Browsing by Subject "Crk"

Adherens junctions are calcium-dependent cell-cell contacts formed during epithelial morphogenesis that link neighboring cells via cadherin receptors. Coordinated regulation of the actin cytoskeleton by the Rho GTPases is required for the formation and dissolution of adherens junctions, however the pathways that link cadherin signaling to cytoskeletal regulation remain poorly defined. The Abl family of tyrosine kinases have been shown to modulate cytoskeletal reorganization downstream of various extracellular signals including growth factor receptors and integrins.

Here we use pharmacological inhibition and RNA interference to identify the Abl family kinases as critical mediators of cadherin-mediated adhesion. Endogenous Abl family kinases, Abl and Arg, are activated and are required for Rac activation following cadherin engagement, and regulate the formation and maintenance of adherens junctions in mammalian cells. Significantly, we show that Abl-dependent regulation of the Rho-ROCK-myosin signaling pathway is critical for the maintenance of adherens junctions. Inhibition of the Abl kinases in epithelial sheets results in activation of Rho and its downstream target ROCK, leading to enhanced phosphorylation of the myosin regulatory light chain. These signaling events result in enhanced stress fiber formation and increased acto-myosin contractility, thereby disrupting adherens junctions. Conversely, Arg gain-of-function promotes adherens junction formation through a Crk-dependent pathway in cells with weak junctions. These data identify the Abl kinases as a novel regulatory link between the cadherin/catenin adhesion complex and the actin cytoskeleton through regulation of Rac and Rho during adherens junction formation.

Unexpectedly, we identified a requirement for Abl and Crk downstream of Rac in the regulation of adherens junctions. Therefore, Abl functions both upstream and downstream of Rac in regulating adherens junctions, which suggests the possibility of a positive feedback loop consisting of Abl-Crk-Rac.

Finally, we identified the Abl kinases as critical mediators of epithelial cell response to HGF. Pharmacological inhibition of Abl kinase activity resulted in impaired dissolution of adherens junctions downstream of HGF stimulation of the Met receptor. Additionally, we observed decreased phosphorylation of the Met receptor itself, along with Gab1 and Crk, downstream effectors of Met signaling. Taken together, these data suggest a requirement for Abl kinases in both adherens junctions formation and turnover.

Desmosomes are a class of cell-cell adhesions whose primary physiological function is to maintain the mechanical integrity of tissues. Morphologically, desmosomes are very distinct protein complexes. They are dense pairs of plaques that straddle the interface between two plasma membranes of adjacent cells. In the extracellular space between each plaque are series of desmosome receptors that mediate the cell-cell attachment events and drive cellular cohesion throughout tissues. Emanating from the intracellular face of desmosomes are networks of intermediate filaments to which desmosome plaques are anchored. It is through this molecular chain that desmosomes traditionally bear mechanical load and promote tissue integrity. Molecular roles of the core components sufficient for completing this chain have been well described. However, insights from desmosome pathologies, genetic interactions, and biochemical fractionation have introduced perspectives that strongly suggest that desmosome composition and function extends beyond what is currently known. It is within this general context that I began my exploration into desmosome biology and employed global approaches towards uncovering a novel proteome that revealed unexpected interactions important for tissue function.

First, we used targeted proximity labeling approaches to elaborate the desmosome proteome in epidermal keratinocytes. Quantitative mass spectrometry analysis uncovered a diverse array of new constituents with broad molecular functions. We validated a number of novel desmosome-associated proteins and found that many are membrane proximal proteins that show a dependence on functional desmosomes for their cortical localization. We further explored the mechanism of localization and function of two adaptor proteins enriched in the desmosome proteome, Crk and CrkL. Epidermal deletion of both Crk and CrkL resulted in perinatal lethality with defects in desmosome morphology and keratin organization, thus demonstrating the utility of this dataset in identifying novel proteins required for desmosome-dependent epidermal integrity.

