The Shb scaffold binds the Nck adaptor protein, p120 RasGAP, and Chimaerins and thereby facilitates heterotypic cell segregation by the receptor EphB2.

Eph receptors are a family of receptor tyrosine kinases that control directional cell movement during various biological processes, including embryogenesis, neuronal pathfinding, and tumor formation. The biochemical pathways of Eph receptors are context-dependent in part because of the varied composition of a heterotypic, oligomeric, active Eph receptor complex. Downstream of the Eph receptors, little is known about the essential phosphorylation events that define the context and instruct cell movement. Here, we define a pathway that is required for Eph receptor B2 (EphB2)-mediated cell sorting and is conserved among multiple Eph receptors. Utilizing a HEK293 model of EphB2+/ephrinB1+ cell segregation, we found that the scaffold adaptor protein SH2 domain-containing adaptor protein B (Shb) is essential for EphB2 functionality. Further characterization revealed that Shb interacts with known modulators of cytoskeletal rearrangement and cell mobility, including Nck adaptor protein (Nck), p120-Ras GTPase-activating protein (RasGAP), and the α- and ß-Chimaerin Rac GAPs. We noted that phosphorylation of Tyr297, Tyr246, and Tyr336 of Shb is required for EphB2-ephrinB1 boundary formation, as well as binding of Nck, RasGAP, and the chimaerins, respectively. Similar complexes were formed in the context of EphA4, EphA8, EphB2, and EphB4 receptor activation. These results indicate that phosphotyrosine-mediated signaling through Shb is essential in EphB2-mediated heterotypic cell segregation and suggest a conserved function for Shb downstream of multiple Eph receptors.

Temporal regulation of EGF signalling networks by the scaffold protein Shc1.

Cell-surface receptors frequently use scaffold proteins to recruit cytoplasmic targets, but the rationale for this is uncertain. Activated receptor tyrosine kinases, for example, engage scaffolds such as Shc1 that contain phosphotyrosine (pTyr)-binding (PTB) domains. Using quantitative mass spectrometry, here we show that mammalian Shc1 responds to epidermal growth factor (EGF) stimulation through multiple waves of distinct phosphorylation events and protein interactions. After stimulation, Shc1 rapidly binds a group of proteins that activate pro-mitogenic or survival pathways dependent on recruitment of the Grb2 adaptor to Shc1 pTyr sites. Akt-mediated feedback phosphorylation of Shc1 Ser 29 then recruits the Ptpn12 tyrosine phosphatase. This is followed by a sub-network of proteins involved in cytoskeletal reorganization, trafficking and signal termination that binds Shc1 with delayed kinetics, largely through the SgK269 pseudokinase/adaptor protein. Ptpn12 acts as a switch to convert Shc1 from pTyr/Grb2-based signalling to SgK269-mediated pathways that regulate cell invasion and morphogenesis. The Shc1 scaffold therefore directs the temporal flow of signalling information after EGF stimulation.

Cancer proteome and metabolite changes linked to SHMT2.

Serine hydroxymethyltransferase 2 (SHMT2) converts serine plus tetrahydrofolate (THF) into glycine plus methylene-THF and is upregulated at the protein level in lung and other cancers. In order to better understand the role of SHMT2 in cancer a model system of HeLa cells engineered for inducible over-expression or knock-down of SHMT2 was characterized for cell proliferation and changes in metabolites and proteome as a function of SHMT2. Ectopic over-expression of SHMT2 increased cell proliferation in vitro and tumor growth in vivo. Knockdown of SHMT2 expression in vitro caused a state of glycine auxotrophy and accumulation of phosphoribosylaminoimidazolecarboxamide (AICAR), an intermediate of folate/1-carbon-pathway-dependent de novo purine nucleotide synthesis. Decreased glycine in the HeLa cell-based xenograft tumors with knocked down SHMT2 was potentiated by administration of the anti-hyperglycinemia agent benzoate. However, tumor growth was not affected by SHMT2 knockdown with or without benzoate treatment. Benzoate inhibited cell proliferation in vitro, but this was independent of SHMT2 modulation. The abundance of proteins of mitochondrial respiration complexes 1 and 3 was inversely correlated with SHMT2 levels. Proximity biotinylation in vivo (BioID) identified 48 mostly mitochondrial proteins associated with SHMT2 including the mitochondrial enzymes Acyl-CoA thioesterase (ACOT2) and glutamate dehydrogenase (GLUD1) along with more than 20 proteins from mitochondrial respiration complexes 1 and 3. These data provide insights into possible mechanisms through which elevated SHMT2 in cancers may be linked to changes in metabolism and mitochondrial function.

