Engineering Improved Antiphosphotyrosine Antibodies Based on an Immunoconvergent Binding Motif.

Phosphotyrosine (pY) is one of the most highly studied posttranslational modifications that is responsible for tightly regulating many signaling pathways in eukaryotes. Pan-specific pY antibodies have emerged as powerful tools for understanding the role of these modifications. Nevertheless, structures have not been reported for pan-specific pY antibodies, greatly impeding the further development of tools for integrating this ubiquitous posttranslational modification using structure-guided designs. Here, we present the first crystal structures of two widely utilized pan-specific pY antibodies, PY20 and 4G10. The two antibodies, although developed independently from animal immunizations, have surprisingly similar modes of recognition of the phosphate group, implicating a generic binding structure among pan-specific pY antibodies. Sequence alignments revealed that many pY binding residues are predominant in the mouse V germline genes, which consequently led to the convergent antibodies. On the basis of the convergent structure, we designed a phage display library by lengthening the CDR-L3 loop with the aid of computational modeling. Panning with this library resulted in a series of 4G10 variants with 4 to 11-fold improvements in pY binding affinities. The crystal structure of one improved variant showed remarkable superposition to the computational model, where the lengthened CDR-L3 loop creates an additional hydrogen bond indirectly bound to the phosphate group via a water molecule. The engineered variants exhibited superior performance in Western blot and immunofluorescence.

Monoclonal Antibody That Recognizes Diethoxyphosphotyrosine-Modified Proteins and Peptides Independent of Surrounding Amino Acids.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are irreversibly inhibited by organophosphorus pesticides through formation of a covalent bond with the active site serine. Proteins that have no active site serine, for example albumin, are covalently modified on tyrosine and lysine. Chronic illness from pesticide exposure is not explained by inhibition of AChE and BChE. Our goal was to produce a monoclonal antibody that recognizes proteins diethoxyphosphorylated on tyrosine. Diethoxyphosphate-tyrosine adducts for 13 peptides were synthesized. The diethoxyphosphorylated (OP) peptides cross-linked to four different carrier proteins were used to immunize, boost, and screen mice. Monoclonal antibodies were produced with hybridoma technology. Monoclonal antibody depY was purified and characterized by ELISA, western blotting, Biacore, and Octet technology to determine binding affinity and binding specificity. DepY recognized diethoxyphosphotyrosine independent of the amino acid sequence around the modified tyrosine and independent of the identity of the carrier protein or peptide. It had an IC50 of 3 × 10-9 M in a competition assay with OP tubulin. Kd values measured by Biacore and OctetRED96 were 10-8 M for OP-peptides and 1 × 10-12 M for OP-proteins. The limit of detection measured on western blots hybridized with 0.14 µg/mL of depY was 0.025 µg of human albumin conjugated to YGGFL-OP. DepY was specific for diethoxyphosphotyrosine (chlorpyrifos oxon adduct) as it failed to recognize diethoxyphospholysine, phosphoserine, phosphotyrosine, phosphothreonine, dimethoxyphosphotyrosine (dichlorvos adduct), dimethoxyphosphoserine, monomethoxyphosphotyrosine (aged dichlorvos adduct), and cresylphosphoserine. In conclusion, a monoclonal antibody that specifically recognizes diethoxyphosphotyrosine adducts has been developed. The depY monoclonal antibody could be useful for identifying new biomarkers of OP exposure.

Selective Targeting of SH2 Domain-Phosphotyrosine Interactions of Src Family Tyrosine Kinases with Monobodies.

