The Cbl-interacting 85-kDa protein (CIN85) plays an important role as a negative regulator of signaling pathways induced by receptor tyrosine kinases. By assembling multiprotein complexes this versatile adaptor enhances receptor tyrosine kinase-activated clathrin-mediated endocytosis and reduces phosphatidylinositol-3-kinase-induced phosphatidylinositol-3,4,5-trisphosphate production. Here we report the expression of CIN85 in primary splenic B lymphocytes and the B-lymphoma cell lines WEHI 231 and Ba/F3. Cross-linking of the B cell antigen receptor resulted in an increased association of CIN85 with the ubiquitin ligase Cbl. Through a systematic pull-down proteomics approach we identified 51 proteins that interact with CIN85 in B cells, including proteins not shown previously to be CIN85-associated. Among these proteins, the SH2-containing inositol phosphatase 1 (SHIP-1) co-precipitated with both the fulllength CIN85 and each of its three SH3 domains. We also showed that this association is constitutive and depends on a region of 79 amino acids near the carboxyl terminus of SHIP-1, a region rich in potential SH3 domain binding sites. Because SHIP-1 is a major negative regulator of the phosphatidylinositol-3-kinase pathway in lymphocytes, we hypothesize that the interaction between SHIP-1 and CIN85 might synergistically facilitate the down-regulation of phosphatidylinositol-3,4,5-trisphosphate levels. Molecular &

On examining different proteomics approaches for the investigation of structure-function relationships of erythropoietin (EPO) receptor signaling, it was found that two-dimensional gel electrophoresis/mass spectrometry procedures are clearly limited in their ability to detect low-expressed signaling proteins. Instead it was found that a strategy involving anti-phosphotyrosine immunoprecipitation, one-dimensional gel electrophoresis (1DE), and capillary liquid chromatography/tandem mass spectrometry (LC/MS/MS) provides the sensitivity required for identification of signaling proteins. In the present work the immunoprecipitation/1DE/LC/MS approach was combined with an in-gel 18O-labeling technique to analyze EPO receptor-dependent proteins. Identification and relative quantification of more than 180 EPO receptor-dependent proteins were achieved directly based on the in-gel 18O-labeling approach.

Proteomic techniques provide new tools for the global analysis of protein profiles but also for the investigation of specific protein functions. The analysis of signaling cascades has traditionally been performed by the determination of enzymatic or transcription factor activities representing a certain pathway. Functional proteomics now allows more comprehensive approaches to study cellular responses induced during ligand/receptor interactions. In this study we evaluated proteomic strategies for the investigation of structure-function relationships in the erythropoietin receptor signalling complex. After expression of epidermal growth factor/erythropoietin receptor mutant molecules in an identical cellular background we characterized their potential to induce cellular activities. Using this system we focused our efforts on post-translational modifications of signalling proteins reflecting a substantial part of receptor-dependent signaling events. Although tyrosine phosphorylated proteins were enriched by immunoprecipitation the analysis using the classical approach combining two-dimensional gel electrophoresis and identification by matrix assisted laser desorption/ionization-time of flight-mass spectrometry revealed that low expressed signaling proteins cannot be detected by this technique. An alternative strategy using one-dimensional gel separation of phosphoproteins and liquid chromatography-tandem mass spectrometry, however, allowed us to identify multiple proteins involved in intracellular signalling representing already established pathways but also proteins which have not been linked to EPO-induced signaling so far. This approach offers the potential to extend functional proteomic studies to complex signaling processes.

In mammals, erythropoietin regulates the development and differentiation of erythrocytes. Although hematopoietic cells of bony fish correspond in their ontogeneic development, morphology, and function to their mammalian counterparts, an erythropoietin (EPO)-like molecule has not been identified. In this study we present evidence for a mitogenic response of blood and head kidney leukocytes of rainbow trout after stimulation by recombinant human EPO (rhu EPO). The modulation of cellular activities is accompanied by the induction of DNA-binding activities in nuclear extracts of these cells. In addition, flow cytometric analysis of intracellular Ca2+ concentrations revealed a long-lasting and rhu EPO dose-dependent increase, which was shown to be abrogated by cross-aggregation of surface IgM using anti-trout-IgM monoclonal antibodies (mabs). In flow cytometric dual-labeling experiments using rhu EPO/anti-EPO antiserum and mabs specific for trout leukocyte subpopulations, it was shown that a subpopulation of trout B-cells binds rhu EPO. Moreover, in a modified Ca2+ activation assay, it was demonstrated that this blood B-cell subpopulation is the rhu EPO responder population. In conclusion, the data suggest the existence of EPO-binding receptors in trout that are able to trigger Ca(2+)-independent intracellular signaling in hematopoietic cells of head kidney and Ca(2+)-dependent activation of a subpopulation of B-lymphocytes.

The development of erythroid progenitor cells is triggered via the expression of the erythropoietin receptor (EPOR) and its activation by erythropoietin. The function of the resulting receptor complex depends critically on the presence of activated JAK2, and the complex contains a large number of signaling molecules recruited to eight phosphorylated tyrosine residues. Studies using mutant receptor forms have demonstrated that truncated receptors lacking all tyrosines are able to support red blood cell development with low efficiency, whereas add-back mutants containing either Tyr 343 or Tyr 479 reconstitute EPOR signaling and erythropoiesis in vivo. To study the contribution of tyrosines to receptor function, we analyzed the activation of essential signaling pathways and early gene induction promoted by different receptor structural variants using human epidermal growth factor receptor/murine EPOR hybrids. In our experiments, receptors lacking all tyrosine residues or the JAK2-binding site did not induce mitogenic and anti-apoptotic signaling, whereas add-back mutant receptors containing single tyrosine residues (Try 343 and Tyr 479 ) supported the activation of these functions efficiently. Profiling of early gene expression using cDNA array hybridization revealed that (i) the high redundancy in the activation of signaling pathways is continued at the level of transcription; (ii) the expression of many genes targeted by the wild-type receptor is not supported by add-back mutants; and (iii) a small set of genes are exclusively induced by add-back receptors. We report the identification of several early genes that have not been implicated in the EPOR-dependent response so far. Erythropoietin (EPO),2 the prime growth factor regulating the erythroid lineage, induces the proliferation of immature red blood cells and their differentiation into mature erythrocytes (1). The corresponding EPO receptor (EPOR), a member of the type 1 cytokine receptor family, is expressed on the surface of erythroid burst-and colony-forming units (2). In contrast to other receptor types, these receptors lack any intrinsic activity and are activated by the induction of a special conformation of pre-existing homodimers after ligand binding (3). The recruitment and activation of the cytosolic tyrosine kinase JAK2 have been shown to be the first and crucial steps in the formation of a multiprotein receptor complex that is responsible for the initiation of diverse signaling pathways (4). Activated JAK2 mediates the phosphorylation of tyrosine residues within the intracellular receptor domain, providing docking sites for Src homology 2 (SH2) domain-encoding effector molecules, including the JAK2 substrate STAT5, a transcription factor that is rapidly translocated to the nucleus (5). Among additional proteins that have been shown to be physically associated with the EPOR following ligand binding are phospholipase C␥1 (6) and phosphatidylinositol 3-kinase (7). Furthermore, negative regulators are also engaged and terminate receptor-dependent signa...