Kinome and phosphoproteome reprogramming underlies the aberrant immune responses in critically ill COVID-19 patients.

SARS-CoV-2 infection triggers extensive host immune reactions, leading to severe diseases in certain individuals. However, the molecular basis underlying the excessive yet non-productive immune responses in severe COVID-19 remains incompletely understood. In this study, we conducted a comprehensive analysis of the peripheral blood mononuclear cell (PBMC) proteome and phosphoproteome in sepsis patients positive or negative for SARS-CoV-2 infection, as well as healthy subjects, using quantitative mass spectrometry. Our findings demonstrate dynamic changes in the COVID-19 PBMC proteome and phosphoproteome during disease progression, with distinctive protein or phosphoprotein signatures capable of distinguishing longitudinal disease states. Furthermore, SARS-CoV-2 infection induces a global reprogramming of the kinome and phosphoproteome, resulting in defective adaptive immune response mediated by the B and T lymphocytes, compromised innate immune responses involving the SIGLEC and SLAM family of immunoreceptors, and excessive cytokine-JAK-STAT signaling. In addition to uncovering host proteome and phosphoproteome aberrations caused by SARS-CoV-2, our work recapitulates several reported therapeutic targets for COVID-19 and identified numerous new candidates, including the kinases PKG1, CK2, ROCK1/2, GRK2, SYK, JAK2/3, TYK2, DNA-PK, PKCδ, and the cytokine IL-12.

The spatial transcriptomic landscape of non-small cell lung cancer brain metastasis.

Brain metastases (BrMs) are a common occurrence in lung cancer with a dismal outcome. To understand the mechanism of metastasis to inform prognosis and treatment, here we analyze primary and metastasized tumor specimens from 44 non-small cell lung cancer patients by spatial RNA sequencing, affording a whole transcriptome map of metastasis resolved with morphological markers for the tumor core, tumor immune microenvironment (TIME), and tumor brain microenvironment (TBME). Our data indicate that the tumor microenvironment (TME) in the brain, including the TIME and TBME, undergoes extensive remodeling to create an immunosuppressive and fibrogenic niche for the BrMs. Specifically, the brain TME is characterized with reduced antigen presentation and B/T cell function, increased neutrophils and M2-type macrophages, immature microglia, and reactive astrocytes. Differential gene expression and network analysis identify fibrosis and immune regulation as the major functional modules disrupted in both the lung and brain TME. Besides providing systems-level insights into the mechanism of lung cancer brain metastasis, our study uncovers potential prognostic biomarkers and suggests that therapeutic strategies should be tailored to the immune and fibrosis status of the BrMs.

Proteome and phosphoproteome signatures of recurrence for HPV+ head and neck squamous cell carcinoma.

Background: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide and the human papillomavirus (HPV+)-driven subtype is the fastest rising cancer in North America. Although most cases of HPV+ HNSCC respond favorably to the treatment via surgery followed by radiochemotherapy, up to 20% recur with a poor prognosis. The molecular and cellular mechanisms of recurrence are not fully understood. Methods: To gain insights into the mechanisms of recurrence and to inform patient stratification and personalized treatment, we compared the proteome and phosphoproteome of recurrent and non-recurrent tumors by quantitative mass spectrometry. Results: We observe significant differences between the recurrent and non-recurrent tumors in cellular composition, function, and signaling. The recurrent tumors are characterized by a pro-fibrotic and immunosuppressive tumor microenvironment (TME) featuring markedly more abundant cancer-associated fibroblasts, extracellular matrix (ECM), neutrophils, and suppressive myeloid cells. Defective T cell function and increased epithelial-mesenchymal transition potential are also associated with recurrence. These cellular changes in the TME are accompanied by reprogramming of the kinome and the signaling networks that regulate the ECM, cytoskeletal reorganization, cell adhesion, neutrophil function, and coagulation. Conclusions: In addition to providing systems-level insights into the molecular basis of recurrence, our work identifies numerous mechanism-based, candidate biomarkers and therapeutic targets that may aid future endeavors to develop prognostic biomarkers and precision-targeted treatment for recurrent HPV+ HNSCC.

