Figure 1.

Human ASK1 specifically binds 14-3-3 in vivo and in vitro. HeLa cells were transiently transfected with plasmids coding for HA-tagged ASK1^wt, ASK1^S967A, ASK1^K709R, or the control CAB1 (Clan of ARF-Binder 1; A. L. Boman and R. A. Kahn, unpublished data). CAB1 is an ADP ribosylation factor-associated protein that does not bind 14-3-3. Forty-eight hours after transfection, cell lysates were prepared. (A) ASK1 immunocomplexes contain 14-3-3 proteins. ASK1 complexes were immunoprecipitated by using an anti-HA antibody and assayed for the presence of 14-3-3 by its ability to activate the ADP ribosyltransferase activity of ExoS. The 14-3-3-dependent ExoS activity was expressed as pmol of ADP ribose incorporated into the substrate per min per pmol of ExoS. Data shown are representative of three experiments. Error bars represent standard error (n = 3). (B) 14-3-3 immunocomplexes contain ASK1. Endogenous 14-3-3 proteins were isolated from the same HeLa cell lysates as in A by using anti-14-3-3 serum. The 14-3-3 IPs were blotted for HA-ASK1 by using an anti-HA antibody (Upper). Lower shows similar expression levels of the HA-tagged proteins in total cell lysates. (C) Interaction of ASK1 with 14-3-3ζ is disrupted by binding-site mutations of 14-3-3ζ. Immobilized His-tagged 14-3-3ζ or control β-gal proteins (5 μg each) were incubated with ³⁵S-labeled ASK1. After washing, bound proteins were resolved by using SDS/PAGE, and ASK1 was revealed by autoradiography (Upper). Similar amounts of immoblized proteins were used as revealed by Coomassie blue staining (Lower). (D) Peptide ligands of 14-3-3 inhibit ASK1–14-3-3 interactions. Peptides were preincubated with immobilized His–14-3-3ζ (0.2 μg) before adding ³⁵S-labeled ASK1. After washing, 14-3-3ζ-bound ASK1 was quantified by PhosphorImager. The percentage of ASK1 bound to 14-3-3 relative to peptide-free samples is plotted against increasing concentrations of the test peptides. Error bars represent standard error (n = 3).