The binding of Src-homology 2 (SH2) domains to phosphotyrosine (pY) sites is critical for the autoinhibition and substrate recognition of the eight Src family kinases (SFKs). The high sequence conservation of the 120 human SH2 domains poses a significant challenge to selectively perturb the interactions of even the SFK SH2 family against the rest of the SH2 domains. We have developed synthetic binding proteins, termed monobodies, for six of the SFK SH2 domains with nanomolar affinity. Most of these monobodies competed with pY ligand binding and showed strong selectivity for either the SrcA (Yes, Src, Fyn, Fgr) or SrcB subgroup (Lck, Lyn, Blk, Hck). Interactome analysis of intracellularly expressed monobodies revealed that they bind SFKs but no other SH2-containing proteins. Three crystal structures of monobody-SH2 complexes unveiled different and only partly overlapping binding modes, which rationalized the observed selectivity and enabled structure-based mutagenesis to modulate inhibition mode and selectivity. In line with the critical roles of SFK SH2 domains in kinase autoinhibition and T-cell receptor signaling, monobodies binding the Src and Hck SH2 domains selectively activated respective recombinant kinases, whereas an Lck SH2-binding monobody inhibited proximal signaling events downstream of the T-cell receptor complex. Our results show that SFK SH2 domains can be targeted with unprecedented potency and selectivity using monobodies. They are excellent tools for dissecting SFK functions in normal development and signaling and to interfere with aberrant SFK signaling networks in cancer cells.

Sensitive Approaches for the Assay of the Global Protein Tyrosine Phosphorylation in Complex Samples Using a Mutated SH2 Domain.

Temporal tyrosine phosphorylation (pTyr) plays a crucial role in numerous cellular functions. The characterization of the tyrosine phosphorylation states of cells is of great interest for understanding the underlying mechanisms. In this study, we developed sensitive and cost-effective methods for the assay of the global protein tyrosine phosphorylation in complex samples by using a novel engineered pTyr binding protein, Src SH2 domain triple-point mutant (Trm-SH2). Taking the advantage of the pan-specific interaction of Trm-SH2 to pTyr, a high throughput approach was developed to determine the total protein tyrosine phosphorylation level in a sample. This method allowed the detection of 0.025 ng of tyrosine phosphorylated proteins. The Trm-SH2 was further exploited to develop a method to profile the global tyrosine phosphorylation state. When this approach was applied to analyze the tyrosine phosphoproteome upon stimulation, distinct patterns were observed. This approach is readily used in many research and clinical fields for the analysis of tyrosine phosphorylated proteins in complex samples, including classifying aberrant phosphotyrosine-dependent signaling in cancer.

Characterization of Receptor-Associated Protein Complex Assembly in Interleukin (IL)-2- and IL-15-Activated T-Cell Lines.

It remains a paradox that IL-2 and IL-15 can differentially modulate the immune response using the same signaling receptors. We have previously dissected the phosphotyrosine-driven signaling cascades triggered by both cytokines in Kit225 T-cells, unveiling subtle differences that may contribute to their functional dichotomy. In this study, we aimed to decipher the receptor complex assembly in IL-2- and IL-15-activated T-lymphocytes that is highly orchestrated by site-specific phosphorylation events. Comparing the cytokine-induced interactome of the interleukin receptor beta and gamma subunits shared by the two cytokines, we defined the components of the early IL-2 and IL-15 receptor-associated complex discovering novel constituents. Additionally, phosphopeptide-directed analysis allowed us to detect several cytokine-dependent and -independent phosphorylation events within the activated receptor complex including novel phosphorylated sites located in the cytoplasmic region of IL-2 receptor ß subunit (IL-2Rß). We proved that the distinct phosphorylations induced by the cytokines serve for recruiting different types of effectors to the initial receptor/ligand complex. Overall, our study sheds new light into the initial molecular events triggered by IL-2 and IL-15 and constitutes a further step toward a better understanding of the early signaling aspects of the two closely related cytokines in T-lymphocytes.

Systematic analysis of phosphotyrosine antibodies recognizing single phosphorylated EPIYA-motifs in CagA of East Asian-type Helicobacter pylori strains.

