Regulation of Catalytic and Non-catalytic Functions of the Drosophila Ste20 Kinase Slik by Activation Segment Phosphorylation.

Protein kinases carry out important functions in cells both by phosphorylating substrates and by means of regulated non-catalytic activities. Such non-catalytic functions have been ascribed to many kinases, including some members of the Ste20 family. The Drosophila Ste20 kinase Slik phosphorylates and activates Moesin in developing epithelial tissues to promote epithelial tissue integrity. It also functions non-catalytically to promote epithelial cell proliferation and tissue growth. We carried out a structure-function analysis to determine how these two distinct activities of Slik are controlled. We find that the conserved C-terminal coiled-coil domain of Slik, which is necessary and sufficient for apical localization of the kinase in epithelial cells, is not required for Moesin phosphorylation but is critical for the growth-promoting function of Slik. Slik is auto- and trans-phosphorylated in vivo. Phosphorylation of at least two of three conserved sites in the activation segment is required for both efficient catalytic activity and non-catalytic signaling. Slik function is thus dependent upon proper localization of the kinase via the C-terminal coiled-coil domain and activation via activation segment phosphorylation, which enhances both phosphorylation of substrates like Moesin and engagement of effectors of its non-catalytic growth-promoting activity.

Rac-specific guanine nucleotide exchange factor DOCK1 is a critical regulator of HER2-mediated breast cancer metastasis.

Progression of solid tumors to the metastatic stage is accountable for the majority of cancer-related deaths. Further understanding of the molecular mechanisms governing metastasis is essential for the development of antimetastatic regimens. Here, we aimed to identify Rac activators that could promote metastasis downstream of human epithelial growth factor receptor 2 (HER2). We investigated if Dedicator of Cytokinesis 1 (DOCK1), based on its evolutionarily conserved role in receptor tyrosine kinases (RTKs)-mediated Rac activation and cell invasion, could be a regulator of metastasis. We report that high expression of DOCK1 in HER2(+) and basal breast cancer subtypes inversely correlates with human patients' survival. Mechanistically, DOCK1 interacts with HER2 and promotes HER2-induced Rac activation and cell migration. To gain further insight, we developed a HER2 breast cancer mouse model with mammary-gland-specific inactivation of DOCK1. In this in vivo model, a significant decrease in tumor growth and metastasis in lungs was found in animals where DOCK1 is inactivated. Furthermore, we found that DOCK1 is required for maximal activation of two HER2 effectors, c-JUN and STAT3. Using an unbiased gene profiling approach, we identified a mammary tumor DOCK1-associated gene signature enriched for genes implicated in response to IFN type I. This analysis revealed a unique set of genes, including Receptor Transporter Protein 4 (RTP4) and STAT1, for which the expression levels can be used to independently predict breast cancer outcome in HER2(+) patients. Our work demonstrates DOCK1-Rac signaling as an HER2 effector pathway essential for HER2-mediated breast cancer progression to metastasis and offers a therapeutic opportunity to limit the spread of metastatic breast cancers.

Microbial contents of vacuum cleaner bag dust and emitted bioaerosols and their implications for human exposure indoors.

Vacuum cleaners can release large concentrations of particles, both in their exhaust air and from resuspension of settled dust. However, the size, variability, and microbial diversity of these emissions are unknown, despite evidence to suggest they may contribute to allergic responses and infection transmission indoors. This study aimed to evaluate bioaerosol emission from various vacuum cleaners. We sampled the air in an experimental flow tunnel where vacuum cleaners were run, and their airborne emissions were sampled with closed-face cassettes. Dust samples were also collected from the dust bag. Total bacteria, total archaea, Penicillium/Aspergillus, and total Clostridium cluster 1 were quantified with specific quantitative PCR protocols, and emission rates were calculated. Clostridium botulinum and antibiotic resistance genes were detected in each sample using endpoint PCR. Bacterial diversity was also analyzed using denaturing gradient gel electrophoresis (DGGE), image analysis, and band sequencing. We demonstrated that emission of bacteria and molds (Penicillium/Aspergillus) can reach values as high as 1E5 cell equivalents/min and that those emissions are not related to each other. The bag dust bacterial and mold content was also consistent across the vacuums we assessed, reaching up to 1E7 bacterial or mold cell equivalents/g. Antibiotic resistance genes were detected in several samples. No archaea or C. botulinum was detected in any air samples. Diversity analyses showed that most bacteria are from human sources, in keeping with other recent results. These results highlight the potential capability of vacuum cleaners to disseminate appreciable quantities of molds and human-associated bacteria indoors and their role as a source of exposure to bioaerosols.

