Figure 4. JNK-IN-7 is an irreversible inhibitor of IRAK1.

(A) The amino acid sequences of human IRAK1 (residues 294–305) and human JNK1 (residues 108–119) were aligned, and conserved residues are highlighted. Met108 of JNK1 is the ‘gatekeeper’ residue. (B) IRAK1 KO IL-1R cells were transfected with 5 µg or DNA of a control empty vector (EV) (lanes 1 and 2), HA-tagged wild-type IRAK1 (lanes 3–6) or HA-tagged IRAK1[C302L] (lanes 7–10). IRAK1 was immunoprecipitated from 0.5 mg of cell extract protein and assayed with GST-Pellino1 and Mg[γ³²P-ATP] in the presence of IRAK4-IN-1 to inhibit co-immunoprecipitating IRAK4 and in the presence or absence of the IRAK1 inhibitor JNK-IN-7. (C) The autoradiogram from B and two other independent experiments were scanned, and the activity of IRAK1 and IRAK1[C302L] (IRAK1[C/L]) measured without or with 1.0 µM JNK-IN-7. The results are shown as a % of wild-type IRAK1 activity in the absence of JNK-IN-7. (D) IL-1R cells were incubated for 1 h without (−) or with (+) 10 µM JNK-IN-7 to inhibit cellular IRAK1 and then stimulated with IL-1β. IRAK1 was immunoprecipitated from 1 mg of cell extract protein and assayed with GST-Pellino1 and Mg[γ³²P-ATP] as substrates in the presence (+) of 1 µM IRAK4-IN-1 to inhibit co-immunoprecipitating IRAK4.