Head and neck cancer can be caused by the human papillomavirus. While this type of cancer responds well to chemotherapy given simultaneously with radiation, a significant proportion of cases recur within a few years, leading to illness and sometimes death in these patients. It is therefore important to understand the mechanisms of recurrence in order to develop better treatments. By comparing the levels of proteins and protein phosphorylation­a type of modification that affects how proteins work­between tumors from patients with or without recurrence, we found that the cells surrounding recurrent tumors show signs of fibrosis­the development of fibrous connective tissue­and suppression of the body's immune responses. This suggests that therapies directed towards the regulators of fibrosis and immune suppression may help to overcome recurrent head and neck cancer.

Blockade of phosphotyrosine pathways suggesting SH2 superbinder as a novel therapy for pulmonary fibrosis.

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible fibrotic disease with high mortality. Currently, pirfenidone and nintedanib are the only approved drugs for IPF by the U.S. Food and Drug Administration (FDA), but their efficacy is limited. The activation of multiple phosphotyrosine (pY) mediated signaling pathways underlying the pathological mechanism of IPF has been explored. A Src homology-2 (SH2) superbinder, which contains mutations of three amino acids (AAs) of natural SH2 domain has been shown to be able to block phosphotyrosine (pY) pathway. Therefore, we aimed to introduce SH2 superbinder into the treatment of IPF. Methods: We analyzed the database of IPF patients and examined pY levels in lung tissues from IPF patients. In primary lung fibroblasts obtained from IPF patient as well as bleomycin (BLM) treated mice, the cell proliferation, migration and differentiation associated with pY were investigated and the anti-fibrotic effect of SH2 superbinder was also tested. In vivo, we further verified the safety and effectiveness of SH2 superbinder in multiple BLM mice models. We also compared the anti-fibrotic effect and side-effect of SH2 superbinder and nintedanib in vivo. Results: The data showed that the cytokines and growth factors pathways which directly correlated to pY levels were significantly enriched in IPF. High pY levels were found to induce abnormal proliferation, migration and differentiation of lung fibroblasts. SH2 superbinder blocked pY-mediated signaling pathways and suppress pulmonary fibrosis by targeting high pY levels in fibroblasts. SH2 superbinder had better therapeutic effect and less side-effect compare to nintedanib in vivo. Conclusions: SH2 superbinder had significant anti-fibrotic effects both in vitro and in vivo, which could be used as a promising therapy for IPF.

Aptamer-SH2 superbinder-based targeted therapy for pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) exhibits the poorest prognosis of all solid tumors with a 5-year survival rate of less than 10% and a median survival of 6 months after diagnosis. Numerous targeted agents have been developed and evaluated to improve the survival benefit in patients with PDAC. Unfortunately, most agents have been proven futile mainly owing to the dense stroma and the sophisticated signaling pathways of PDAC. Here, we show the potent effectiveness of Aptamer-SH2 superbinder-(Arg)9 conjugate on the treatment of PDAC. In this conjugate, DNA aptamer selected against PDAC cell line confers the function of specifically recognizing and binding to the PDAC cells and activated pancreatic stellate cells (PSCs) in stroma; cell penetrating peptide (Arg)9 facilitates the intracellular delivery of fused proteins; SH2 superbinder conducts the drastic blockade of multiple phosphotyrosines (pY)-based signaling pathways in tumor cells. METHODS: PDAC-associated pY were reanalyzed by bioinformatics screen. XQ-2d and SH2 superbinder-(Arg)9 were crosslinked with BMH to form XQ-2d-SH2 CM-(Arg)9 conjugate. Immunofluorescence was utilized to assess the potency of the conjugate entering cells. MTT and wound healing assays were performed to evaluate the proliferation or migration of PANC-1 and BxPC-3 cells, respectively. Western blot and Pulldown assays revealed that conjugate influenced several pY-based signaling pathways. Tumor-bearing mice were used to validate XQ-2d-SH2 CM-(Arg)9, which restrained the growth and metastasis of cancer cells. RESULTS: XQ-2d-His-SH2 CM-(Arg)9 conjugate restrained proliferation, invasion, and metastasis of PDAC cells with potent efficacy via blocking the activity of several pY-related signaling cascades. XQ-2d-His-SH2 CM-(Arg)9 could eliminate the dense stroma of PDAC and then arrive at tumor tissues. CONCLUSIONS: XQ-2d-SH2 CM-(Arg)9 conjugate may efficiently destroy the pancreatic stroma and show potent antitumor efficacy with minimal toxic effect by regulating tumor cell proliferation and metastasis in vitro and in vivo, which makes it to be a promising targeted therapy of PDAC.

