Actionable Cytopathogenic Host Responses of Human Alveolar Type 2 Cells to SARS-CoV-2.

Human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative pathogen of the COVID-19 pandemic, exerts a massive health and socioeconomic crisis. The virus infects alveolar epithelial type 2 cells (AT2s), leading to lung injury and impaired gas exchange, but the mechanisms driving infection and pathology are unclear. We performed a quantitative phosphoproteomic survey of induced pluripotent stem cell-derived AT2s (iAT2s) infected with SARS-CoV-2 at air-liquid interface (ALI). Time course analysis revealed rapid remodeling of diverse host systems, including signaling, RNA processing, translation, metabolism, nuclear integrity, protein trafficking, and cytoskeletal-microtubule organization, leading to cell cycle arrest, genotoxic stress, and innate immunity. Comparison to analogous data from transformed cell lines revealed respiratory-specific processes hijacked by SARS-CoV-2, highlighting potential novel therapeutic avenues that were validated by a high hit rate in a targeted small molecule screen in our iAT2 ALI system.

Phosphoproteomics Analysis Identifies Novel Candidate Substrates of the Nonreceptor Tyrosine Kinase, Src-related Kinase Lacking C-terminal Regulatory Tyrosine and N-terminal Myristoylation Sites (SRMS).

SRMS (Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristoylation sites), also known as PTK 70 (Protein tyrosine kinase 70), is a non-receptor tyrosine kinase that belongs to the BRK family of kinases (BFKs). To date less is known about the cellular role of SRMS primarily because of the unidentified substrates or signaling intermediates regulated by the kinase. In this study, we used phosphotyrosine antibody-based immunoaffinity purification in large-scale label-free quantitative phosphoproteomics to identify novel candidate substrates of SRMS. Our analyses led to the identification of 1258 tyrosine-phosphorylated peptides which mapped to 663 phosphoproteins, exclusively from SRMS-expressing cells. DOK1, a previously characterized SRMS substrate, was also identified in our analyses. Functional enrichment analyses revealed that the candidate SRMS substrates were enriched in various biological processes including protein ubiquitination, mitotic cell cycle, energy metabolism and RNA processing, as well as Wnt and TNF signaling. Analyses of the sequence surrounding the phospho-sites in these proteins revealed novel candidate SRMS consensus substrate motifs. We utilized customized high-throughput peptide arrays to validate a subset of the candidate SRMS substrates identified in our MS-based analyses. Finally, we independently validated Vimentin and Sam68, as bona fide SRMS substrates through in vitro and in vivo assays. Overall, our study identified a number of novel and biologically relevant SRMS candidate substrates, which suggests the involvement of the kinase in a vast array of unexplored cellular functions.

Global phosphoproteomic analysis identifies SRMS-regulated secondary signaling intermediates.

BACKGROUND: The non-receptor tyrosine kinase, SRMS (Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristoylation sites) is a member of the BRK family kinases (BFKs) which represents an evolutionarily conserved relative of the Src family kinases (SFKs). Tyrosine kinases are known to regulate a number of cellular processes and pathways via phosphorylating substrate proteins directly and/or by partaking in signaling cross-talks leading to the indirect modulation of various signaling intermediates. In a previous study, we profiled the tyrosine-phosphoproteome of SRMS and identified multiple candidate substrates of the kinase. The broader cellular signaling intermediates of SRMS are unknown. METHODS: In order to uncover the broader SRMS-regulated phosphoproteome and identify the SRMS-regulated indirect signaling intermediates, we performed label-free global phosphoproteomics analysis on cells expressing wild-type SRMS. Using computational database searching and bioinformatics analyses we characterized the dataset. RESULTS: Our analyses identified 60 hyperphosphorylated (phosphoserine/phosphothreonine) proteins mapped from 140 hyperphosphorylated peptides. Bioinfomatics analyses identified a number of significantly enriched biological and cellular processes among which DNA repair pathways were found to be upregulated while apoptotic pathways were found to be downregulated. Analyses of motifs derived from the upregulated phosphosites identified Casein kinase 2 alpha (CK2α) as one of the major potential kinases contributing to the SRMS-dependent indirect regulation of signaling intermediates. CONCLUSIONS: Overall, our phosphoproteomics analyses identified serine/threonine phosphorylation dynamics as important secondary events of the SRMS-regulated phosphoproteome with implications in the regulation of cellular and biological processes.

Understanding the cellular roles of Fyn-related kinase (FRK): implications in cancer biology.