BACKGROUND: Highly virulent strains of the gastric pathogen Helicobacter pylori encode a type IV secretion system (T4SS) that delivers the effector protein CagA into gastric epithelial cells. Translocated CagA undergoes tyrosine phosphorylation by members of the oncogenic c-Src and c-Abl host kinases at EPIYA-sequence motifs A, B and D in East Asian-type strains. These phosphorylated EPIYA-motifs serve as recognition sites for various SH2-domains containing human proteins, mediating interactions of CagA with host signaling factors to manipulate signal transduction pathways. Recognition of phospho-CagA is mainly based on the use of commercial pan-phosphotyrosine antibodies that were originally designed to detect phosphotyrosines in mammalian proteins. Specific anti-phospho-EPIYA antibodies for each of the three sites in CagA are not forthcoming. RESULTS: This study was designed to systematically analyze the detection preferences of each phosphorylated East Asian CagA EPIYA-motif by pan-phosphotyrosine antibodies and to determine a minimal recognition sequence. We synthesized phospho- and non-phosphopeptides derived from each predominant EPIYA-site, and determined the recognition patterns by seven different pan-phosphotyrosine antibodies using Western blotting, and also investigated representative East Asian H. pylori isolates during infection. The results indicate that a total of only 9-11 amino acids containing the phosphorylated East Asian EPIYA-types are required and sufficient to detect the phosphopeptides with high specificity. However, the sequence recognition by the different antibodies was found to bear high variability. From the seven antibodies used, only four recognized all three phosphorylated EPIYA-motifs A, B and D similarly well. Two of the phosphotyrosine antibodies preferentially bound primarily to the phosphorylated motif A and D, while the seventh antibody failed to react with any of the phosphorylated EPIYA-motifs. Control experiments confirmed that none of the antibodies reacted with non-phospho-CagA peptides and in accordance were able to recognize phosphotyrosine proteins in human cells. CONCLUSIONS: The results of this study disclose the various binding preferences of commercial anti-phosphotyrosine antibodies for phospho-EPIYA-motifs, and are valuable in the application for further characterization of CagA phosphorylation events during infection with H. pylori and risk prediction for gastric disease development.

A general assay for monitoring the activities of protein tyrosine phosphatases in living eukaryotic cells.

Protein tyrosine phosphatases (PTPs) are key signal-transduction regulators and have emerged as potential drug targets for inhibitor design. Here we report a yeast-based assay that provides a general means of assessing the activity and/or inhibition of essentially any classical PTP in living cells. The assay uses the activity of an exogenously expressed PTP to counter the activity of a coexpressed and toxic tyrosine kinase, such that only active PTPs are capable of rescuing growth. PTP activity gives rise to both increased growth and decreased phosphotyrosine levels; cellular PTP activity can therefore be monitored by either yeast-growth curves or anti-phosphotyrosine Western blots. We show that four PTPs (TCPTP, Shp2, PEST, PTPα) are capable of rescuing the effects of v-Src toxicity. Since these PTPs are chosen from four distinct subfamilies, it is likely that biologically and medicinally important PTPs from other subfamilies can similarly function in the cellular PTP assay. Because many small-molecule PTP inhibitors fail to penetrate cell membranes effectively, this cell-based assay has the potential to serve as a useful screening tool for determining the cellular efficacy of candidate inhibitors in a more biologically relevant context than can be provided by an in vitro PTP assay.

HemITAM signaling by CEACAM3, a human granulocyte receptor recognizing bacterial pathogens.

Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) belong to the immunoglobulin superfamily and contribute to cell-cell adhesion and signal modulation in various tissues. In humans, several CEACAMs are targeted by pathogenic bacteria. One peculiar member of this family, CEACAM3, is exclusively expressed by human granulocytes and functions as an opsonin-independent phagocytic receptor for CEACAM-binding bacteria. Here, we will discuss CEACAM3-dependent processes by summarizing recent insight into the phosphotyrosine-based signaling complex formed upon CEACAM3 engagement. Compared to different well-studied phagocytic receptors, such as Fcγ receptors and Dectin-1, CEACAM3 appears as an example of a hemITAM-containing innate immune receptor, which promotes rapid internalization and intracellular destruction of a diverse group of CEACAM-binding bacteria. The particular efficiency of CEACAM3 arises from the direct coupling of upstream activators and downstream effectors of the small GTPase Rac by the cytoplasmic domain of CEACAM3, which co-ordinates actin cytoskeleton re-arrangements and bactericidal effector mechanisms of granulocytes.