Phosphorylation-dependent substrate selectivity of protein kinase B (AKT1).

Protein kinase B (AKT1) is a central node in a signaling pathway that regulates cell survival. The diverse pathways regulated by AKT1 are communicated in the cell via the phosphorylation of perhaps more than 100 cellular substrates. AKT1 is itself activated by phosphorylation at Thr-308 and Ser-473. Despite the fact that these phosphorylation sites are biomarkers for cancers and tumor biology, their individual roles in shaping AKT1 substrate selectivity are unknown. We recently developed a method to produce AKT1 with programmed phosphorylation at either or both of its key regulatory sites. Here, we used both defined and randomized peptide libraries to map the substrate selectivity of site-specific, singly and doubly phosphorylated AKT1 variants. To globally quantitate AKT1 substrate preferences, we synthesized three AKT1 substrate peptide libraries: one based on 84 "known" substrates and two independent and larger oriented peptide array libraries (OPALs) of ∼1011 peptides each. We found that each phospho-form of AKT1 has common and distinct substrate requirements. Compared with pAKT1T308, the addition of Ser-473 phosphorylation increased AKT1 activities on some, but not all of its substrates. This is the first report that Ser-473 phosphorylation can positively or negatively regulate kinase activity in a substrate-dependent fashion. Bioinformatics analysis indicated that the OPAL-activity data effectively discriminate known AKT1 substrates from closely related kinase substrates. Our results also enabled predictions of novel AKT1 substrates that suggest new and expanded roles for AKT1 signaling in regulating cellular processes.

Phospho-Form Specific Substrates of Protein Kinase B (AKT1).

Protein kinase B (AKT1) is hyper-activated in diverse human tumors. AKT1 is activated by phosphorylation at two key regulatory sites, Thr308 and Ser473. Active AKT1 phosphorylates many, perhaps hundreds, of downstream cellular targets in the cytosol and nucleus. AKT1 is well-known for phosphorylating proteins that regulate cell survival and apoptosis, however, the full catalog of AKT1 substrates remains unknown. Using peptide arrays, we recently discovered that each phosphorylated form of AKT1 (pAKT1S473, pAKT1T308, and ppAKT1S473,T308) has a distinct substrate specificity, and these data were used to predict potential new AKT1 substrates. To test the high-confidence predictions, we synthesized target peptides representing putative AKT1 substrates. Peptides substrates were synthesized by solid phase synthesis and their purity was confirmed by mass spectrometry. Most of the predicted peptides showed phosphate accepting activity similar to or greater than that observed with a peptide derived from a well-established AKT1 substrate, glycogen synthase kinase 3ß (GSK-3ß). Among the novel substrates, AKT1 was most active with peptides representing PIP3-binding protein Rab11 family-interacting protein 2 and cysteinyl leukotriene receptor 1, indicating their potential role in AKT1-dependent cellular signaling. The ppAKT1S473,T308 enzyme was highly selective for peptides containing a patch of basic residues at -5, -4, -3 and aromatic residues (Phe/Tyr) at +1 positions from the phosphorylation site. The pAKT1S473 variant preferred more acidic peptides, Ser or Pro at +4, and was agnostic to the residue at -5. The data further support our hypothesis that Ser473 phosphorylation plays a key role in modulating AKT1 substrate selectivity.

