Structure-function analysis of the WIP role in T cell receptor-stimulated NFAT activation: evidence that WIP-WASP dissociation is not required and that the WIP NH2 terminus is inhibitory.
WASP and its binding partner WIP play important roles in T cells both in actin polymerization and in interleukin-2 transcription. Aberrations thereof contribute to the pathology of Wiskott-Aldrich syndrome (WAS). To directly evaluate the cooperativity of WIP and WASP in interleukin-2 transcription, we investigated how the WIP-WASP complex regulates NF-AT-mediated gene transcription. We developed an improved model system for analysis, using WIP and WASP cotransfection into Jurkat cells, in which strong induction of NFAT reporter activation is observed with anti-T cell receptor (TCR) antibody without the phorbol 12-myristate 13-acetate usually used previously. Using this system, our findings contradict a prevailing conceptual model of TCR-induced WIP-WASP dissociation by showing in three ways that the WIP-WASP complex mediates TCR-induced NFAT activation without dissociation. First, phosphorylation of WIP Ser(488) does not cause dissociation of the WIP-WASP complex. Second, WIP-WASP complexes do not dissociate demonstrably after TCR stimulation. Third, a fusion protein of WIP to WASP efficiently mediates NFAT activation. Next, our studies clarify that WIP stabilization of WASP explains otherwise unexpected results in TCR-induced NFAT activation. Finally, we find that the NH(2) terminus of WIP is a highly inhibitory region for TCR-mediated transcriptional activation in which at least two elements contribute: the NH(2)-terminal polyproline and the NH(2)-terminal actin-binding WH2 domain. This suggests that WIP, like WASP, is subject to autoinhibition. Our data indicate that the WIP-WASP complex plays an important role in WASP stabilization and NFAT activation.
Dong
Xiaoyun
X
Experimental Immunology Branch, NCI, National Institutes of Health, Bethesda, Maryland 20892-1360, USA.
Patino-Lopez
Genaro
G
Candotti
Fabio
F
Shaw
Stephen
S
D000199:Actins / Q000737:chemistry; D017252:CD3 Complex / Q000096:biosynthesis; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D006801:Humans; D007376:Interleukin-2 / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D019169:Jurkat Cells; D007700:Kinetics; D008954:Models, Biological; D050778:NFATC Transcription Factors / Q000737:chemistry / Q000502:physiology*; D017434:Protein Structure, Tertiary; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D013329:Structure-Activity Relationship; D013755:Tetradecanoylphorbol Acetate / Q000737:chemistry; D014158:Transcription, Genetic; D014923:Wiskott-Aldrich Syndrome / Q000378:metabolism; D051316:Wiskott-Aldrich Syndrome Protein / Q000378:metabolism*
D000199:Actins; D017252:CD3 Complex; D003598:Cytoskeletal Proteins; D007376:Interleukin-2; D047908:Intracellular Signaling Peptides and Proteins; D050778:NFATC Transcription Factors; D011948:Receptors, Antigen, T-Cell; C495767:WAS protein, human; C109903:WIPF1 protein, human; D051316:Wiskott-Aldrich Syndrome Protein; D013755:Tetradecanoylphorbol Acetate
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