FIGURE 1.

Inhibitor-3 is cleaved by caspase-3 in vitro at a DTVD cleavage site. A, domain map of human Inh3. The diagram shows the location of the putative caspase-3 cleavage site (⁴⁶DTVD⁴⁹), the nuclear localization signal (NLS), the nucleolar targeting signal (NTS) (19), the PP1 binding motif (³⁹KKVEW⁴³) (18), and the inhibitory domain that lies between residues 64 and 77. B, in vitro cleavage of Inh3 by caspase-3. Purified recombinant His₆-Inh3 was incubated with recombinant human caspase-3 for the indicated times and Western blotted with a rabbit polyclonal antibody against Inh3 (see “Experimental Procedures”). Inh3-CT is the 17.5-kDa cleavage product. C, purified recombinant Inh3 was treated with caspase-3 as in B, except that a peptide-specific antibody to amino acids 69–83 of Inh3 was used. D, mutation of the caspase-3 site in Inh3 renders it resistant to cleavage. Recombinant Inh3(D49A) was treated with caspase-3 as in B, and the digests were analyzed by Western blotting with a polyclonal antibody against Inh3. One representative experiment of three is shown in A–D. E, the Inh3-CT cleavage product does not immunoprecipitate with PP1. Purified Inh3 or Inh3 predigested with caspase-3 was incubated with PP1α for 1 h at 4 °C, immunoprecipitated with PP1α antibody, and Western blotted with anti-Inh3 antibody (see “Experimental Procedures”). Lane 1, Inh3 input; lane 1′, immunoprecipitate of PP1α plus Inh3; lane 2, caspase-3-cleaved Inh3 input; lane 2′, immunoprecipitate of PP1α plus caspase-3-cleaved Inh3. F, overlay assay. Left, Western blot of Inh3 and caspase-3-treated Inh3 with Inh3 antibody. Lane 1, Inh3; lane 2, caspase-3-cleaved Inh3. Right, overlay blot of Inh3 and caspase-3-treated Inh3 with PP1. Lanes 1′ and 2′ correspond to lanes 1 and 2 in the left panel. The membranes were blocked with 5% nonfat milk proteins and then incubated with purified PP1α. The membrane was washed and then probed with anti-PP1 antibody (see “Experimental Procedures”). Essentially identical results were obtained in two independent experiments for the data shown in E and F. a.a., amino acids; WB, Western blot.