Laboratory of Cell Biology: Miaczynska Laboratory
Description of Current Research
We study the ways in which intracellular signal transduction and membrane trafficking in endocytosis are integrated at the molecular level. We focus on proteins that have well known roles in endocytosis to investigate their involvement in various signaling pathways and their impact on patterns of gene expression. Initially, our efforts were focused on studying the endosomal and nuclear roles of APPL proteins. In recent years, we broadened our interests to other multifunctional proteins that act in endocytosis and signaling. The specific projects that are developed by our group seek to answer the following questions:
- What is the role of endosomal compartments in the trafficking and signaling of growth factors and cytokines?
- How does the endocytic trafficking of receptors impinge on the patterns of gene expression in different signaling pathways?
Endocytosis was first viewed simply as a mechanism of signal termination through the downregulation and degradation of surface receptors. However, more recent data indicate that endosomal compartments and their resident proteins play an important role in transmitting intracellular signals by transporting ligand-receptor complexes and affecting their activity inside the cell (Hupalowska and Miaczynska, Traffic, 2012; Miaczynska, Cold Spring Harb Perspect Biol, 2013; Miaczynska and Bar-Sagi, Curr Opin Cell Biol, 2010; Sadowski et al., Exp Cell Res, 2009). Moreover, several endocytic proteins can undergo nucleocytoplasmic shuttling and interact with nuclear molecules that are involved in transcription or chromatin remodeling, changing their localization or activity, and thus may directly modulate the levels or specificity of gene transcription (Pilecka et al., Eur J Cell Biol, 2007). Importantly, some such dual-function endocytic and nuclear proteins affect cell proliferation or act as tumor suppressors, or their expression changes in human cancers (Pyrzyńska et al., Mol Oncol, 2009).
To systematically study the possible mechanisms by which endocytic proteins may contribute to transcriptional regulation, we recently established and performed small-scale, targeted RNAi screens. We sought to identify the endocytic proteins that affect transcriptional responses in selected signaling pathways, such as those that activate TCF/LEF, AP-1, NF-κB, and STAT transcription factors. All of these pathways can be induced by extracellular ligands that bind appropriate plasma membrane receptors that undergo internalization, but the way in which endocytosis affects the ultimate signaling responses remains poorly investigated and controversial. Luciferase-based reporter tests were used as a primary screening assay to measure transcription that depends on the chosen factors upon knockdown of the genes that encode endocytic proteins. The screens led to the identification of candidate regulators that function as activators or inhibitors of a given pathway. After initial validation, we delineated the molecular mechanisms of action of newly identified regulators. We were using cultured mammalian cells as our main model but have also introduced zebrafish embryos as an additional experimental model in some projects.
In 2015, we completed three projects based on the results of the aforementioned RNAi screens and characterized novel regulators of Wnt, AP-1, and NF-B signaling. In the first of these projects, we characterized an endocytic adaptor protein, Tollip, as a novel, evolutionarily conserved inhibitor of canonical Wnt signaling (Toruń et al., PLoS One, 2015). We found that Tollip depletion potentiated the activity of the β-catenin/TCF-dependent transcriptional reporter, whereas its overproduction inhibited reporter activity and the expression of Wnt target genes. These effects were independent of dynamin-mediated endocytosis but required the ubiquitin-binding CUE domain of Tollip. In Wnt-stimulated cells, Tollip counteracted the activation of β-catenin and its nuclear accumulation, without affecting its total levels. Additionally, under conditions of ligand-independent signaling, Tollip inhibited pathway activity after the stage of β-catenin stabilization. We also demonstrated that the regulation of Wnt signaling by Tollip occurred during early the embryonic development of zebrafish. Our results indicate that the function of Tollip in inhibiting the canonical Wnt pathway may contribute to both embryonic development and carcinogenesis.
In the second project, we identified a link between the GTPase activity of dynamin 2 (Dyn2), a major regulator of endocytic internalization, and the activation of AP-1 transcription factors, composed of Jun and Fos proteins (Szymańska et al., Cell Signal, 2016). We showed that the expression of a dominant-negative Dyn2 K44A mutant strongly stimulated the AP-1 pathway, increasing the total levels of c-Jun, its phosphorylation on Ser63/73, and the transcription of AP-1 target genes. Importantly, DNM2 mutations that are implicated in human neurological disorders exerted similar effects on AP-1 signaling. We further found that Dyn2 K44A induced AP-1 by increasing the phosphorylation of several receptor tyrosine kinases. Their activation was required to initiate a Src- and JNK-dependent signaling cascade that converged on c-Jun and stimulated the expression of AP-1 target genes. Our data uncovered a connection between Dyn2 function and JNK signaling that leads to the induction of AP-1.
In the third project, we identified four components of endosomal sorting complexes required for transport (ESCRTs) as novel inhibitors of NF-κB signaling (Mamińska et al., Sci Signal, 2016). We found that the depletion of Tsg101, Vps28, UBAP1, and CHMP4B in the absence of cytokine stimulation potently activated both canonical and noncanonical NF-κB signaling. This led to upregulation of the expression of NF-κB target genes in cultured human cells, zebrafish embryos, and fat bodies in flies. These effects depended on cytokine receptors, such as the lymphotoxin β receptor (LTβR) and tumor necrosis factor receptor 1 (TNFR1). Upon the depletion of ESCRT subunits, both receptors became concentrated on and signaled from endosomes. The endosomal accumulation of LTβR induced its ligand-independent oligomerization and signaling through TRAF2 and TRAF3 adaptor proteins. We propose that ESCRTs constitutively control the distribution of cytokine receptors in their ligand-free state to restrict their signaling (Fig. 1). This may represent a general mechanism to prevent the spurious activation of NF-κB and uncontrolled inflammatory signaling.
Fig. 1. Model of ESCRT function in restricting NF-κB signaling. (A) In the basal state, an unliganded cytokine receptor, such as LTβR, is constitutively internalized, reaches early endosomes, and is sorted to the MVB by ESCRTs for further degradation in lysosomes. The NF-κB transcriptional complex remains inactive in the cytoplasm associated with the IκB inhibitor. (B) Under conditions of the depletion of ESCRT subunits, unliganded LTβR internalizes and reaches early endosomes. The lack of ESCRT-I and CHMP4B activity prevents maturation of the MVB and sorting of the receptor to the ILVs. LTβR accumulates at the limiting membrane of endosomes and oligomerizes, inducing NF-κB signaling. As a result, the NF-κB complex translocates to the nucleus and activates the expression of its target genes. PM, plasma membrane; EE, early endosome; MVB, multivesicular body; ILV, intraluminal vesicle; LE, late endosome. Author: Agnieszka Mamińska