Purification and proteomic analysis of lysosomal integral membrane proteins.

Lysosomes are essential for normal function of cells. This is best illustrated by the occurrence of greater than 40 lysosomal storage diseases. While the enzymes of the luminal compartment have been widely studied usually in the context of these diseases, the composition of the enveloping membrane has received scant attention. Advances in mass spectrometry and proteomics have laid the necessary groundwork to facilitate investigation of membranes such as those of lysosomes, mitochondria, and other organelles to find novel proteins and novel functions. Pure lysosomes are a prerequisite, and we have successfully identified an abundance of membrane proteins from lysosomes of rat liver. Here, we describe two comparable and easy methods to isolate lysosomes from mouse or rat liver in sufficient quantities for proteomics studies. Also included is a comparison of the soluble, luminal proteins obtained from each of the two preparations separated by 2D immobilized pH gradient (IPG) sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).

Functional post-translational proteomics approach to study the role of N-glycans in the development of Caenorhabditis elegans.

Glycosylation is one of the most common post-translational protein modifications. Carbohydrate-mediated interactions between cells and their environment are important in differentiation, embryogenesis, inflammation, cancer and metastasis and other processes. Humans and mice with mutations that prevent normal N-glycosylation show multi-systemic defects in embryogenesis, thereby proving that these molecules are essential for normal development; however, a large number of proteins undergo defective glycosylation in these human and mouse mutants, and it is therefore difficult to determine the precise molecular roles of specific N-glycans on individual proteins. We describe here a 'functional post-translational proteomics' approach that is designed to determine the role of N-glycans on individual glycoproteins in the development of Caenorhabditis elegans.

The in vivo brain interactome of the amyloid precursor protein.

Despite intense research efforts, the physiological function and molecular environment of the amyloid precursor protein has remained enigmatic. Here we describe the application of time-controlled transcardiac perfusion cross-linking, a method for the in vivo mapping of protein interactions in intact tissue, to study the interactome of the amyloid precursor protein (APP). To gain insights into the specificity of reported protein interactions the study was extended to the mammalian amyloid precursor-like proteins (APLP1 and APLP2). To rule out sampling bias as an explanation for differences in the individual datasets, a small scale quantitative iTRAQ (isobaric tags for relative and absolute quantitation)-based comparison of APP, APLP1, and APLP2 interactomes was carried out. An interactome map was derived that confirmed eight previously reported interactions of APP and revealed the identity of more than 30 additional proteins that reside in spatial proximity to APP in the brain. Subsequent validation studies confirmed a physiological interaction between APP and leucine-rich repeat and Ig domain-containing protein 1, demonstrated a strong influence of Ig domain-containing protein 1 on the proteolytic processing of APP, and consolidated similarities in the biology of APP and p75.

A method for proteomic identification of membrane-bound proteins containing Asn-linked oligosaccharides.

Glycosylated proteins on the cell surface have been shown to be essential for cell-cell interactions in development and differentiation. Our ultimate goal is to identify Asn-linked oligosaccharides that are directly involved in these critical in vivo functions. Because such oligosaccharides would be expected to reside on the integral plasma membrane proteins, and conventional two-dimensional gel techniques are ineffective at separating such proteins, we have developed a new approach to their identification on a proteomics scale from Caenorhabditis elegans. Membrane proteins are solubilized in guanidine-HCl, precipitated, and digested with trypsin. The glycopeptides are then separated by lectin chromatography. Next, glycopeptidase F digestion removes the oligosaccharides from the peptides and converts to Asp each Asn to which one was attached. The peptides are then analyzed by matrix-assisted laser desorption/ionization quadrupole time-of-flight (MALDI-Q-TOF) mass spectrometry. Thus, the membrane glycoproteins are identified through the sequence tags of these peptides and the conversion of at least one deduced Asn residue to Asp at the Asn-X-Ser/Thr consensus sequence. To validate the utility of this approach, we have identified 13 membrane-bound N-glycosylated proteins from the major peaks observed on MALDI-Q-TOF analysis of our total glycopeptide fraction.