title: Characterization of Cbl-Nck and Nck-Pak1 interactions in myeloid FcgammaRII signaling.
abstract: Fc receptors modulate inflammatory processes, including phagocytosis, serotonin and histamine release, superoxide production, and secretion of cytokines. Aggregation of FcgammaRIIa, the low-affinity receptor for monomeric IgG, activates nonreceptor protein tyrosine kinases such as Lyn, Hck, and Syk, potentially driving the phosphorylation of the downstream adaptor proteins, including Cbl and/or Nck. Previous work from our laboratory using interferon-gamma-differentiated U937 (U937IF) myeloid cells investigated mechanisms which regulate Fcgamma receptor-induced assembly of adaptor complexes. Herein we report that FcgammaRII receptor signaling in U937IF and HEL cells involves Cbl and Nck, suggesting that Cbl-Nck interactions may link FcgammaRII to downstream activation of Pak kinase. FcgammaRII crosslinking induced the phosphorylation of Cbl and Nck on tyrosine. The alphaCbl immunoprecipitations revealed constitutive binding of Nck and Grb2 to Cbl and FcgammaRII-inducible binding of CrkL to Cbl. The interactions of Cbl with Nck and CrkL were phosphorylation dependent since dephosphorylation of cellular proteins with potato acid phosphatase abrogated binding. GST-Nck fusion protein pulldown experiments show that Cbl and Pak1 bind to the second SH3 domain of Nck. A specific Src inhibitor, PP1, was shown to completely abrogate the FcgammaR-induced superoxide response, correlating with a decrease in Cbl and Nck tyrosine phosphorylation. Our results provide the first evidence that Src is required for FcgammaR activation of the respiratory burst in myeloid cells and suggest that Cbl-Nck, Cbl-Pak1, and Nck-Pak1 interactions may regulate this response.
authors.affiliation: The Neil Bogart Memorial Laboratories, Division of Hematology-Oncology, Los Angeles, California, 90027, USA.
authors.forename: K D
authors.initials: KD
authors.lastname: Izadi
authors.forename: A
authors.initials: A
authors.lastname: Erdreich-Epstein
authors.forename: Y
authors.initials: Y
authors.lastname: Liu
authors.forename: D L
authors.initials: DL
authors.lastname: Durden
mesh_terms: D000135:Acid Phosphatase / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D015703:Antigens, CD / Q000378:metabolism*; D002460:Cell Line; D051380:GRB2 Adaptor Protein; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D050722:Oncogene Protein v-cbl; D015513:Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000037:antagonists & inhibitors / Q000502:physiology; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D017452:Receptors, IgG / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016897:Respiratory Burst / Q000187:drug effects; D015682:Retroviridae Proteins, Oncogenic / Q000378:metabolism*; D015398:Signal Transduction; D020298:U937 Cells; D054462:p21-Activated Kinases; D018909:src Homology Domains
chemical_list: C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; D048868:Adaptor Proteins, Signal Transducing; D015703:Antigens, CD; C082869:CRKL protein; C080571:Fc gamma receptor IIB; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; C078332:Nck protein; D009687:Nuclear Proteins; D050722:Oncogene Protein v-cbl; D015513:Oncogene Proteins; D011506:Proteins; D011720:Pyrazoles; D011743:Pyrimidines; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins; D015682:Retroviridae Proteins, Oncogenic; D019000:Phosphotyrosine; D016392:Proto-Oncogene Proteins pp60(c-src); C515903:PAK1 protein, human; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D000135:Acid Phosphatase; D017027:Protein Tyrosine Phosphatases