Gld2 activity is regulated by phosphorylation in the N-terminal domain.

The de-regulation of microRNAs (miRNAs) is associated with multiple human diseases, yet cellular mechanisms governing miRNA abundance remain largely elusive. Human miR-122 is required for Hepatitis C proliferation, and low miR-122 abundance is associated with hepatic cancer. The adenylyltransferase Gld2 catalyses the post-transcriptional addition of a single adenine residue (A + 1) to the 3'-end of miR-122, enhancing its stability. Gld2 activity is inhibited by binding to the Hepatitis C virus core protein during HepC infection, but no other mechanisms of Gld2 regulation are known. We found that Gld2 activity is regulated by site-specific phosphorylation in its disordered N-terminal domain. We identified two phosphorylation sites (S62, S110) where phosphomimetic substitutions increased Gld2 activity and one site (S116) that markedly reduced activity. Using mass spectrometry, we confirmed that HEK 293 cells readily phosphorylate the N-terminus of Gld2. We identified protein kinase A (PKA) and protein kinase B (Akt1) as the kinases that site-specifically phosphorylate Gld2 at S116, abolishing Gld2-mediated nucleotide addition. The data demonstrate a novel phosphorylation-dependent mechanism to regulate Gld2 activity, revealing tumour suppressor miRNAs as a previously unknown target of Akt1-dependent signalling.

NUMB regulates the endocytosis and activity of the anaplastic lymphoma kinase in an isoform-specific manner.

NUMB is an evolutionarily conserved protein that plays an important role in cell adhesion, migration, polarity, and cell fate determination. It has also been shown to play a role in the pathogenesis of certain cancers, although it remains controversial whether NUMB functions as an oncoprotein or tumor suppressor. Here, we show that NUMB binds to anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase aberrantly activated in several forms of cancer, and this interaction regulates the endocytosis and activity of ALK. Intriguingly, the function of the NUMB-ALK interaction is isoform-dependent. While both p66-NUMB and p72-NUMB isoforms are capable of mediating the endocytosis of ALK, the former directs ALK to the lysosomal degradation pathway, thus decreasing the overall ALK level and the downstream MAP kinase signal. In contrast, the p72-NUMB isoform promotes ALK recycling back to the plasma membrane, thereby maintaining the kinase in its active state. Our work sheds light on the controversial role of different isoforms of NUMB in tumorigenesis and provides mechanistic insight into ALK regulation.

Drugging RB1 Deficiency: Synthetic Lethality with Aurora Kinases.

Two recent reports describe pharmacologic approaches to specifically treat RB1-mutant cancers. The basis of this approach is a synthetic lethal relationship between RB1 mutations and inhibition of Aurora kinases A or B.See related article by Oser et al., p. 230.See related article by Gong et al., p. 248.

Increased IGFBP-1 phosphorylation in response to leucine deprivation is mediated by CK2 and PKC.

Insulin-like growth factor binding protein-1 (IGFBP-1), secreted by fetal liver, is a key regulator of IGF-I bioavailability and fetal growth. IGFBP-1 phosphorylation decreases IGF-I bioavailability and diminishes its growth-promoting effects. Growth-restricted fetuses have decreased levels of circulating essential amino acids. We recently showed that IGFBP-1 hyperphosphorylation (pSer101/119/169) in response to leucine deprivation is regulated via activation of the amino acid response (AAR) in HepG2 cells. Here we investigated nutrient-sensitive protein kinases CK2/PKC/PKA in mediating IGFBP-1 phosphorylation in leucine deprivation. We demonstrated that leucine deprivation stimulated CK2 activity (enzymatic assay) and induced IGFBP-1 phosphorylation (immunoblotting/MRM-MS). Inhibition (pharmacological/siRNA) of CK2/PKC, but not PKA, prevented IGFBP-1 hyperphosphorylation in leucine deprivation. PKC inhibition also prevented leucine deprivation-stimulated CK2 activity. Functionally, leucine deprivation decreased IGF-I-induced-IGF-1R autophosphorylation when CK2/PKC were not inhibited. Our data strongly support that PKC promotes leucine deprivation-induced IGFBP-1 hyperphosphorylation via CK2 activation, mechanistically linking decreased amino acid availability and reduced fetal growth.