Ubiquitin-activating enzyme (UBA1) is required for sperm capacitation, acrosomal exocytosis and sperm-egg coat penetration during porcine fertilization.

Protein ubiquitination is a stable, covalent post-translational modification that alters protein activity and/or targets proteins for proteolysis by the 26S proteasome. The E1-type ubiquitin-activating enzyme (UBA1) is responsible for ubiquitin activation, the initial step of ubiquitin-protein ligation. Proteasomal proteolysis of ubiquitinated spermatozoa and oocyte proteins occurs during mammalian fertilization, particularly at the site of sperm acrosome contact with oocyte zona pellucida. However, it is not clear whether the substrates are solely proteins ubiquitinated during gametogenesis or if de novo ubiquitination also occurs during fertilization supported by ubiquitin-activating and -conjugating enzymes present in the sperm acrosome. Along this line of inquiry, UBA1 was detected in boar sperm-acrosomal extracts by Western blotting (WB). Immunofluorescence revealed accumulation of UBA1 in the nuclei of spermatogonia, spermatocytes and spermatids, and in the acrosomal caps of round and elongating spermatids. Thiol ester assays utilizing biotinylated ubiquitin and isolated sperm acrosomes confirmed the enzymatic activity of the resident UBA1. A specific UBA1 inhibitor, PYR-41, altered the remodelling of the outer acrosomal membrane (OAM) during sperm capacitation, monitored using flow cytometry of fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Although viable and motile, the spermatozoa capacitated in the presence of PYR-41, showed significantly reduced fertilization rates during in vitro fertilization (IVF; p < 0.05). Similarly, the fertilization rate was lowered by the addition of PYR-41 directly into fertilization medium during IVF. In WB, high Mr bands, suggestive of protein ubiquitination, were detected in non-capacitated spermatozoa by antibodies against ubiquitin; WB with anti-phosphotyrosine antibodies and antibodies against acrosomal proteins SPINK2 (acrosin inhibitor) and AQN1 (spermadhesin) revealed that the capacitation-induced modification of those proteins was altered by PYR-41. In summary, it appears that de novo protein ubiquitination involving UBA1 contributes to sperm capacitation and acrosomal function during fertilization.

A strategy for identification of protein tyrosine phosphorylation.

To develop methods for studying phosphorylation of protein tyrosine residues is an important task since this protein modification regulates many cellular functions and often is involved in oncogenesis. An optimal protocol includes enrichment of tyrosine phosphorylated (pTyr) peptides or proteins, followed by a high resolving analytical method for identification of the enriched components. In this Methods paper, we describe a working strategy on how immunoaffinity enrichments, using anti-pTyr antibodies, combined with mass spectrometric (MS) analysis can be used to study the pTyr proteome. We describe in detail how our procedure was used to characterize the pTyr proteome of K562 leukemia cells. Important questions concerning the use of different anti-pTyr antibodies, enrichments performed at the peptide and/or the protein level, pooling of enrichments and requirements for the MS characterization are discussed.

Development of unique antibodies directed against each of the six different phosphotyrosine residues within the T cell receptor CD3ζ chain.

Signal transduction from the T cell antigen receptor (TCR)/CD3 complex involves six different immunoreceptor tyrosine-based activation motifs (ITAM) located within the cytoplasmic tails of the CD3 chains. Each ITAM possesses two conserved tyrosine residues that can undergo phosphorylation upon TCR/CD3 crosslinking and become a docking site for SH2-containing effector molecules. Specificity of the SH2 domains is determined by their ability to bind a phosphorylated tyrosine in the context of a longer peptide motif within the target protein. As a result, phosphorylation of different tyrosines within the CD3 cytoplasmic tails creates docking sites for distinct SH2-containing signaling proteins that differentially impact on the quality of the T cell response. In the present study, we prepared antibodies specific for each of the six different phosphotyrosines of the mouse CD3ζ chain. The antibodies were characterized with respect to their cross-reactivity, ability to recognize the phosphorylated versus non-phosphorylated forms of tyrosine-containing motifs, and cross-reactivity with the homologous phospho-motifs on the human CD3ζ protein. The antibodies were found to be specific and selective for phospho-CD3ζ. They can serve as useful tools for distinguishing between the six potential tyrosine phosphorylation sites on the CD3ζ chain, and for correlating the phosphorylation of specific CD3ζ tyrosine residues with activation of signaling pathways that dictate T cell differentiation into responding, anergic, or apoptotic cells.

