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Figure 2.
Fig. 2. The VIVIT peptide is a potent inhibitor of the
NFAT-calcineurin interaction, and its substitution into the
calcineurin docking site enhances the calcineurin responsiveness
of NFAT1. (A) Inhibition of the NFAT-calcineurin interaction
(9). Calcineurin (Cn) was activated with calmodulin (CaM) and
CaCl[2] (Ca^2+), and its binding to GST (lane 1) and GST-NFAT1
(residues 1 through 415) (lanes 2 through 11) was evaluated by
protein immunoblotting. Cn A, calcineurin A chain. (B and C)
Inhibition of the calcineurin-mediated dephosphorylation of NFAT
proteins (9). Lysates of HeLa cells expressing HA-NFAT1 (B) or
lysates from HEK 293T cells expressing HA-NFAT1, HA-NFAT2, or
HA-NFAT4 (C) were incubated with the phosphatase inhibitor
sodium pyrophosphate (NaPPi, lane 1) or with activated
calcineurin (Cn+CaM+Ca^2+) in the absence or presence of
peptides at the indicated micromolar concentrations. The
phosphorylation status of NFAT proteins was evaluated by protein
immunoblotting with anti-HA. The positions of phospho- and
dephospho-NFAT are indicated by arrows in (B). (D) Inhibition of
NFAT-dependent gene expression. (Left panel) Jurkat cells were
cotransfected with a 3xNFAT-Luc reporter plasmid and with
expression plasmids encoding murine NFAT1, GFP, GFP-SPRIEIT, or
GFP-VIVIT as indicated (13). (Right panel) Jurkat cells were
cotransfected with a 3xNFAT-Luc reporter plasmid and with
expression plasmids encoding GFP, GFP-VIVIT, and murine NFAT1,
human NFAT2, and human NFAT4 as indicated (11). Twenty-four
hours after transfection, luciferase activity induced by
endogenous NFAT (Endog.) or by overexpressed NFAT proteins was
measured in unstimulated cells and in cells stimulated for 6
hours with PMA and ionomycin. (E) Substitution of the VIVIT
sequence into NFAT1 (12). Cl.7W2 murine T cells were transfected
with wild-type HA-NFAT1-GFP or with the mutant
HA-NFAT1[VIVIT]-GFP, in which HPVIVITGP replaces SPRIEITPS.
Cells were stimulated with ionomycin (Iono) at the
concentrations indicated in the absence or presence of 1
µM CsA, and the phosphorylation status of NFAT1 was
assessed by protein immunoblotting with anti-HA. (F)
Localization of NFAT1 and NFAT1[VIVIT] in cells (12). HeLa cells
transiently expressing wild-type HA-NFAT1-GFP or
HA-NFAT1[VIVIT]-GFP were left untreated or were treated with 10
µM CsA (for 16 hours). NFAT1 proteins were visualized in
fixed cells by GFP fluorescence.
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