Hypoxia Increases IGFBP-1 Phosphorylation Mediated by mTOR Inhibition.

In fetal growth restriction (FGR), fetal growth is limited by reduced nutrient and oxygen supply. Insulin-like growth factor I (IGF-I) is a key regulator of fetal growth and IGF binding protein -1(IGFBP-1) is the principal regulator of fetal IGF-I bioavailability. Phosphorylation enhances IGFBP-1's affinity for IGF-I. Hypoxia induces IGFBP-1 hyperphosphorylation, markedly decreasing IGF-I bioavailability. We recently reported that fetal liver IGFBP-1 hyperphosphorylation is associated with inhibition of the mechanistic target of rapamycin (mTOR) in a nonhuman primate model of FGR. Here, we test the hypothesis that IGFBP-1 hyperphosphorylation in response to hypoxia is mediated by mTOR inhibition. We inhibited mTOR either by rapamycin or small interfering RNA (siRNA) targeting raptor (mTOR complex [mTORC]1) and/or rictor (mTORC2) in HepG2 cells cultured under hypoxia (1% O2) or basal (20% O2) conditions. Conversely, we activated mTORC1 or mTORC1+mTORC2 by silencing endogenous mTOR inhibitors (tuberous sclerosis complex 2/DEP-domain-containing and mTOR-interacting protein). Immunoblot analysis demonstrated that both hypoxia and inhibition of mTORC1 and/or mTORC2 induced similar degrees of IGFBP-1 phosphorylation at Ser101/119/169 and reduced IGF-I receptor autophosphorylation. Activation of mTORC1+mTORC2 or mTORC1 alone prevented IGFBP-1 hyperphosphorylation in response to hypoxia. Multiple reaction monitoring-mass spectrometry showed that rapamycin and/or hypoxia increased phosphorylation also at Ser98 and at a novel site Ser174. In silico structural analysis indicated that Ser174 was in close proximity to the IGF-binding site. Together, we demonstrate that signaling through the mTORC1 or mTORC2 pathway is sufficient to induce IGFBP-1 hyperphosphorylation in response to hypoxia. This study provides novel understanding of the cellular mechanism that controls fetal IGFBP-1 phosphorylation in hypoxia, and we propose that mTOR inhibition constitutes a mechanistic link between hypoxia, reduced IGF-I bioavailability and FGR.

A Comprehensive Immunoreceptor Phosphotyrosine-based Signaling Network Revealed by Reciprocal Protein-Peptide Array Screening.

Cells of the immune system communicate with their environment through immunoreceptors. These receptors often harbor intracellular tyrosine residues, which, when phosphorylated upon receptor activation, serve as docking sites to recruit downstream signaling proteins containing the Src Homology 2 (SH2) domain. A systematic investigation of interactions between the SH2 domain and the immunoreceptor tyrosine-based regulatory motifs (ITRM), including inhibitory (ITIM), activating (ITAM), or switching (ITSM) motifs, is critical for understanding cellular signal transduction and immune function. Using the B cell inhibitory receptor CD22 as an example, we developed an approach that combines reciprocal or bidirectional phosphopeptide and SH2 domain array screens with in-solution binding assays to identify a comprehensive SH2-CD22 interaction network. Extending this approach to 194 human ITRM sequences and 78 SH2 domains led to the identification of a high-confidence immunoreceptor interactome containing 1137 binary interactions. Besides recapitulating many previously reported interactions, our study uncovered numerous novel interactions. The resulting ITRM-SH2 interactome not only helped to fill many gaps in the immune signaling network, it also allowed us to associate different SH2 domains to distinct immune functions. Detailed analysis of the NK cell ITRM-mediated interactions led to the identification of a network nucleated by the Vav3 and Fyn SH2 domains. We showed further that these SH2 domains have distinct functions in cytotoxicity. The bidirectional protein-peptide array approach described herein may be applied to the numerous other peptide-binding modules to identify potential protein-protein interactions in a systematic and reliable manner.

