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Summary:

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 in part 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 the signals’ 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. We are focusing on this particularly intriguing phenomenon.

As our studies have shown, the endocytic adaptor proteins APPL1 and APPL2 are examples of endosomes playing a role in signaling and, in parallel, endocytic proteins can be active in the cell nucleus. Through their interactions with a small GTPase Rab5, APPL proteins primarily localize to a subpopulation of endosomes that appear segregated from the well characterized canonical early endosomes and participate in signaling events. However, both APPL proteins can also undergo nucleocytoplasmic shuttling and reside in the cell nucleus. To elucidate their function in this compartment, we identified and characterized interactions of APPL proteins with other binding partners.

APPL proteins were found to bind to the nuclear co-repressor complex NuRD, which contains nucleosome-remodeling and histone deacetylase activities. We characterized the binding between APPL1 and NuRD in more detail, identifying histone deacetylase 2 (HDAC2) as the key NuRD subunit responsible for this association. APPL1 interacts with the NuRD complex, which contains enzymatically active HDAC2, but not HDAC1, as the only deacetylase, even though the cellular levels of HDAC1 can modulate the extent of APPL1-NuRD interactions. Importantly, binding to the NuRD complex regulates the nucleocytoplasmic distribution of APPL1, which has no classical nuclear localization signal and must be transported to the nucleus in association with other partners. We demonstrated that increased binding of APPL1 to NuRD upon silencing of HDAC1 promotes the nuclear localization of APPL1, while HDAC1 overexpression exerts the opposite effect.

We also uncovered a NuRD-independent interaction of HDAC1 with APPL proteins, which appeared to be related to their function as activators of β-catenin/TCF-mediated transcription in canonical Wnt signaling. We found that both APPL proteins interact directly with Reptin, a transcriptional repressor that binds to HDAC1 and β-catenin. As a result, APPL proteins are present in an endogenous complex containing Reptin, β-catenin, HDAC1, and HDAC2. We demonstrated that overexpression of APPL1 or APPL2 protein stimulates the activity of a β-catenin/TCF-dependent reporter construct whereas silencing of APPL1 reduces the activity. Increased levels of either APPL protein are able to relieve Reptin-dependent transcriptional repression and correlate with the reduced amounts of HDACs and β-catenin associated with Reptin, as well as with the lower levels of Reptin and HDAC1 on the promoters of β-catenin target genes. We propose that APPL proteins exert their stimulatory effects on β-catenin/TCF-dependent transcription by decreasing the activity of a Reptin-containing repressive complex. These data may point to a link between Rab5-dependent endocytic processes and canonical Wnt signaling and provide an interesting example of the involvement of endocytic proteins in transcriptional control. We are currently extending our studies to other proteins implicated in endocytosis and capable of nucleocytoplasmic shuttling.

Last updated: February 2010