF is localized to the late endosome/lysosomal compartment in a variety of cell lines. We also show that Itch, a WW-domain containing protein, and LITAF strongly interact and that this interaction depends on the two PPXY motifs in the N-terminus of LITAF. Interestingly, co-expression of LITAF with Itch induces major changes in Itch intracellular localization, bringing Itch from the trans-Golgi network to lysosomes. We show that this re-localization is dependent upon the interaction with the PPXY sequences of LITAF, since disruption of these binding motifs completely abrogates Itch re-localization.

The ubiquitin ligase Itch mediates the antiapoptotic activity of epidermal growth factor by promoting the ubiquitylation and degradation of the truncated C-terminal portion of Bid.

The truncated C‐terminal portion of Bid (tBid) is an important intermediate in ligand‐induced apoptosis. tBid has been shown to be sensitive to proteasomal inhibitors and downregulated by activation of the epidermal growth factor (EGF) pathway. Here, we provide evidence that tBid is a substrate of the ubiquitin ligase Itch, which can specifically interact with and ubiquitinate tBid, but not intact Bid. Consistently, overexpression of Itch increases cell survival and inhibits caspase 3 activity, whereas downregulation of Itch by RNA interference has the opposite effect, increasing cell death and apoptosis. Treatment with EGF increases Itch phosphorylation and activity, and Itch expression is important for the ability of EGF to increase cell survival after tumour necrosis factor‐related apoptosis‐inducing ligand treatment. Our findings identify Itch as a key molecule between EGF signalling and resistance to apoptosis through downregulation of tBid, providing further details on how EGF receptor and proteasome inhibitors can contribute to the induction of apoptosis and the treatment of cancer.

Adaptation shifts preferred orientation of tuning curve in the mouse visual cortex.

In frontalized mammals it has been demonstrated that adaptation produces shift of the peak of the orientation tuning curve of neuron following frequent or lengthier presentation of a non-preferred stimulus. Depending on the duration of adaptation the shift is attractive (toward the adapter) or repulsive (away from the adapter). Mouse exhibits a salt-and-pepper cortical organization of orientation maps, hence this species may respond differently to adaptation. To examine this question, we determined the effect of twelve minutes of adaptation to one particular orientation on neuronal orientation tuning curves in V1 of anesthetized mice. Multi-unit activity of neurons in V1 was recorded in a conventional fashion. Cells were stimulated with sine-wave drifting gratings whose orientation tilted in steps. Results revealed that similarly to cats and monkeys, majority of cells shifted their optimal orientation in the direction of the adapter while a small proportion exhibited a repulsive shift. Moreover, initially untuned cells showing poor tuning curves reacted to adaptation by displaying sharp orientation selectivity. It seems that modification of the cellular property following adaptation is a general phenomenon observed in all mammals in spite of the different organization pattern of the visual cortex. This study is of pertinence to comprehend the mechanistic pathways of brain plasticity.

The Ubiquitin Ligase Itch and Ubiquitination Regulate BFRF1-Mediated Nuclear Envelope Modification for Epstein-Barr Virus Maturation.

ABSTRACT The cellular endosomal sorting complex required for transport (ESCRT) was recently found to mediate important morphogenesis processes at the nuclear envelope (NE). We previously showed that the Epstein-Barr virus (EBV) BFRF1 protein recruits the ESCRT-associated protein Alix to modulate NE structure and promote EBV nuclear egress. Here, we uncover new cellular factors and mechanisms involved in this process. BFRF1-induced NE vesicles are similar to those observed following EBV reactivation. BFRF1 is ubiquitinated, and elimination of possible ubiquitination by either lysine mutations or fusion of a deubiquitinase hampers NE-derived vesicle formation and virus maturation. While it interacts with multiple Nedd4-like ubiquitin ligases, BFRF1 preferentially binds Itch ligase. We show that Itch associates with Alix and BFRF1 and is required for BFRF1-induced NE vesicle formation. Our data demonstrate that Itch, ubiquitin, and Alix control the BFRF1-mediated modulation of the NE and EBV maturation, uncovering novel regulatory mechanisms of nuclear egress of viral nucleocapsids. IMPORTANCE The nuclear envelope (NE) of eukaryotic cells not only serves as a transverse scaffold for cellular processes, but also as a natural barrier for most DNA viruses that assemble their nucleocapsids in the nucleus. Previously, we showed that the cellular endosomal sorting complex required for transport (ESCRT) machinery is required for the nuclear egress of EBV. Here, we further report the molecular interplay among viral BFRF1, the ESCRT adaptor Alix, and the ubiquitin ligase Itch. We found that BFRF1-induced NE vesicles are similar to those observed following EBV reactivation. The lysine residues and the ubiquitination of BFRF1 regulate the formation of BFRF1-induced NE-derived vesicles and EBV maturation. During the process, a ubiquitin ligase, Itch, preferably associates with BFRF1 and is required for BFRF1-induced NE vesicle formation. Therefore, our data indicate that Itch, ubiquitin, and Alix control the BFRF1-mediated modulation of the NE, suggesting novel regulatory mechanisms for ESCRT-mediated NE modulation.

Cellular LITAF interacts with frog virus 3 75L protein and alters its subcellular localization.

