Fig. 4.

Substitutions mimicking phosphorylation at Ser-65, Thr-69, and Ser-70 inhibit PARL β-cleavage. (A) Constructs expressing the indicated mutant PARL protein were transfected in either HEK 293 or HeLa cells. The effect of mutations abolishing (Ala) or mimicking (Asp) phosphorylation (17, 18) is monitored by the amount of β-cleaved form of PARL detected, PACT (Fig. 1A). Note that Asp but not Ala substitutions block β-cleavage. IP, immunoprecipitation; WB, Western blotting. (B) Scheme of the deletions (Δ) within PARL Pβ domain that have been tested in this work. (C) The Δ84–87 mutant is constitutively cleaved at the β-cleavage site. (D) Asp substitutions at positions Ser-65, Thr-69, and Ser-70 in the Δ84–87 dominantly reestablish the block of β-cleavage. (E) Ala and Asp substitutions at the phosphorylated Ser-65, Thr-69, and Ser-70 residues do not affect PARL protease activity. HEK 293 cells were cotransfected with a catalytically dead PARL protein (PARL-Myc-CT S277G) and the indicated FLAG-tagged mutant, whose enzymatic activity is monitored by its ability to cleave the inactive PARL in trans and produce PACT (11).