Syk protein tyrosine kinase involves PECAM-1 signaling through tandem immunotyrosine inhibitory motifs in human THP-1 macrophages.

Although recent evidence supports a functional relationship between platelet endothelial cell adhesion molecule (PECAM-1) and Syk tyrosine kinase, little is known about the interaction of Syk with PECAM-1. We report that down-regulation of Syk inhibits the spreading of human THP-1 macrophage cells. Moreover, our data indicate that Syk binds PECAM-1 through its immune tyrosine-based inhibitory motif (ITIM), and dual phosphorylation of the ITIM domain of PECAM-1 leads to activation of Syk. Our results indicate that the distance between the phosphotyrosines could be up to 22 amino acids in length, depending on the conformational flexibility, and that the dual ITIM tyrosine motifs of PECAM-1 facilitate immunoreceptor tyrosine-based activation motif-like signaling. The preferential binding of PECAM-1 to Src homology region 2 domain-containing phosphatase-2 or Syk may depend on their relative affinities, and could provide a mechanism by which signal transduction from PECAM-1 is internally regulated by both positive and negative signaling enzymes.

Rapid and reproducible single-stage phosphopeptide enrichment of complex peptide mixtures: application to general and phosphotyrosine-specific phosphoproteomics experiments.

Reversible protein phosphorylation is an essential regulatory component of virtually every cellular process and is frequently dysregulated in cancer. However, significant analytical barriers persist that hamper the routine application of phosphoproteomics in translational settings. Here, we present a straightforward and reproducible approach for the broadscale analysis of protein phosphorylation that relies on a single phosphopeptide enrichment step using titanium dioxide microspheres from whole cell lysate digests and compared it to the well-established SCX-TiO(2) workflow for phosphopeptide purification on a proteome-wide scale. We demonstrate the scaleabilty of our approach from 200 µg to 5 mg of total NCI-H23 non-small cell lung adenocarcinoma cell lysate digest and determine its quantitative reproducibility by label-free analysis of phosphopeptide peak areas from replicate purifications (median CV: 20% RSD). Finally, we combine this approach with immunoaffinity phosphotyrosine enrichment, enabling the identification of 3168 unique nonredundant phosphotyrosine peptides in two LC-MS/MS runs from 8 mg of HeLa peptides, each with 80% phosphotyrosine selectivity, at a peptide FDR of 0.2%. Taken together, we establish and validate a robust approach for proteome-wide phosphorylation analysis in a variety of scenarios that is easy to implement in biomedical research and translational settings.

Issues associated with the use of phosphospecific antibodies to localise active and inactive pools of GSK-3 in cells.

BACKGROUND: Glycogen synthase kinase-3 (GSK-3) is a ubiquitously expressed serine/threonine (Ser/Thr) kinase comprising two isoforms, GSK-3α and GSK-3ß. Both enzymes are similarly inactivated by serine phosphorylation (GSK-3α at Ser21 and GSK-3ß at Ser9) and activated by tyrosine phosphorylation (GSK-3α at Tyr279 and GSK-3ß at Tyr216). Antibodies raised to phosphopeptides containing the sequences around these phosphorylation sites are frequently used to provide an indication of the activation state of GSK-3 in cell and tissue extracts. These antibodies have further been used to determine the subcellular localisation of active and inactive forms of GSK-3, and the results of those studies support roles for GSK-3 phosphorylation in diverse cellular processes. However, the specificity of these antibodies in immunocytochemistry has not been addressed in any detail. RESULTS: Taking advantage of gene silencing technology, we examined the specificity of several commercially available anti-phosphorylated GSK-3 antibodies. We show that antibodies raised to peptides containing the phosphorylated Ser21/9 epitope crossreact with unidentified antigens that are highly expressed by mitotic cells and that mainly localise to spindle poles. In addition, two antibodies raised to peptides containing the phosphorylated Tyr279/216 epitope recognise an unidentified protein at focal contacts, and a third antibody recognises a protein found in Ki-67-positive cell nuclei. While the phosphorylated Ser9/21 GSK-3 antibodies also recognise other proteins whose levels increase in mitotic cells in western blots, the phosphorylated Tyr279/216 antibodies appear to be specific in western blotting. However, we cannot rule out the posssibility that they recognise very large or very small proteins that might not be detected using a standard western blotting approach. CONCLUSIONS: Our findings indicate that care should be taken when examining the subcellular localisation of active or inactive GSK-3 and, furthermore, suggest that the role of GSK-3 phosphorylation in some cellular processes be reassessed.

