Many viral, protozoal, and fungal pathogens represent major human and animal health problems due to their great potential of causing infectious diseases. Research on these pathogens has contributed substantially to our current understanding of both microbial virulence determinants and host key factors during infection. Countless studies have also shed light on the molecular mechanisms of host-pathogen interactions that are employed by these microbes. For example, actin cytoskeletal dynamics play critical roles in effective adhesion, host cell entry, and intracellular movements of intruding pathogens. Cortactin is an eminent host cell protein that stimulates actin polymerization and signal transduction, and recently emerged as fundamental player during host-pathogen crosstalk. Here we review the important role of cortactin as major target for various prominent viral, protozoal and fungal pathogens in humans, and its role in human disease development and cancer progression. Most if not all of these important classes of pathogens have been reported to hijack cortactin during infection through mediating up- or downregulation of cortactin mRNA and protein expression as well as signaling. In particular, pathogen-induced changes in tyrosine and serine phosphorylation status of cortactin at its major phospho-sites (Y-421, Y-470, Y-486, S-113, S-298, S-405, and S-418) are addressed. As has been reported for various Gram-negative and Gram-positive bacteria, many pathogenic viruses, protozoa, and fungi also control these regulatory phospho-sites, for example, by activating kinases such as Src, PAK, ERK1/2, and PKD, which are known to phosphorylate cortactin. In addition, the recruitment of cortactin and its interaction partners, like the Arp2/3 complex and F-actin, to the contact sites between pathogens and host cells is highlighted, as this plays an important role in the infection process and internalization of several pathogens. However, there are also other ways in which the pathogens can exploit the function of cortactin for their needs, as the cortactin-mediated regulation of cellular processes is complex and involves numerous different interaction partners. Here, the current state of knowledge is summarized.

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Expression levels of the lactate-H+ cotransporter MCT4 (also known as SLC16A3) and its chaperone CD147 (also known as basigin) are upregulated in breast cancers, correlating with decreased patient survival. Here, we test the hypothesis that MCT4 and CD147 favor breast cancer invasion through interdependent effects on extracellular matrix (ECM) degradation. MCT4 and CD147 expression and membrane localization were found to be strongly reciprocally interdependent in MDA-MB-231 breast cancer cells. Overexpression of MCT4 and/or CD147 increased, and their knockdown decreased, migration, invasion and the degradation of fluorescently labeled gelatin. Overexpression of both proteins led to increases in gelatin degradation and appearance of the matrix metalloproteinase (MMP)-generated collagen-I cleavage product reC1M, and these increases were greater than those observed upon overexpression of each protein alone, suggesting a concerted role in ECM degradation. MCT4 and CD147 colocalized with invadopodia markers at the plasma membrane. They also colocalized with MMP14 and the lysosomal marker LAMP1, as well as partially with the autophagosome marker LC3, in F-actin-decorated intracellular vesicles. We conclude that MCT4 and CD147 reciprocally regulate each other and interdependently support migration and invasiveness of MDA-MB-231 breast cancer cells. Mechanistically, this involves MCT4-CD147-dependent stimulation of ECM degradation and specifically of MMP-mediated collagen-I degradation. We suggest that the MCT4-CD147 complex is co-delivered to invadopodia with MMP14.

