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Signaling from Endosomes to the Nucleus: The Role of APPL Proteins and Their Interacting Partners
Summary: Marta Miaczynska is interested in the integration of endocytic membrane transport with intracellular signal transduction. She focuses on the mechanisms underlying the signaling from endosomal compartments to the cell nucleus.
Signal transduction in response to extracellular stimuli and membrane transport between intracellular compartments represent two important aspects of basic cell physiology. Although both have been studied for a number of years, we have only recently begun to appreciate the extensive interdependence and coordinated regulation of these processes. Intracellular compartmentalization of signal transduction processes is the major interest of our research. We focus on the role of endocytic organelles in regulating intracellular signaling, in particular how signals from endosomes may be transmitted to the nucleus, leading to changes in gene expression. Signaling molecules such as growth factors initiate cellular responses by ligand-dependent activation of specific receptors at the plasma membrane, which in turn triggers a series of protein-protein interactions and phosphorylation events executed by downstream kinase cascades. The ultimate step in the process, activation of transcription factors in the nucleus, leads to the reprogramming of gene expression, which represents an adaptive response of a cell to the physiological cue. Traditionally, it was thought that signal transduction between the plasma membrane and the nucleus required no intracellular organelles; instead, it was believed that signal transduction occurred via cytoplasmic diffusion.
After ligands initiate signaling, ligand-receptor complexes formed at the plasma membrane are cleared from the cell surface by endocytic internalization. Endocytosis enables uptake of extracellular molecules and plasma membrane proteins in vesicles that pinch off from the cell surface and are transported intracellularly between various endosomal compartments (such as early, late, or recycling endosomes), leading to cargo degradation in lysosomes or recycling back to the plasma membrane. By directing ligand-receptor complexes for lysosomal degradation, endocytosis has long been recognized as a mechanism for terminating signals initiated at the plasma membrane. Strikingly, recent evidence from a number of groups indicates that endocytosis may also play an important active role in the propagation of signals. First, the mode of receptor internalization (such as clathrin- or caveolae-dependent endocytosis) can determine different signaling outputs. Second, even after their internalization, activated ligand-receptor complexes continue to emit signals during their transport between various endosomal compartments. Third, some downstream cascades are preferentially activated only after receptor internalization, and signaling complexes assembled on endosomes are to some extent distinct from those formed at the plasma membrane. Fourth, the concept of 'signaling endosomes' has been particularly well documented in neurons, where signals from axon terminals must travel long distances to the cell body and where cytoplasmic diffusion is insufficient for transmission. Overall, it appears that the endocytic transport of signaling molecules can actively influence a cellular response to an extracellular stimulus; conversely, signal transduction cascades can modulate endocytic trafficking. These data indicate that, as an important parameter determining the ultimate cellular response, signaling events have to be analyzed in the context of their intracellular localization. Moreover, endosomes can be considered intracellular platforms for active signal propagation, contributing to a precise spatial and temporal control of cellular responses.
The relaying of signals from the plasma membrane via endosomes to the nucleus requires signal mediators to be transported between different cellular locations. A growing number of studies indicate that some clathrin adaptors and endosomal proteins can undergo nucleocytoplasmic shuttling. This process is often based on intrinsic sequence motifs and requires active transport mechanisms. Endocytic proteins can associate with nuclear molecules, changing their localization and activity, and may modulate the levels and specificity of gene transcription.
While in the group of Marino Zerial at the Max Planck Institute of Molecular Cell Biology and Genetics in Dresden, I carried out studies on APPL proteins that provided important evidence for the role of endosomal constituents in the regulation of nuclear signal transduction. APPL1 and APPL2 proteins localize to a subpopulation of endosomes that appear segregated from the well-characterized canonical early endosomes. Both proteins can also reside in the cell nucleus, and APPL1 has been shown to undergo nucleocytoplasmic shuttling in response to epidermal growth factor or oxidative stress. Both APPL proteins are required for cell proliferation. Strikingly, they interact on endosomes with the small GTPase Rab5, an endocytosis regulator, and in the nucleus with NuRD/MeCP1, a histone deacetylase complex involved in chromatin remodeling. Thus, APPL proteins appear to act as signal transducers functioning both on endosomes and in the nucleus, possibly serving as a direct molecular link between the processes of endocytosis and chromatin remodeling.
Identification of a novel pathway involving an APPL-positive endosomal compartment as an intermediate in signaling between the plasma membrane and the nucleus raised several new and fascinating questions. The long-term objective of our studies is to understand molecular mechanisms underlying the processes of signaling from endosomes to the nucleus. To this end, we are characterizing both the nuclear and endosomal pools of APPL proteins, using several approaches: subcellular fractionation, biochemical assays of protein-protein interactions, microscopy studies of intracellular distribution, and transcriptional activity analyses of APPL1/2 and their interacting proteins.
To define the functions of APPL proteins in the nucleus, we are characterizing further the proteins' interactions with nuclear partners such as the NuRD complex. We also intend to clarify the intracellular topology of such interactions by analyzing the distribution and trafficking of APPL1 and its nuclear binding partners using microscopy techniques. We are also attempting to determine the molecular identity of the endosomal compartment occupied by APPL proteins, both in terms of its biochemical composition and the cargo trafficking routes connecting it to other endosomal membranes. The compartment's physical segregation from the canonical early endosomes may indicate some specialized functions, likely related to signal transduction. Finally, we would also like to understand the mechanisms responsible for cellular APPL1 shuttling and the roles of various intracellular pools of APPL1 (endosomal, cytoplasmic, and nuclear). For this purpose, we are searching for compartment-specific determinants (interacting partners and/or posttranslational modifications) that localize APPL1 to various organelles. We wish to clarify to what extent these pools are interconnected or interchangeable and which is related to APPL function in the regulation of cell proliferation. In the long term, such studies should lead to conceptual advances in our understanding of how intracellular compartmentalization affects signaling processes and how molecular communication between endosomes and the nucleus is achieved.
Last updated September 2008
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