A dynamic immunological synapse mediates homeostatic TCR-dependent and -independent signaling.

For homeostasis, T cells integrate non-cognate TCR-dependent and -independent signals to survive and weakly proliferate. In contrast to antigen-specific, stable, and long-lived contacts, signaling in short-lived homeostatic interactions depends upon the coordination of ongoing T-cell migration on the surface of DC and signaling at the cell-cell junction. To mimic peripheral tissues and analyze how T-cell migration and cell-cell signaling are integrated, we used live-cell imaging and 3-D reconstruction of fixed conjugates between DO11.10 T cells and DC in 3-D low-density collagen matrices. T cells simultaneously maintained amoeboid migration and polarized towards the DC, leading to a fully dynamic interaction plane that delivered signals for homeostatic T-cell survival and proliferation. The contact plane comprised three zones, the actin-rich leading edge poor in signal but driving migration, a mid-zone mediating TCR/MHC-induced signal associated with proliferation, and the rear uropod mediating predominantly MHC-independent signals. Thus a dynamic immunological synapse with distinct signaling sectors enables moving T cells to serially sample resident tissue cells and acquire molecular information "en passant".

Detecting tyrosine-phosphorylated proteins by Western blot analysis.

The development of monoclonal antibodies (mAbs) that recognize nearly all of the phosphorylated tyrosine residues, irrespective of the surrounding sequences, enables researchers to detect the phosphorylation state of proteins through the use of anti-phosphotyrosine western blotting. The availability of this simple, reliable, nonradioactive and yet sensitive method created a boom in signal transduction research. While the methodology of how to perform an anti-phosphotyrosine western blot remains unchanged since the procedure became widely used in the early part of 1990s, steady improvements in reagents and detection technologies have allowed researchers to detect tyrosine phosphorylation quantitatively, at unprecedented sensitivity. In addition to the improvements in the western blot-based systems, powerful new phosphotyrosine detection platforms, based on proteomic technologies, are emerging rapidly. This unit will describe in detail the steps needed to perform the standard anti-phosphotyrosine western blot analysis.

Screening kinase inhibitors with a microarray-based fluorescent and resonance light scattering assay.

In this technical note, a microarray-based spectroscopic assay with two readout principles, fluorescence and resonance light scattering (RLS), for screening kinase inhibitors has been reported. In this assay, the phosphorylation and inhibition events are marked by biotinylated antiphosphoserinen/antiphosphotyrosine antibodies, and gold nanoparticles are attached to the antibodies by standard avidin-biotin chemistry followed by silver deposition for RLS signal enhancement. The avidin conjugated fluorescein is used as a fluorescent probe. Assays for both serine kinase, the alpha-catalytic subunit of cyclic adenosine 5'-monophosphate (cAMP) dependent protein kinase (PKA), and tyrosine kinase, leukocyte-specific protein tyrosine kinase (LCK), have been developed. The utility of this assay to high-throughput screening was demonstrated with a commercial inhibitor library, a collection of 80 kinase inhibitors, and satisfactory results were obtained. In addition, quantitative determination of binding strength and the inhibiting type (type I) of these inhibitors are also demonstrated by the adenosine 5'-triphosphate (ATP) competing assays.

High-throughput kinase assay based on surface plasmon resonance.