Large-scale interaction profiling of PDZ domains through proteomic peptide-phage display using human and viral phage peptidomes.

The human proteome contains a plethora of short linear motifs (SLiMs) that serve as binding interfaces for modular protein domains. Such interactions are crucial for signaling and other cellular processes, but are difficult to detect because of their low to moderate affinities. Here we developed a dedicated approach, proteomic peptide-phage display (ProP-PD), to identify domain-SLiM interactions. Specifically, we generated phage libraries containing all human and viral C-terminal peptides using custom oligonucleotide microarrays. With these libraries we screened the nine PSD-95/Dlg/ZO-1 (PDZ) domains of human Densin-180, Erbin, Scribble, and Disks large homolog 1 for peptide ligands. We identified several known and putative interactions potentially relevant to cellular signaling pathways and confirmed interactions between full-length Scribble and the target proteins ß-PIX, plakophilin-4, and guanylate cyclase soluble subunit α-2 using colocalization and coimmunoprecipitation experiments. The affinities of recombinant Scribble PDZ domains and the synthetic peptides representing the C termini of these proteins were in the 1- to 40-µM range. Furthermore, we identified several well-established host-virus protein-protein interactions, and confirmed that PDZ domains of Scribble interact with the C terminus of Tax-1 of human T-cell leukemia virus with micromolar affinity. Previously unknown putative viral protein ligands for the PDZ domains of Scribble and Erbin were also identified. Thus, we demonstrate that our ProP-PD libraries are useful tools for probing PDZ domain interactions. The method can be extended to interrogate all potential eukaryotic, bacterial, and viral SLiMs and we suggest it will be a highly valuable approach for studying cellular and pathogen-host protein-protein interactions.

Dynamic methylation of Numb by Set8 regulates its binding to p53 and apoptosis.

Although Numb exhibits its tumor-suppressive function in breast cancer in part by binding to and stabilizing p53, it is unknown how the Numb-p53 interaction is regulated in cells. We found that Numb is methylated in its phosphotyrosine-binding (PTB) domain by the lysine methyltransferase Set8. Moreover, methylation uncouples Numb from p53, resulting in increased p53 ubiquitination and degradation. While Numb promotes apoptosis in a p53-dependent manner, the apoptotic function is abolished when Numb is methylated by Set8 or the Lys methylation sites in Numb are mutated. Conversely, the Numb-p53 interaction and Numb-mediated apoptosis are significantly enhanced by depletion of Set8 from cancer cells or by treating the cells with doxorubicin, a chemotherapeutic drug that causes a reduction in the mRNA and protein levels of Set8. Our work identifies the Set8-Numb-p53 signaling axis as an important regulatory pathway for apoptosis and suggests a therapeutic strategy by targeting Numb methylation.

A method for systematic mapping of protein lysine methylation identifies functions for HP1ß in DNA damage response.

Lysine methylation occurs on both histone and nonhistone proteins. However, our knowledge on the prevalence and function of nonhistone protein methylation is poor. We describe an approach that combines peptide array, bioinformatics, and mass spectrometry to systematically identify lysine methylation sites and map methyllysine-driven protein-protein interactions. Using this approach, we identified a high-confidence and high-resolution interactome of the heterochromatin protein 1ß (HP1ß) and uncovered, simultaneously, numerous methyllysine sites on nonhistone proteins. We found that HP1ß binds to DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and regulates its localization to double-strand breaks (DSBs) during DNA damage response (DDR). Mutation of the methylation sites in DNA-PKcs or depletion of HP1ß in cells caused defects in DDR. Furthermore, we showed that the methylation of DNA-PKcs and many other proteins in the HP1ß interactome undergoes large changes in response to DNA damage, indicating that Lys methylation is a highly dynamic posttranslational modification.

