Ras-dependent, Ca2+-stimulated activation of nuclear factor of activated T cells by a constitutively active Cbl mutant in T cells.
T cell receptor (TCR) stimulation induces rapid tyrosine phosphorylation of cellular proteins, including Cbl, a protooncogene product whose function remains unclear. As a first step toward elucidating the function of Cbl in TCR-initiated signaling, we evaluated the ability of wild-type Cbl or a transforming Cbl mutant (70Z/3) to induce transcriptional activation of a nuclear factor of activated T cells (NFAT) element derived from the interleukin 2 (IL2) promoter in transiently cotransfected Jurkat-TAg T cells. 70Z/3, but not Cbl, caused NFAT activation which was significantly enhanced by stimulation with calcium ionophore, and was drastically reduced by cyclosporin A pretreatment. A point mutation of a potential phosphatidylinositol 3-kinase (PI3-K) binding site (Y731EAM to Y731EAC) in 70Z/3 disrupted the association of PI3-K with 70Z/3, but did not reduce the induction of NFAT activity, suggesting that the interaction between Cbl and PI3-K is not required in the 70Z/3-mediated induction of NFAT. Additional mapping studies indicated that defined deletions of C-terminal 70Z/3 sequences affected to a variable degree its ability to stimulate NFAT activity. Strikingly, deletion of 346 C-terminal residues augmented this activity, whereas removal of 20 additional residues abolished it. Coexpression of dominant negative Ras abrogated the basal or ionomycin-stimulated, 70Z/3-mediated NFAT activation, suggesting a functional Ras is required for this activation. These results implicate Cbl in Ras-dependent signaling pathways which lead to NFAT activation.
Liu
Y C
YC
Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121, USA.
Elly
C
C
Langdon
W Y
WY
Altman
A
A
D000818:Animals; D019703:Calcineurin; D002118:Calcium / Q000502:physiology*; D002148:Calmodulin-Binding Proteins / Q000502:physiology; D004268:DNA-Binding Proteins / Q000502:physiology*; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D008213:Lymphocyte Activation; D051379:Mice; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D050722:Oncogene Protein v-cbl; D019869:Phosphatidylinositol 3-Kinases; D010749:Phosphoprotein Phosphatases / Q000502:physiology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000502:physiology*; D050721:Proto-Oncogene Proteins c-cbl; D016283:Proto-Oncogene Proteins p21(ras) / Q000502:physiology*; D015682:Retroviridae Proteins, Oncogenic / Q000378:metabolism; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D013601:T-Lymphocytes / Q000378:metabolism*; D014157:Transcription Factors / Q000502:physiology*; D014407:Tumor Cells, Cultured; D044767:Ubiquitin-Protein Ligases
D002148:Calmodulin-Binding Proteins; D004268:DNA-Binding Proteins; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D050722:Oncogene Protein v-cbl; D011518:Proto-Oncogene Proteins; D015682:Retroviridae Proteins, Oncogenic; D014157:Transcription Factors; D015759:Ionomycin; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); C067419:CBL protein, human; C493849:Cbl protein, mouse; D002118:Calcium