Desmosomes role in mechanical integrity is paramount in the epidermis as skin-blistering pathologies represent one of the primary hallmarks of diseases caused by desmosome disruption. Pemphigus Vulgaris (PV) is a series of autoimmune syndromes where patients produce autoantibodies that target desmosome receptors, desmoglein 3 and desmoglein 1. PV is characterized by weakening cell adhesions that result in widespread epidermal cell separations. While pathologies that characterize PV have been long documented, molecular mechanisms that drive initial desmosome disruption are still not fully understood. We sought to address initial molecular signaling events induced by PV antibodies using targeted proteomic analysis to identify early phosphorylation events. We performed time course phosphoproteomic analysis on keratinocytes early after exposure to PV antibody, AK23. With this approach we observed large coverage of the keratinocyte phosphoproteome that included significant shifts of abundant phosphorylation events after AK23 treatment. To identify the functional relevance of novel phosphorylation events, we followed the localization of constructs containing tagged protein candidates harboring phosphomimetic or non-phosphorylatable mutations. Altogether, these results provide an initial analysis of global phosphorylation events that occur in keratinocytes during the early stages of PV.

Finally, we sought to understand what was an unusual enrichment of RNA-binding proteins in our desmosome proteome. Amongst enriched protein classes were regulators of protein translation. Using immunofluorescent approaches, we were able to visualize distinct desmosome localization of representative members of protein translation machinery as well as assembled ribosomes. Considering this unusual abundance, I was interested in finding out the types of RNAs associated with these desmosome-associated RBPs. To do this we combined our proteomic approach with RNA sequencing and developed new techniques to enrich for specific RNA subpopulations localized at desmosomes. By probing for candidates of interest from this list I was able to observe enrichment of specific transcripts at desmosomes, suggesting an intimate link between RNA regulation and cell-cell adhesion. Taken together, these studies represent the first comprehensive proteomic analyses of desmosome adhesions and highlight novel desmosome interactions that may broaden mechanistic roles for desmosomes in epithelial biology.

Chlamydia trachomatis is a bacterial pathogen with a large socioeconomic impact: it is the leading cause of preventable blindness worldwide, and the most prevalent sexually transmitted infection in the United States. Despite its importance, relatively little is known about the molecular mechanisms that Chlamydia employs to invade epithelial cells, manipulate the secretory pathway, evade innate immune responses and acquire nutrients from its host. Chlamydia, like many other intracellular pathogens, is known to use a type III secretion mechanism to deliver bacterial effector proteins directly to the host cell cytoplasm. These effectors are thought to be the principle actors involved in co-opting host cell functions. TepP is an effector protein that is pre-loaded into infectious Chlamydia particles, and that is secreted early during infection, but whose function is unknown. We took large-scale, unbiased approaches to identify genes whose transcription is modified during the course of infection in a TepP-dependent manner (microarrays), and proteins that interact with TepP and and/or whose phosphorylation is altered by the absence of TepP (proteomics). We used biochemical techniques, cell biology, and molecular techniques to validate interactions identified using large-scale methods, and to further probe the molecular mechanism underlying these connections. In sum, we have determined that TepP contributes to four major phenotypes: changes in the host cell cytoskeleton, modification of the host cell phosphoproteome, bacterial replication, and interferon-dependent gene activation. We have additionally determined that TepP interacts with the Crk family of host cell adaptor proteins, and the class 1 phosphoinositol-3-kinase (PI3K). Cell lines where the levels or activity of TepP interacting partners were modified by deletion, knockdown, or inhibitors, showed that these host proteins are important for the growth of Chlamydia during infection, but are not required for all TepP-dependent phenotypes. TepP not only interacts with PI3K but also induces its activation during infection. Finally, we have determined that the requirements for phosphorylation of TepP are complex, but that the Src kinases are largely responsible for its phosphorylation. Additionally, Src kinases are required for some TepP-dependent phenotypes, but are not required for the recruitment of TepP-interacting proteins during infection.