Structural basis for endosomal trafficking of diverse transmembrane cargos by PX-FERM proteins.

Transit of proteins through the endosomal organelle following endocytosis is critical for regulating the homeostasis of cell-surface proteins and controlling signal transduction pathways. However, the mechanisms that control these membrane-transport processes are poorly understood. The Phox-homology (PX) domain-containing proteins sorting nexin (SNX) 17, SNX27, and SNX31 have emerged recently as key regulators of endosomal recycling and bind conserved Asn-Pro-Xaa-Tyr-sorting signals in transmembrane cargos via an atypical band, 4.1/ezrin/radixin/moesin (FERM) domain. Here we present the crystal structure of the SNX17 FERM domain bound to the sorting motif of the P-selectin adhesion protein, revealing both the architecture of the atypical FERM domain and the molecular basis for recognition of these essential sorting sequences. We further show that the PX-FERM proteins share a promiscuous ability to bind a wide array of putative cargo molecules, including receptor tyrosine kinases, and propose a model for their coordinated molecular interactions with membrane, cargo, and regulatory proteins.

Differential regulation of the activity of deleted in liver cancer 1 (DLC1) by tensins controls cell migration and transformation.

The epithelial growth factor receptor plays an important role in cell migration and cancer metastasis, but the underlying molecular mechanism is not fully understood. We show here that differential regulation of the Ras-homology-GTPase-activating protein [corrected] (Rho-GAP) activity of deleted in liver cancer 1 (DLC1) by tensin3 and COOH-terminal tensin-like protein (cten) controls EGF-driven cell migration and transformation. Tensin3 binds DLC1 through its actin-binding domain, a region that is missing in cten, and thereby releases an autoinhibitory interaction between the sterile alpha motif and Rho-GAP domains of DLC1. Consequently, tensin3, but not cten, promotes the activation of DLC1, which, in turn, leads to inactivation of RhoA and decreased cell migration. Depletion of endogenous tensin3, but not cten, augmented the formation of actin stress fibers and focal adhesions and enhanced cell motility. These effects were, however, ablated by an inhibitor of the Rho-associated protein kinase. Importantly, activation of DLC1 by tensin3 or its actin-binding domain drastically reduced the anchorage-independent growth of transformed cells. Our study therefore links dynamic regulation of tensin family members by EGF to Rho-GAP through DLC1 and suggests that the tensin-DLC1-RhoA signaling axis plays an important role in tumorigenesis and cancer metastasis, and may be explored for cancer intervention.

The human phosphotyrosine signaling network: evolution and hotspots of hijacking in cancer.

Phosphotyrosine (pTyr) signaling, which plays a central role in cell-cell and cell-environment interactions, has been considered to be an evolutionary innovation in multicellular metazoans. However, neither the emergence nor the evolution of the human pTyr signaling system is currently understood. Tyrosine kinase (TK) circuits, each of which consists of a TK writer, a kinase substrate, and a related reader, such as Src homology (SH) 2 domains and pTyr-binding (PTB) domains, comprise the core machinery of the pTyr signaling network. In this study, we analyzed the evolutionary trajectories of 583 literature-derived and 50,000 computationally predicted human TK circuits in 19 representative eukaryotic species and assigned their evolutionary origins. We found that human TK circuits for intracellular pTyr signaling originated largely from primitive organisms, whereas the inter- or extracellular signaling circuits experienced significant expansion in the bilaterian lineage through the "back-wiring" of newly evolved kinases to primitive substrates and SH2/PTB domains. Conversely, the TK circuits that are involved in tissue-specific signaling evolved mainly in vertebrates by the back-wiring of vertebrate substrates to primitive kinases and SH2/PTB domains. Importantly, we found that cancer signaling preferentially employs the pTyr sites, which are linked to more TK circuits. Our work provides insights into the evolutionary paths of the human pTyr signaling circuits and suggests the use of a network approach for cancer intervention through the targeting of key pTyr sites and their associated signaling hubs in the network.