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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 Ca 2+ 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 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.
Selected Publications:
Puertollano R. Clathrin-mediated transport: assembly required. EMBO Reports. 2004; 5: 942-946.
Puertollano R. Interactions of TOM1L1 with the multivesicular body sorting machinery. Journal of
Biological Chemistry. 2005; 280: 9258-9264.
Vergarajauregui S. and Puertollano R. Two Di-leucine Motifs Regulate Trafficking of Mucolipin-1
to Lysosomes . 2006. Traffic. 2006; In press.
Questions, comments and suggestions about this page may be addressed to
Rosa Puertollano
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