title: In vivo regulation of human CrkII by cyclophilin A and FK506-binding protein.
abstract: Members of the Crk family of adaptor proteins are key players in signal transduction from a variety of cell surface receptors. CrkI and CrkII are two alternative-spliced forms of a single gene which possess an N-terminal SH2 domain and an SH3 domain that mediate interaction with other proteins. CrkII possesses an additional C-terminal linker region plus an extra SH3 domain, which does not interact with other proteins, but operates as regulatory moiety. Utilizing human Jurkat T cells, we demonstrate that CrkII-SH3N binding of C3G is inhibited by cyclosporin A (CsA) plus FK506 that inhibit the cyclophilin A (CypA) and FK506 binding protein (FKBP) peptidyl-prolyl cis-trans isomerases (PPIases; also termed immunophilins), respectively. Jurkat T cells were found to express ∼ 5-fold lower levels of CrkI protein compared to CrkII, but the efficiency of C3G binding by CrkI was ∼ 5-fold higher than that of CrkII, suggesting that the majority of cellular CrkII proteins adopt a conformation that is inaccessible for C3G. Treatment of Jurkat T cells with CsA plus FK506 led to a time-dependent conformational change in overexpressed human CrkII1-236 protein-containing FRET-based biosensor, supporting the accumulation of cis conformers of human CrkII1-236 in the presence of PPIase inhibitors. Our data suggest that the Gly(219)-Pro-Tyr motif in the human CrkII linker region serves as the recognition and isomerization site of PPIases, and raise the possibility that CsA and FK506 might interfere with selected effector T cell functions via a CrkII-, but not CrkI-dependent mechanisms.
authors.affiliation: The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences and the Cancer Research Center, Ben Gurion University of the Negev, P.O.B. 653, Beer Sheva 84105, Israel.
authors.forename: Pulak Ranjan
authors.initials: PR
authors.lastname: Nath
authors.affiliation: The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences and the Cancer Research Center, Ben Gurion University of the Negev, P.O.B. 653, Beer Sheva 84105, Israel.
authors.forename: Guangyu
authors.initials: G
authors.lastname: Dong
authors.affiliation: The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences and the Cancer Research Center, Ben Gurion University of the Negev, P.O.B. 653, Beer Sheva 84105, Israel.
authors.forename: Alex
authors.initials: A
authors.lastname: Braiman
authors.affiliation: The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences and the Cancer Research Center, Ben Gurion University of the Negev, P.O.B. 653, Beer Sheva 84105, Israel. Electronic address: noah@bgu.ac.il.
authors.forename: Noah
authors.initials: N
authors.lastname: Isakov
mesh_terms: D021984:Cyclophilin A / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D020104:Immunophilins / Q000378:metabolism*; D019169:Jurkat Cells; D050737:Proto-Oncogene Proteins c-crk / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D022021:Tacrolimus Binding Proteins / Q000378:metabolism*
chemical_list: D050737:Proto-Oncogene Proteins c-crk; D021984:Cyclophilin A; D022021:Tacrolimus Binding Proteins; D020104:Immunophilins
keywords: Crk adaptor protein; Cyclophilin A; Cyclosporine A; Immunophilins; PPIase; Peptidyl-prolyl cis-trans isomerase