A dynamic constitutive and inducible binding of c-Cbl by PLCgamma1 SH3 and SH2 domains (negatively) regulates antigen receptor-induced PLCgamma1 activation in lymphocytes.
We investigated the structural requirements for c-Cbl-mediated inhibition of Ag receptor-induced PLCgamma1 activation. Analysis of site-specific c-Cbl mutants indicated that tyrosine phosphorylation of c-Cbl was required for down-regulation of the PLCgamma1/Ca2+ pathway. Coprecipitation experiments indicated that c-Cbl and PLCgamma1 constitutively interact through a PLCgamma1 SH3 domain-dependent mechanism and that c-Cbl and PLCgamma1 can inducibly interact through the SH2(C) domain of PLCgamma1. Additional data indicate that the SH3 domain of PLCgamma1 binds to both canonical and noncanonical SH3 domain-binding sites in the proline-rich region of c-Cbl. Overexpression of c-Cbl in a PLCgamma-deficient B cell line, P10-14, stably reconstituted with wild-type PLCgamma1 led to a significant decrease in B cell receptor-induced NF-AT-dependent transcription, a PLCgamma- and Ca(2+)-dependent event. In contrast, c-Cbl overexpression in P10-14 cells reconstituted with a PLCgamma1 SH3 domain mutant had little effect on receptor-induced NF-AT activation. These data suggest that c-Cbl-mediated regulation of PLCgamma1 requires an interaction between c-Cbl and PLCgamma1 that is primarily mediated by the SH3 domain of PLCgamma1. The interaction of c-Cbl with PLCgamma1 may negatively effect events required for PLCgamma1 activation.
Rellahan
Barbara L
BL
Laboratory of Immunobiology, Division of Monoclonal Antibodies, Center for Biologics Evaluation and Research, NIH, Bethesda, MD 20892, USA. rellahan@cber.fda.gov
Graham
Laurie J
LJ
Tysgankov
Alexander Y
AY
DeBell
Karen E
KE
Veri
Maria-Concetta
MC
Noviello
Cristiana
C
Bonvini
Ezio
E
D000818:Animals; D001665:Binding Sites / Q000276:immunology; D004268:DNA-Binding Proteins / Q000378:metabolism; D015536:Down-Regulation / Q000276:immunology; D025461:Feedback, Physiological / Q000276:immunology; D006801:Humans; D019169:Jurkat Cells; D008214:Lymphocytes / Q000276:immunology / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D051966:Phospholipase C gamma; D010766:Phosphorylation; D011392:Proline / Q000276:immunology; D011485:Protein Binding / Q000276:immunology; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl; D011946:Receptors, Antigen / Q000276:immunology / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation / Q000276:immunology; D010738:Type C Phospholipases / Q000276:immunology / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D018909:src Homology Domains / Q000276:immunology*
D004268:DNA-Binding Proteins; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D011946:Receptors, Antigen; D014157:Transcription Factors; D014443:Tyrosine; D011392:Proline; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; C067419:CBL protein, human