De Novo Multi-Omics Pathway Analysis Designed for Prior Data Independent Inference of Cell Signaling Pathways.

New tools for cell signaling pathway inference from multi-omics data that are independent of previous knowledge are needed. Here, we propose a new de novo method, the de novo multi-omics pathway analysis (DMPA), to model and combine omics data into network modules and pathways. DMPA was validated with published omics data and was found accurate in discovering reported molecular associations in transcriptome, interactome, phosphoproteome, methylome, and metabolomics data, and signaling pathways in multi-omics data. DMPA was benchmarked against module discovery and multi-omics integration methods and outperformed previous methods in module and pathway discovery especially when applied to datasets of relatively low sample sizes. Transcription factor, kinase, subcellular location, and function prediction algorithms were devised for transcriptome, phosphoproteome, and interactome modules and pathways, respectively. To apply DMPA in a biologically relevant context, interactome, phosphoproteome, transcriptome, and proteome data were collected from analyses carried out using melanoma cells to address gamma-secretase cleavage-dependent signaling characteristics of the receptor tyrosine kinase TYRO3. The pathways modeled with DMPA reflected the predicted function and its direction in validation experiments.

STAT5b is a key effector of NRG-1/ERBB4-mediated myocardial growth.

The growth factor Neuregulin-1 (NRG-1) regulates myocardial growth and is currently under clinical investigation as a treatment for heart failure. Here, we demonstrate in several in vitro and in vivo models that STAT5b mediates NRG-1/EBBB4-stimulated cardiomyocyte growth. Genetic and chemical disruption of the NRG-1/ERBB4 pathway reduces STAT5b activation and transcription of STAT5b target genes Igf1, Myc, and Cdkn1a in murine cardiomyocytes. Loss of Stat5b also ablates NRG-1-induced cardiomyocyte hypertrophy. Dynamin-2 is shown to control the cell surface localization of ERBB4 and chemical inhibition of Dynamin-2 downregulates STAT5b activation and cardiomyocyte hypertrophy. In zebrafish embryos, Stat5 is activated during NRG-1-induced hyperplastic myocardial growth, and chemical inhibition of the Nrg-1/Erbb4 pathway or Dynamin-2 leads to loss of myocardial growth and Stat5 activation. Moreover, CRISPR/Cas9-mediated knockdown of stat5b results in reduced myocardial growth and cardiac function. Finally, the NRG-1/ERBB4/STAT5b signaling pathway is differentially regulated at mRNA and protein levels in the myocardium of patients with pathological cardiac hypertrophy as compared to control human subjects, consistent with a role of the NRG-1/ERBB4/STAT5b pathway in myocardial growth.

The guanine nucleotide exchange factor VAV3 participates in ERBB4-mediated cancer cell migration.

ERBB4 is a member of the epidermal growth factor receptor (EGFR)/ERBB subfamily of receptor tyrosine kinases that regulates cellular processes including proliferation, migration, and survival. ERBB4 signaling is involved in embryogenesis and homeostasis of healthy adult tissues, but also in human pathologies such as cancer, neurological disorders, and cardiovascular diseases. Here, an MS-based analysis revealed the Vav guanine nucleotide exchange factor 3 (VAV3), an activator of Rho family GTPases, as a critical ERBB4-interacting protein in breast cancer cells. We confirmed the ERBB4-VAV3 interaction by targeted MS and coimmunoprecipitation experiments and further defined it by demonstrating that kinase activity and Tyr-1022 and Tyr-1162 of ERBB4, as well as the intact phosphotyrosine-interacting SH2 domain of VAV3, are necessary for this interaction. We found that ERBB4 stimulates tyrosine phosphorylation of the VAV3 activation domain, known to be required for guanine nucleotide exchange factor (GEF) activity of VAV proteins. In addition to VAV3, the other members of the VAV family, VAV1 and VAV2, also coprecipitated with ERBB4. Analyses of the effects of overexpression of dominant-negative VAV3 constructs or shRNA-mediated down-regulation of VAV3 expression in breast cancer cells indicated that active VAV3 is involved in ERBB4-stimulated cell migration. These results define the VAV GEFs as effectors of ERBB4 activity in a signaling pathway relevant for cancer cell migration.

An extracellular receptor tyrosine kinase motif orchestrating intracellular STAT activation.

The ErbB4 receptor isoforms JM-a and JM-b differ within their extracellular juxtamembrane (eJM) domains. Here, ErbB4 isoforms are used as a model to address the effect of structural variation in the eJM domain of receptor tyrosine kinases (RTK) on downstream signaling. A specific JM-a-like sequence motif is discovered, and its presence or absence (in JM-b-like RTKs) in the eJM domains of several RTKs is demonstrated to dictate selective STAT activation. STAT5a activation by RTKs including the JM-a like motif is shown to involve interaction with oligosaccharides of N-glycosylated cell surface proteins such as ß1 integrin, whereas STAT5b activation by JM-b is dependent on TYK2. ErbB4 JM-a- and JM-b-like RTKs are shown to associate with specific signaling complexes at different cell surface compartments using analyses of RTK interactomes and super-resolution imaging. These findings provide evidence for a conserved mechanism linking a ubiquitous extracellular motif in RTKs with selective intracellular STAT signaling.

Gamma-secretase-dependent signaling of receptor tyrosine kinases.

Human genome harbors 55 receptor tyrosine kinases (RTK). At least half of the RTKs have been reported to be cleaved by gamma-secretase-mediated regulated intramembrane proteolysis. The two-step process involves releasing the RTK ectodomain to the extracellular space by proteolytic cleavage called shedding, followed by cleavage in the RTK transmembrane domain by the gamma-secretase complex resulting in release of a soluble RTK intracellular domain. This intracellular domain, including the tyrosine kinase domain, can in turn translocate to various cellular compartments, such as the nucleus or proteasome. The soluble intracellular domain may interact with transcriptional regulators and other proteins to induce specific effects on cell survival, proliferation, and differentiation, establishing an additional signaling mode for the cleavable RTKs. On the other hand, the same process can facilitate RTK turnover and proteasomal degradation. In this review we focus on the regulation of RTK shedding and gamma-secretase cleavage, as well as signaling promoted by the soluble RTK ICDs. In addition, therapeutic implications of increased knowledge on RTK cleavage on cancer drug development are discussed.

Genome-wide screen of gamma-secretase-mediated intramembrane cleavage of receptor tyrosine kinases.

Receptor tyrosine kinases (RTKs) have been demonstrated to signal via regulated intramembrane proteolysis, in which ectodomain shedding and subsequent intramembrane cleavage by gamma-secretase leads to release of a soluble intracellular receptor fragment with functional activity. For most RTKs, however, it is unknown whether they can exploit this new signaling mechanism. Here we used a system-wide screen to address the frequency of susceptibility to gamma-secretase cleavage among human RTKs. The screen covering 45 of the 55 human RTKs identified 12 new as well as all nine previously published gamma-secretase substrates. We biochemically validated the screen by demonstrating that the release of a soluble intracellular fragment from endogenous AXL was dependent on the sheddase disintegrin and metalloprotease 10 (ADAM10) and the gamma-secretase component presenilin-1. Functional analysis of the cleavable RTKs indicated that proliferation promoted by overexpression of the TAM family members AXL or TYRO3 depends on gamma-secretase cleavage. Taken together, these data indicate that gamma-secretase-mediated cleavage provides an additional signaling mechanism for numerous human RTKs.