ABSTRACT Iridoviruses are a family of large double-stranded DNA (dsDNA) viruses that are composed of 5 genera, including the Lymphocystivirus, Ranavirus, Megalocytivirus, Iridovirus, and Chloriridovirus genera. The frog virus 3 (FV3) 75L gene is a nonessential gene that is highly conserved throughout the members of the Ranavirus genus but is not found in other iridoviruses. FV3 75L shows high sequence similarity to a conserved domain found in the C terminus of LITAF, a small cellular protein with unknown function. Here we show that FV3 75L localizes to early endosomes, while LITAF localizes to late endosomes/lysosomes. Interestingly, when FV3 75L and LITAF are cotransfected into cells, LITAF can alter the subcellular localization of FV3 75L to late endosomes/lysosomes, where FV3 75L then colocalizes with LITAF. In addition, we demonstrated that virally produced 75L colocalizes with LITAF. We confirmed a physical interaction between LITAF and FV3 75L but found that this interaction was not mediated by two PPXY motifs in the N terminus of LITAF. Mutation of two PPXY motifs in LITAF did not affect the colocalization of LITAF and FV3 75L but did change the location of the two proteins from late endosomes/lysosomes to early endosomes.

Alternative splicing and genomic organization of the L5-67 gene of Aplysia californica.

The L5-67 gene was first identified on the basis of its high expression level in the LUQ neurons, a group of four giant cells located in the left upper quadrant of the abdominal ganglion of Aplysia californica. Its mRNA and peptides were later shown to be present in these cells, as well as in about 100 other smaller neurons in the CNS. L5-67 propeptide and/or mature peptides are also present in peripheral organs, particularly in the kidney, which is the target of most LUQ processes. Using RT-PCR, we show the presence of an alternatively spliced L5-67 transcript arising from the exclusion of the fourth exon from the mature mRNA. This alternative splicing event occurs specifically in the kidney, although we could not identify the cells in which it takes place. Translation of this transcript generates a 52 amino acid (aa) propeptide in which the first N-terminal 45 aa are identical to the original L5-67 propeptide. The last seven C-terminal aa are unrelated to the previously characterized L5-67 peptides due to a change in the open reading frame.

Interactions between nuclear genes and a foreign mitochondrial genome in the redbelly dace Chrosomus eos.

Given the coevolution process occurring between nuclear and mitochondrial genomes, the effects of introgressive hybridization remain puzzling. In this study, we take advantage of the natural co-occurrence of two biotypes bearing a similar nuclear genome (Chrosomus eos) but harbouring mitochondria from different species (wild type: C. eos; cybrids: Chrosomus neogaeus) to determine the extent of phenotype changes linked to divergence in the mitochondrial genome. Changes were assessed through differences in gene expression, enzymatic activity, proteomic and swimming activity. Our data demonstrate that complex IV activity was significantly higher in cybrids compared to wild type. This difference could result from one variable amino acid on the COX3 mitochondrial subunit and/or from a tremendous change in the proteome. We also show that cybrids present a higher swimming performance than wild type. Ultimately, our results demonstrate that the absence of coevolution for a period of almost ten million years between nuclear and mitochondrial genomes does not appear to be necessarily deleterious but could even have beneficial effects. Indeed, the capture of foreign mitochondria could be an efficient way to circumvent the selection process of genomic coevolution, allowing the rapid accumulation of new mutations in C. eos cybrids.

Multiple Src Homology 3 Binding to the Ubiquitin Ligase Itch Conserved Proline-Rich Region

Itch is a member of the C2-WW-HECT (CWH) family of ubiquitin ligases involved in the control of inflammatory signaling pathways, several transcription factors, and sorting of surface receptors to the degradative pathway. In addition to these common domains, Itch also contains a conserved proline-rich region (PRR) allowing its interaction with Src homology 3 (SH3) domain-containing proteins. This region is composed of 20 amino acids and contains one consensus class I and three class II SH3-binding motifs. Several SH3 domain-containing partners have been shown to recognize the Itch PRR, but their binding properties have been poorly defined. Here we compare a subset of endocytic SH3 domain-containing proteins using bioluminescence resonance energy transfer, isothermal titration calorimetry, and pull-down assays. Results indicate that Endophilin is a high-affinity binding partner of Itch both in vivo and in vitro, with a calculated KD placing this complex among the highest-affinity SH3 domain-mediated interactions reported to date. All of the SH3 domains tested here bind to Itch with a 1:1 stoichiometry, except for β-PIX that binds with a 2:1 stoichiometry. Together, these results indicate that Itch PRR is a versatile binding module that can accommodate several different SH3 domain-containing proteins but has a preference for Endophilin. Interestingly, the catalytic activity of Itch toward different SH3 domain-containing proteins was similar, except for β-PIX that was not readily ubiquitylated even though it could interact with an affinity comparable to those of other substrates tested.

Gene products from LUQ neurons in the abdominal ganglion are present at the renal pore of Aplysia californica

The L2-4,6 and L5 cells located in the left upper quadrant of the abdominal ganglion of Aplysia californica express the L5-67 and LUQ-1 genes, respectively, in a nonoverlapping manner. These cells send major neurites to the kidney and at least some of them were shown to innervate the renal pore closer muscle, and thereby control its function. By using in-situ hybridization and immunofluorescence, the presence of L5-67 and LUQ-1 mRNAs and peptides was studied in the kidney, with emphasis on the region of the renal pore. We detected immunoreactive materials in many small varicose nerve fibers running along the central epithelium in the inner parts of the kidney, and in neurites located within a large nerve associated with muscles inside the renal pore. Our observations represent the first direct evidence of the presence of gene products from LUQ cells at the renal pore, suggesting that they may be responsible for mediating LUQ cell signals. Furthermore, mRNAs coding for the L5-67 and LUQ-1 peptides were also found in the nerve structure inside the renal pore. Our report documents a striking example of neuropeptide mRNA targeting nerve terminals that are very distant from their cell bodies.