Spatiotemporal regulation of the GPCR activity of BAI3 by C1qL4 and Stabilin-2 controls myoblast fusion.

Myoblast fusion is tightly regulated during development and regeneration of muscle fibers. BAI3 is a receptor that orchestrates myoblast fusion via Elmo/Dock1 signaling, but the mechanisms regulating its activity remain elusive. Here we report that mice lacking BAI3 display small muscle fibers and inefficient muscle regeneration after cardiotoxin-induced injury. We describe two proteins that repress or activate BAI3 in muscle progenitors. We find that the secreted C1q-like1-4 proteins repress fusion by specifically interacting with BAI3. Using a proteomic approach, we identify Stabilin-2 as a protein that interacts with BAI3 and stimulates its fusion promoting activity. We demonstrate that Stabilin-2 activates the GPCR activity of BAI3. The resulting activated heterotrimeric G-proteins contribute to the initial recruitment of Elmo proteins to the membrane, which are then stabilized on BAI3 through a direct interaction. Collectively, our results demonstrate that the activity of BAI3 is spatiotemporally regulated by C1qL4 and Stabilin-2 during myoblast fusion.

The Receptor Tyrosine Kinase AXL Is Required at Multiple Steps of the Metastatic Cascade during HER2-Positive Breast Cancer Progression.

AXL is activated by its ligand GAS6 and is expressed in triple-negative breast cancer cells. In the current study, we report AXL expression in HER2-positive (HER2+) breast cancers where it correlates with poor patient survival. Using murine models of HER2+ breast cancer, Axl, but not its ligand Gas6, was found to be essential for metastasis. We determined that AXL is required for intravasation, extravasation, and growth at the metastatic site. We found that AXL is expressed in HER2+ cancers displaying epithelial-to-mesenchymal transition (EMT) signatures where it contributes to sustain EMT. Interfering with AXL in a patient-derived xenograft (PDX) impaired transforming growth factor ß (TGF-ß)-induced cell invasion. Last, pharmacological inhibition of AXL specifically decreased the metastatic burden of mice developing HER2+ breast cancer. Our data identify AXL as a potential anti-metastatic co-therapeutic target for the treatment of HER2+ breast cancers.

Opening up on ELMO regulation: New insights into the control of Rac signaling by the DOCK180/ELMO complex.

GTPases are central hubs for directing cytoskeletal reorganization and cell migration. The DOCK family enforces positive regulation of certain GTPases, leading to their activation in discrete areas of the cell. ELMO, a well-known DOCK180 binding partner, has been cast with the role of potentiating DOCK180-mediated Rac activation. Exactly how ELMO contributes to Rac signaling is only beginning to be understood. Here, we discuss our most recent research investigating ELMO regulation of the DOCK180/Rac pathway. Interestingly, we found that ELMO is autoinhibited via intramolecular contacts at basal levels and we explore the novel domains that we identified at the heart of the auto-regulatory switch. We propose that the closed ELMO molecule masks protein-protein interfaces or domains with novel uncharacterized function; cell stimulation and GTPase binding to ELMO is proposed to activate (open) the protein and/or target the ELMO/DOCK180 complex to the cell membrane. In this manner, promiscuous signaling/activity downstream of ELMO/DOCK180 can be controlled for both spatially and temporally. Additionally, we report new data highlighting that DOCK proteins can form heterodimers, and we discuss possible mechanisms that could be implicated in controlling the ELMO activation state.

An alpha-helical extension of the ELMO1 pleckstrin homology domain mediates direct interaction to DOCK180 and is critical in Rac signaling.

The mammalian DOCK180 protein belongs to an evolutionarily conserved protein family, which together with ELMO proteins, is essential for activation of Rac GTPase-dependent biological processes. Here, we have analyzed the DOCK180-ELMO1 interaction, and map direct interaction interfaces to the N-terminal 200 amino acids of DOCK180, and to the C-terminal 200 amino acids of ELMO1, comprising the ELMO1 PH domain. Structural and biochemical analysis of this PH domain reveals that it is incapable of phospholipid binding, but instead structurally resembles FERM domains. Moreover, the structure revealed an N-terminal amphiphatic alpha-helix, and point mutants of invariant hydrophobic residues in this helix disrupt ELMO1-DOCK180 complex formation. A secondary interaction between ELMO1 and DOCK180 is conferred by the DOCK180 SH3 domain and proline-rich motifs at the ELMO1 C-terminus. Mutation of both DOCK180-interaction sites on ELMO1 is required to disrupt the DOCK180-ELMO1 complex. Significantly, although this does not affect DOCK180 GEF activity toward Rac in vivo, Rac signaling is impaired, implying additional roles for ELMO in mediating intracellular Rac signaling.