We have designed a novel high-throughput (HTP) kinase assay using an array-based surface plasmon resonance (SPR) apparatus. For high flexibility and performance, the kinase assay procedure is divided into an in vitro phosphorylation part and a phospho-detection part on a sensor chip. Not only biotinylated peptides but also recombinant proteins fused with FLAG-GST tandem tag can be used as native substrates. The substrate is selectively captured by a capture antibody immobilized on a sensor chip, and phospho-tyrosine (pTyr) residues are detected by an anti-pTyr antibody. The level of tyrosine phosphorylation is calculated from the capture level of the substrates and the binding level of the anti-pTyr antibody monitored by SPR. A wide dynamic range and real-time monitoring of SPR contribute to improved data reliability, and optimization of the procedure for an array-based apparatus achieved multiple sample processing (1,000 samples/day).

Proteomic analysis of Src family kinases signaling complexes in Golgi/endosomal fractions using a site-selective anti-phosphotyrosine antibody: identification of LRP1-insulin receptor complexes.

A role for Src Family Kinases (SFKs) in the dynamics of endocytic and secretory pathways has previously been reported. Identification of low-abundance compartmentalized complexes still remains challenging, highlighting the need for novel tools. Here we describe analysis of SFK-signaling complexes of hepatic Golgi/endosomes (G/E) fractions by sequential affinity enrichment of proteins. Mouse G/E permeabilized membranes were first validated in terms of electron microscopy, 1-D electrophoresis (1-DE), insulin-mediated endocytosis and protein content. With the use of quantitative N-terminal labeling of tryptic peptides (iTRAQ), 1-DE and IEF tryptic peptides separation methods, a total of 666 proteins were identified, including the SFK Lyn. Following insulin injection, a series of proteins were recognized by an anti-phosphotyrosine antibody (alpha P42-2) raised against the residue most frequently phosphorylated by SFK on the adenoviral protein E4orf4 and that cross-reacts with endosomal SFK targets. By using affinity chromatography coupled with mass spectrometry, we identified 16 proteins classified as (1) recycling receptors, (2) vesicular trafficking proteins, (3) actin network proteins, (4) metabolism proteins, or (5) signaling proteins. One of these proteins, low density lipoprotein-related protein 1 (LRP1), which is a known SFK substrate, was found to associate with the internalized insulin receptor (IR), suggesting the presence of a co-internalization process. The identification of these proteomes should, thus, contribute to a better understanding of the molecular mechanisms that regulate trafficking events and insulin clearance.

IGF-1 receptors in Xenopus laevis ovarian follicle cells support the oocyte maturation response.

Insulin-like growth factor 1 (IGF-1)-stimulated amphibian oocyte maturation has been studied extensively by a number of laboratory groups, but in previous studies possible effects of IGF-1 on ovarian follicle cells had not been tested directly. In the study reported here, biochemical and immunofluorescent techniques were used to test Xenopus ovarian follicle cells for the presence of hormone-sensitive IGF-1 receptor. Anti-xIGF-1 receptor beta-subunit antibodies detected a 90- and 98-kDa protein doublet in manually dissected oocyte cortices (plasma membrane-vitelline envelope complexes) by protein immunoblotting both before and after removal of follicle cells from oocytes by sandpaper rolling. The 90-kDa IGF-1 receptor beta-subunit was also detected in follicle cell pellets. Tyrosine phosphorylation of the receptor beta-subunits was increased by treatment of cortices with 10 nM IGF-1 both in the presence and absence of associated follicle cells, was reduced by removal of follicle cells, and was detected in follicle cell pellets. Treatment of follicle cell pellets with nanomolar concentrations of IGF-1 stimulated receptor tyrosine phosphorylation in a dose-dependent fashion that correlated with dose-dependent stimulation of oocyte maturation. IGF-1 receptor was also detected in cultured follicle cells by immunofluorescence. Removal of follicle cells significantly reduced the IGF-1-stimulated oocyte maturation response. These results offer the first direct evidence for hormone-responsive IGF-1 receptors in Xenopus laevis ovarian follicle cells and demonstrate that follicle cells somehow support IGF-1-stimulated oocyte maturation.