Cortactin, a cytoskeletal protein and substrate of src kinase, is implicated in tumor aggressiveness. However, its role in bone cell differentiation remains unknown. The current study revealed that cortactin was upregulated during osteoblast and adipocyte differentiation. Functional experiments demonstrated that cortactin promoted the differentiation of mesenchymal stem/progenitor cells into osteogenic and adipogenic lineages. Mechanistically, cortactin was able to stabilize the protein level of mechanistic target of rapamycin kinase (mTOR), leading to the activation of mTOR signaling. In-depth investigation revealed that cortactin could bind with casitas B lineage lymphoma-c (c-CBL) and counteract the function of c-CBL, a known E3 ubiquitin ligase responsible for the proteasomal degradation of mTOR. Silencing c-Cbl alleviated the impaired differentiation of osteoblasts and adipocytes caused by cortactin siRNA, while silencing mTOR mitigated the stimulation of osteoblast and adipocyte differentiation induced by cortactin overexpression. Notably, transplantation of cortactin-silenced bone marrow stromal cells (BMSCs) into the marrow of mice led to a reduction in trabecular bone mass, accompanied by a decrease in osteoblasts and an increase in osteoclasts. Furthermore, cortactin-silenced BMSCs expressed higher levels of RANKL than control BMSCs did, and promoted osteoclast differentiation when cocultured with bone marrow-derived osteoclast precursor cells. This study provides evidence that cortactin favors osteoblast differentiation by counteracting the c-CBL-induced degradation of mTOR and inhibits osteoclast differentiation by downregulating the expression of RANKL. It also suggests that maintaining an appropriate level of cortactin expression may be advantageous for maintaining bone homeostasis.

Cell polarity is crucial for gastric mucosal barrier integrity and mainly regulated by polarity-regulating kinase partitioning-defective 1b (Par1b). During infection, the carcinogen Helicobacter pylori hijacks Par1b via the bacterial oncoprotein CagA leading to loss of cell polarity, but the precise molecular mechanism is not fully clear. Here we discovered a novel function of the actin-binding protein cortactin in regulating Par1b, which forms a complex with cortactin and the tight junction protein zona occludens-1 (ZO-1). We found that serine phosphorylation at S405/418 and the SH3 domain of cortactin are important for its interaction with both Par1b and ZO-1. Cortactin knockout cells displayed disturbed Par1b cellular localization and exhibited morphological abnormalities that largely compromised transepithelial electrical resistance, epithelial cell polarity, and apical microvilli. H. pylori infection promoted cortactin/Par1b/ZO-1 abnormal interactions in the tight junctions in a CagA-dependent manner. Infection of human gastric organoid-derived mucosoids supported these observations. We therefore hypothesize that CagA disrupts gastric epithelial cell polarity by hijacking cortactin, and thus Par1b and ZO-1, suggesting a new signaling pathway for the development of gastric cancer by Helicobacter.

Nipah virus (NiV) is a highly lethal zoonotic virus with a potential large-scale outbreak, which poses a great threat to world health and security. In order to explore more potential factors associated with NiV, a proximity labeling method was applied to investigate the F, G, and host protein interactions systematically. We screened 1996 and 1524 high-confidence host proteins that interacted with the NiV fusion (F) glycoprotein and attachment (G) glycoprotein in HEK293T cells by proximity labeling technology, and 863 of them interacted with both F and G. The results of GO and KEGG enrichment analysis showed that most of these host proteins were involved in cellular processes, molecular binding, endocytosis, tight junction, and other functions. Cytoscape software (v3.9.1) was used for visual analysis, and the results showed that Cortactin (CTTN), Serpine mRNA binding protein 1 (SERBP1), and stathmin 1 (STMN1) were the top 20 proteins and interacted with F and G, and were selected for further validation. We observed colocalization of F-CTTN, F-SERBP1, F-STMN1, G-CTTN, G-SERBP1, and G-STMN1 using confocal fluorescence microscopy, and the results showed that CTTN, SERBP1, and STMN1 overlapped with NiV F and NiV G in HEK293T cells. Further studies found that CTTN can significantly inhibit the infection of the Nipah pseudovirus (NiVpv) into host cells, while SERBP1 and STMN1 had no significant effect on pseudovirus infection. In addition, CTTN can also inhibit the infection of the Hendra pseudovirus (HeVpv) in 293T cells. In summary, this study revealed that the potential host proteins interacted with NiV F and G and demonstrated that CTTN could inhibit NiVpv and HeVpv infection, providing new evidence and targets for the study of drugs against these diseases.