The non-receptor tyrosine kinase Fyn-related kinase (FRK) is a member of the BRK family kinases (BFKs) and is distantly related to the Src family kinases (SFKs). FRK was first discovered in 1993, and studies pursued thereafter attributed a potential tumour-suppressive function to the enzyme. In recent years, however, further functional characterization of the tyrosine kinase in diverse cancer types suggests that FRK may potentially play an oncogenic role as well. Specifically, while ectopic expression of FRK suppresses cell proliferation and migration in breast and brain cancers, knockdown or catalytic inhibition of FRK suppresses these cellular processes in pancreatic and liver cancer. Such functional paradox is therefore evidently exhibited in a tissue-specific context. This review sheds light on the recent developments emerged from investigations on FRK which include: (a) a review of the expression pattern of the protein in mammalian cells/tissues, (b) underlying genomic perturbations and (c) a mechanistic function of the enzyme across different cellular environments. Given its functional heterogeneity observed across different cancers, we also discuss the therapeutic significance of FRK.

Tracing the footprints of the breast cancer oncogene BRK - Past till present.

Twenty years have passed since the non-receptor tyrosine kinase, Breast tumor kinase (BRK) was cloned. While BRK is evolutionarily related to the Src family kinases it forms its own distinct sub-family referred here to as the BRK family kinases. The detection of BRK in over 60% of breast carcinomas two decades ago and more remarkably, its absence in the normal mammary gland attributed to its recognition as a mammary gland-specific potent oncogene and led BRK researchers on a wild chase to characterize the role of the enzyme in breast cancer. Where has this chase led us? An increasing number of studies have been focused on understanding the cellular roles of BRK in breast carcinoma and normal tissues. A majority of such studies have proposed an oncogenic function of BRK in breast cancers. Thus far, the vast evidence gathered highlights a regulatory role of BRK in critical cellular processes driving tumor formation such as cell proliferation, migration and metastasis. Functional characterization of BRK has identified several signaling proteins that work in concert with the enzyme to sustain such a malignant phenotype. As such targeting the non-receptor tyrosine kinase has been proposed as an attractive approach towards therapeutic intervention. Yet much remains to be explored about (a) the discrepant expression levels of BRK in cancer versus normal conditions, (b) the dependence on the enzymatic activity of BRK to promote oncogenesis and (c) an understanding of the normal physiological roles of the enzyme. This review outlines the advances made towards understanding the cellular and physiological roles of BRK, the mechanisms of action of the protein and its therapeutic significance, in the context of breast cancer.

Seeking a better understanding of the non-receptor tyrosine kinase, SRMS.

SRMS (Src-Related kinase lacking C-terminal regulatory tyrosine and N-terminal Myristoylation Sites) is a non-receptor tyrosine kinase first reported in a 1994 screen for genes regulating murine neural precursor cells. SRMS, pronounced "Shrims", lacks the C-terminal regulatory tyrosine critical for the regulation of the enzymatic activity of Src-family kinases (SFKs). Another remarkable characteristic of SRMS is its localization into distinct SRMS cytoplasmic punctae (SCPs) or GREL (Goel Raghuveera-Erique Lukong) bodies, a pattern not observed in the SFKs. This unique subcellular localization of SRMS could dictate its cellular targets, proteome, and potentially, substrates. However, the function of SRMS is still relatively unknown. Further, how is its activity regulated and by what cellular targets? Studies have emerged highlighting the potential role of SRMS in autophagy and in regulating the activation of BRK/PTK6. Potential novel cellular substrates have also been identified, including DOK1, vimentin, Sam68, FBKP51, and OTUB1. Recent studies have also demonstrated the potential role of the kinase in various cancers, including gastric and colorectal cancers and platinum resistance in ovarian cancer. This review discusses the advancements made in SRMS-related biology to date and the path to understanding the cellular and physiological significance of the kinase.

Scalable multiplex co-fractionation/mass spectrometry platform for accelerated protein interactome discovery.

Co-fractionation/mass spectrometry (CF/MS) enables the mapping of endogenous macromolecular networks on a proteome scale, but current methods are experimentally laborious, resource intensive and afford lesser quantitative accuracy. Here, we present a technically efficient, cost-effective and reproducible multiplex CF/MS (mCF/MS) platform for measuring and comparing, simultaneously, multi-protein assemblies across different experimental samples at a rate that is up to an order of magnitude faster than previous approaches. We apply mCF/MS to map the protein interaction landscape of non-transformed mammary epithelia versus breast cancer cells in parallel, revealing large-scale differences in protein-protein interactions and the relative abundance of associated macromolecules connected with cancer-related pathways and altered cellular processes. The integration of multiplexing capability within an optimized workflow renders mCF/MS as a powerful tool for systematically exploring physical interaction networks in a comparative manner.

