In the last several years an increasing number of genes associated with different human diseases have been identified. Interestingly, many of these genes have been demonstrated to encode components of the intracellular sorting machinery that mediates the selective trafficking of lipids and proteins in the secretory and endocytic pathways. Two different projects in my laboratory try to address how defects in intracellular trafficking might lead to human diseases: Project 1. Mucolipidosis type IV (MLIV) is an autosomal recessive lysosome storage disorder characterized by severe psychomotor retardation and ophthalmologic abnormalities, including corneal opacity, retinal degeneration, and strabismus. Unlike the situation in other lysosomal disorders, the accumulation of heterogeneous storage material observed in MLIV does not result from the block in the catabolic pathways, but is due to a transport defect in the late steps of endocytosis. MCOLN1, the gene mutated in MLIV patients, encodes a protein called mucolipin-1 that might function as a Ca2+ permeable channel and has been implicated in the biogenesis of lysosomes. To gain information on the mechanisms underlying this pathology we are trying to identify proteins that interact with or regulate the function of mucolipin-1 by using a combination of pull-down, yeast two hybrid screening, and siRNA techniques. In addition we are seeking to identify the sorting motifs that regulate trafficking of mucolipin-1 within the cell. So far we have found that mucolipin-1 can reach lysosomes through both a direct (from Golgi to lysosomes) and an indirect (through the plasma membrane) route. The direct route appears to be dependent on a dileucine motif located at the N-terminal cytosolic tail of the protein that mediates interaction with the clathrin adaptors AP-1 and AP-3. In contrast, the indirect pathway is dependent on an internalization motif positioned at the end of the mucolipin-1 C-terminal cytosolic tail. This sequence binds AP-2 and promotes the endocytosis of the protein from the plasma membrane through clathrin-coated vesicles. Interestingly, palmitoylation of three cysteine residues seems to regulate the efficiency of mucolipin-1 internalization. Project 2. Growth factors and their transmembrane receptor tyrosine kinases (RTK) play important roles during embryonic development and in the regulation of several cellular processes including proliferation, survival, migration and differentiation. Binding of growth factors to their receptors activates a myriad of signaling pathways that permit cells to respond to changes in the environment. In many cases the termination of these signaling events is mediated by receptor internalization and degradation. Defects in receptor down-regulation might lead to sustained signaling and transformation. The epithelial growth factor receptor (EGFR) is considered the prototypal member of the RTK family and its activation by EGF and trafficking has been exhaustively characterized. However, there are still some aspects that remain to be addressed in more detail. One of these aspects is the role played by kinases in the regulation of EGFR trafficking. Our results show that activation of p38 MAP kinase by anisomycin is sufficient to induce internalization of EGFR. Anisomycin and EGF employ different mechanisms to promote EGFR endocytosis as anisomycin-induced internalization does not require tyrosine kinase activity or ubiquitination of the receptor. Incubation with a specific inhibitor of p38, or depletion of endogenous p38 by small interfering RNA (siRNA), abolished anisomycin-induced internalization of EGFR while having no effect on transferrin endocytosis. Interestingly, inhibition of p38 activation also abolished endocytosis of EGFR induced by UV radiation. These results suggest that stimulation of EGFR internalization by p38 might represent a general mechanism to prevent generation of proliferative or anti-apoptotic signals under stress conditions. While further studies will be required to assess the specific mechanisms used by p38 to regulate EGFR internalization, our results strengthen the idea that there is a clear intercommunication between signaling and intracellular traffic and suggest that p38 is a key player in this process. Another protein that might coordinate the connection between trafficking and signaling is Tom-like1 (Tom1L1). Tom1L1, and related proteins Tom1 (Target of Myb1) and Tom1L2 (Tom1-like2), constitute a new family of proteins that are characterized by the presence of a VHS (Vps27p/Hrs/Stam) domain in the N-terminal portion followed by a GAT (GGA and Tom) domain. Recently it was demonstrated that the GAT domain of both Tom1 and Tom1L1 bind ubiquitin, suggesting that these proteins might participate in the sorting of ubiquitinated proteins into multivesicular bodies (MVBs). We have identified a novel interaction between Tom1L1 and members of the MVB sorting machinery. Specifically, we found that the VHS domain of Tom1L1 interacts with Hrs (Hepatocyte growth factor?regulated tyrosine kinase substrate), while a PTAP motif, located between the VHS and GAT domain of Tom1L1, is responsible for the binding to TSG101 (tumor susceptibility gene 101). Myc-epitope tagged Tom1L1 showed a cytosolic distribution but was recruited to endosomes following Hrs expression. In addition, Tom1L1 possesses several tyrosine motifs at the C-terminal region that mediate interactions with members of the Src family kinases and other signaling proteins such as Grb2 and p85. We showed that a fraction of Fyn kinase localizes at endosomes and that this distribution becomes more evident after EGF internalization. Moreover, expression of a constitutive active form of Fyn also promoted the recruitment of Tom1L1 to enlarged endosomes. Taken together we propose that Tom1L1 could act as an intermediary between signaling and degradative pathways.