Neuromuscular junctions transmit signals from the nervous system to skeletal muscles, triggering their contraction, and their proper organization is essential for breathing and voluntary movements. αDystrobrevin-1 is a cytoplasmic component of the dystrophin-glycoprotein complex and has pivotal functions in regulating the integrity of muscle fibers and neuromuscular junctions. Previous studies identified that αDystrobrevin-1 functions in the organization of the neuromuscular junction and that its phosphorylation in the C-terminus is required in this process. Our proteomic screen identified several putative αDystrobrevin-1 interactors recruited to the Y730 site in phosphorylated and unphosphorylated states. Amongst various actin-modulating proteins, we identified the Arp2/3 complex regulator cortactin. We showed that similarly to αDystrobrevin-1, cortactin is strongly enriched at the neuromuscular postsynaptic machinery and obtained results suggesting that these two proteins interact in cell homogenates and at the neuromuscular junctions. Analysis of synaptic morphology in cortactin knockout mice showed abnormalities in the slow-twitching soleus muscle and not in the fast-twitching tibialis anterior. However, muscle strength examination did not reveal apparent deficits in knockout animals.

Colorectal cancer (CRC) ranks as the third most prevalent cancer type globally. Nevertheless, the fundamental mechanisms driving CRC progression remain ambiguous, and the prognosis for the majority of patients diagnosed at an advanced stage is dismal. YWHA/14-3-3 proteins serve as central nodes in several signaling pathways and are closely related to tumorigenesis and progression. However, their exact roles in CRC are still poorly elucidated. In this study, we revealed that YWHAG was the most significantly upregulated member of the YWHA/14-3-3 family in CRC tissues and was associated with a poor prognosis. Subsequent phenotypic experiments showed that YWHAG promoted the proliferation, migration, and invasion of CRC cells. Mechanistically, RNA-seq data showed that multiple signaling pathways, including Wnt and epithelial-mesenchymal transition, were potentially regulated by YWHAG. CTTN was identified as a YWHAG-associated protein, and mediated its tumor-promoting functions by activating the Wnt/β-catenin signaling in CRC cells. In summary, our data indicate that YWHAG facilitates the proliferation, migration, and invasion of CRC cells by modulating the CTTN-Wnt/β-catenin signaling pathway, which offers a novel perspective for the treatment of CRC.

It is a challenge for the traditional affinity methods to capture transient interactions of enzyme-post-translational modification (PTM) substrates in vivo. Herein we presented a strategy termed proximity labeling-based orthogonal trap approach (ProLORT), relying upon APEX2-catalysed proximity labeling and an orthogonal trap pipeline as well as quantitative proteomics to directly investigate the transient interactome of enzyme-PTM substrates in living cells. As a proof of concept, ProLORT allows for robust evaluation of a known HDAC8 substrate, histone H3K9ac. By leveraging this approach, we identified numerous of putative acetylated proteins targeted by HDAC8, and further confirmed CTTN as a bona fide substrate in vivo. Next, we demonstrated that HDAC8 facilitates cell motility via deacetylation of CTTN at lysine 144 that attenuates its interaction with F-actin, expanding the underlying regulatory mechanisms of HDAC8. We developed a general strategy to profile the transient enzyme-substrate interactions mediated by PTMs, providing a powerful tool for identifying the spatiotemporal PTM-network regulated by enzymes in living cells.

The microtubule-associated protein MAP1B has been implicated in axonal growth and brain development. We found that MAP1B is highly expressed in the most aggressive and deadliest breast cancer subtype, triple-negative breast cancer (TNBC), but not in other subtypes. Expression of MAP1B was found to be highly correlated with poor prognosis. Depletion of MAP1B in TNBC cells impairs cell migration and invasion concomitant with a defect in tumorigenesis. We found that MAP1B interacts with key components for invadopodia formation, cortactin, and Tks5, the latter of which is a PtdIns(3,4)P2-binding and scaffold protein that localizes to invadopodia. We also found that Tks5 associates with microtubules and supports the association between MAP1B and α-tubulin. In accordance with their interaction, depletion of MAP1B leads to Tks5 destabilization, leading to its degradation via the autophagic pathway. Collectively, these findings suggest that MAP1B is a convergence point of the cytoskeleton to promote malignancy in TNBC and thereby a potential diagnostic and therapeutic target for TNBC.