Development of a low-cost and portable smart fluorometer for detecting breast cancer cells.

Instruments that allow the detection of fluorescence signal are invaluable tools for biomedical and clinical researchers. The technique is widely used in cell biology to microscopically detect target proteins of interest in mammalian cells. Importantly, fluorescence microscopy finds major applications in cancer biology where cancer cells are chemically labelled for detection. However, conventional fluorescence detection instruments such as fluorescence imaging microscopes are expensive, not portable and entail potentially high maintenance costs. Here we describe the design, development and applicability of a low-cost and portable fluorometer for the detection of fluorescence signal emitted from a model breast cancer cell line, engineered to stably express the green fluorescent protein (GFP). This device utilizes a flashlight which works in the visible range as an excitation source and a photodiode as the detector. It also utilizes an emission filter to mainly allow the fluorescence signal to reach the detector while eliminating the use of an excitation filter and dichroic mirror, hence, making the device compact, low-cost, portable and lightweight. The custom-built sample chamber is fabricated with a 3D printer to house the detector circuitry. We demonstrate that the developed fluorometer is able to distinguish between the cancer cell expressing GFP and the control cell. The fluorometer we developed exhibits immense potential for future applicability in the selective detection of fluorescently-labelled breast cancer cells.

FRK inhibits breast cancer cell migration and invasion by suppressing epithelial-mesenchymal transition.

The human fyn-related kinase (FRK) is a non-receptor tyrosine kinase known to have tumor suppressor activity in breast cancer cells. However, its mechanism of action has not been fully characterized. We generated FRK-stable MDA-MB-231 breast cancer cell lines and analyzed the effect on cell proliferation, migration, and invasiveness. We also used kinome analysis to identify potential FRK-regulated signaling pathways. We employed both immunoblotting and RT-PCR to identify/validate FRK-regulated targets (proteins and genes) in these cells. Finally, we interrogated the TCGA and GENT gene expression databases to determine the correlation between the expression of FRK and epithelial/mesenchymal markers. We observed that FRK overexpression suppressed cell proliferation, migration, and invasiveness, inhibited various JAK/STAT, MAPK and Akt signaling pathways, and suppressed the expression of some STAT3 target genes. Also, FRK overexpression increased the expression of epithelial markers including E-cadherin mRNA and down-regulated the transcript levels of vimentin, fibronectin, and slug. Finally, we observed an inverse correlation between FRK expression and mesenchymal markers in a large cohort of breast cancer cells. Our data, therefore, suggests that FRK represses cell proliferation, migration and invasiveness by suppressing epithelial to mesenchymal transition.

Mesenchymal stromal cells' role in tumor microenvironment: involvement of signaling pathways.

Mesenchymal stromal cells (MSCs) are adult multipotent stem cells residing as pericytes in various tissues and organs where they can differentiate into specialized cells to replace dying cells and damaged tissues. These cells are commonly found at injury sites and in tumors that are known to behave like " wounds that do not heal." In this article, we discuss the mechanisms of MSCs in migrating, homing, and repairing injured tissues. We also review a number of reports showing that tumor microenvironment triggers plasticity mechanisms in MSCs to induce malignant neoplastic tissue formation, maintenance, and chemoresistance, as well as tumor growth. The antitumor properties and therapeutic potential of MSCs are also discussed.

BRK targets Dok1 for ubiquitin-mediated proteasomal degradation to promote cell proliferation and migration.

Breast tumor kinase (BRK), also known as protein tyrosine kinase 6 (PTK6), is a non-receptor tyrosine kinase overexpressed in more that 60% of human breast carcinomas. The overexpression of BRK has been shown to sensitize mammary epithelial cells to mitogenic signaling and to promote cell proliferation and tumor formation. The molecular mechanisms of BRK have been unveiled by the identification and characterization of BRK target proteins. Downstream of tyrosine kinases 1 or Dok1 is a scaffolding protein and a substrate of several tyrosine kinases. Herein we show that BRK interacts with and phosphorylates Dok1 specifically on Y362. We demonstrate that this phosphorylation by BRK significantly downregulates Dok1 in a ubiquitin-proteasome-mediated mechanism. Together, these results suggest a novel mechanism of action of BRK in the promotion of tumor formation, which involves the targeting of tumor suppressor Dok1 for degradation through the ubiquitin proteasomal pathway.