Fig. 6.

Activation of different mutants of Pellino 1. Wild-type and mutated forms of Pellino 1 were phosphorylated with (lanes 2–7) or without (lane 1) IRAK4, and their E3 ligase activity was measured for 3 min as described in Experimental Procedures with Ubc13-Uevla. Pellino 1[10A] is a form of Pellino 1 in which the 10 phosphorylation sites at residues 70, 76, 78, 80, 82, 86, 125, 127, 288, and 293 are all mutated to Ala. (A) Lanes 1 and 2, wild-type Pellino 1; lane 3, Pellino 1[10A] in which Ala-125 and Ala-127 had been back-mutated to Ser and Thr, respectively; lane 4, same as lane 3, except for the additional back mutation of Ala-288 to Thr; lane 5, same as lane 3 except for the additional back mutation of Ala-293 to Ser; lane 6, same as lane 3, except that both Ala-288 and Ala-293 were back-mutated to Thr and Ser, respectively; lane 7, Pellino 1 in which Thr-288 and Ser-293 (but no other residue) was mutated to Ala. (B) Lanes 1 and 2, wild-type Pellino 1; lane 3, Pellino 1[10A] in which Ala-125 and Ala-127 were back-mutated to Ser and Thr, respectively; lanes 4, 5, 6, 7, and 8 show the activities of Pellino 1[10A] in which Ala-76 alone, Ala-78 alone, Ala-80 alone, Ala-82 alone, or Ala-86 alone, respectively were back-mutated to the Ser or Thr present in wild-type Pellino 1; lane 9, Pellino 1[10A] in which Ala-78, Ala-80, and Ala-82 were backmutated to Ser, Thr, and Ser, respectively; lane 10, Pellino 1[10A] in which Ala-78, Ala-80, Ala-82, and Ala-86 were back-mutated to Ser, Thr, Ser, and Thr, respectively. (C) DNA vectors encoding HA-Pellino 2 and either wild-type flag–IRAK1 (lane 1) or the catalytically-inactive flag–IRAK1[K239A] mutant (lane 2) were cotransfected into IRAK1-null IL-1R cells. Twenty-four hours later the cells were lysed and 25 μg of extract protein was subjected to SDS/PAGE and immunoblotted with a phospho-specific antibody that recognizes Thr-288 of Pellino 1 and the equivalent residue in Pellino 2 (Thr-290). NS, nonspecific band recognized by the antibody.