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Abstract
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 full-length 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.
Highlights
B lymphocytes require a precise regulation of their activation status, because aberrances may lead to severe dysfunctions associated with hyper- or hyposensitivity
B cell antigen receptor (BCR) Cross-linking Increases Binding of Casitas B-lineage lymphoma (Cbl)-interacting 85-kDa protein (CIN85) to Cbl—At first we determined if CIN85-related events, such as the RTKinduced association between CIN85 and the ubiquitin ligase Cbl, occur in B cells
Using increasing concentrations of anti-IgM Abs, BCRs were cross-linked in WEHI 231 cells and Cbl was immunoprecipitated from cytosolic extracts and subjected to Western blot analysis using various Abs
Summary
Cell Culture, Stimulation, and Transfection—Murine Ba/F3 B cells (#ACC 300, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were cultured in RPMI 1640 (Biochrom AG, Berlin, Germany) supplemented with 10% fetal calf serum (FCS) (Invitrogen, Karlsruhe, Germany), 50 M -mercaptoethanol and 25 pM recombinant murine IL-3 (rmIL-3, Biomol, Hamburg, Germany) [20]. After 48 h cells (2–3 ϫ 107) were lysed in 1 ml PBS containing 1% (v/v) Triton X-100 and protease inhibitors (“Complete”, Roche Applied Science, Mannheim, Germany) for 30 min at 4 °C. Nuclei were pelleted by centrifugation (18,000 ϫ g) and cytosolic extracts incubated for 3 h at 4 °C with primary Abs specific for Cbl (C-15), SHIP-1 (P1C1), HA-tag (F-7), CIN85 (S-19) (Santa Cruz Biotechnology, Heidelberg, Germany), CIN85 (clone 179.1.E1) or CIN85 (clone 84) from Upstate (Lake Placid, NY, USA). (C-15), Grb (C-23), SHIP-1 (P1C1), -Actin (C4), Dynamin-2 (C-18), BCAP (S-15), N-WASP (H-100), and HA-tag (F-7) from Santa Cruz Biotechnology (Heidelberg, Germany), WASP, HPK1, and PhosphoErk1/2 (E10) from New England Biolabs (Frankfurt am Main, Germany) and GFP from Roche Applied Science (Mannheim, Germany)
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