Mutant cells that do not respond to interleukin-1 (IL-1) reveal a novel role for IL-1 receptor-associated kinase

Abstract

Mutagenized human 293 cells containing an interleukin-1 (IL-1)-regulated herpes thymidine kinase gene, selected in IL-1 and gancyclovir, have yielded many independent clones that are unresponsive to IL-1. The four clones analyzed here carry recessive mutations and represent three complementation groups. Mutant A in complementation group I1 lacks IL-1 receptor-associated kinase (IRAK), while the mutants in the other two groups are defective in unknown components that function upstream of IRAK. Expression of exogenous IRAK in I1A cells (I1A-IRAK) restores their responsiveness to IL-1. Neither NFkappaB nor Jun kinase is activated in IL-1-treated I1A cells, but these responses are restored in I1A-IRAK cells, indicating that IRAK is required for both. To address the role of the kinase activity of IRAK in IL-1 signaling, its ATP binding site was mutated (K239A), completely abolishing kinase activity. In transfected I1A cells, IRAK-K239A was still phosphorylated upon IL-1 stimulation and, surprisingly, still complemented all the defects in the mutant cells. Therefore, IRAK must be phosphorylated by a different kinase, and phospho-IRAK must play a role in IL-1-mediated signaling that does not require its kinase activity.

FIG. 1

A model for the IL-1 signaling pathway. See the text for details. PC, phosphatidylcholine.

FIG. 2

Double drug selection with TK and Zeo under control of the E-selectin promoter. (A) E-selectin–TK and E-selectin–Zeo. An upstream fragment of the E-selectin gene (−730 to +52), containing one ATF and three NF-κB binding sites and a TATA box, was cloned in front of the TK cDNA or the Zeo gene. (B) Drug selection. E-selectin–TK and E-selectin–Zeo were cotransfected into 293 cells, and the transfected cells were selected in Zeo plus IL-1. Individual clones were assayed for survival in gancyclovir (GCV), death in gancyclovir plus IL-1, death in Zeo, and survival in Zeo plus IL-1. One such clone was expanded and subjected to five rounds of mutagenesis. IL-1-unresponsive mutants were isolated by selecting the mutagenized pools in gancyclovir plus IL-1. Putative mutants were then tested for survival in gancyclovir and gancyclovir plus IL-1 and for death in Zeo and Zeo plus IL-1.

FIG. 3

Analyses of IL-1-unresponsive mutants. (A) NF-κB gel shift assay. Cell extracts were made from 293-TK/Zeo cells (WT [wild type]) and the IL-1-unresponsive mutants treated for 15 min with IL-1 (100 U/ml) or TNF-α (20 ng/ml) or untreat. The NF-κB binding site from the IP-10 gene was used as a probe. The two bands in the gel shift assay are due mainly to p50-p65 heterodimers (bottom) and p65-p65 homodimers (top) (63a). (B) Luciferase reporter assay. E-selectin–Luc (1 μg/10-cm-diameter plate) was transiently transfected into 293-TK/Zeo cells (WT) and the IL-1-unresponsive mutants. Thirty-six hours later, the cells were either left untreated or stimulated for 4 h more with IL-1 (100 U/ml; closed bars) or TNF-α (20 ng/ml; hatched bars). Luciferase activities were normalized to β-galactosidase. Data are presented as the fold induction of luciferase activity in the treated cells. Shown are the averages and standard deviations from three independent experiments. (C) Northern analysis of IL-8 gene expression. Total RNAs were made from 293-TK/Zeo cells (WT) and the IL-1-unresponsive mutants treated for 6 h with IL-1 (100 U/ml) or TNF-α (20 ng/ml) or untreated. Human IL-8 cDNA was used as a probe, and the signals were normalized after reprobing with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA.

FIG. 4

NF-κB gel shift assay for dominance and complementation. Extracts were made from 293-TK/Zeo cells (WT [wild type]), clones I1A and I2A, and heterokaryons WT/I1A and I1A/I2A, treated for 15 min with IL-1 (100 U/ml) or TNF-α (20 ng/ml) or untreated. The NF-κB binding site from the IP-10 gene was used as a probe.

FIG. 5

Clone I1A lacks IRAK. (A) Analysis of the IRAK protein. Extracts were made from 293-TK/Zeo (WT [wild type]) cells and from the IL-1-unresponsive mutants. Aliquots were analyzed with anti-IRAK after Western transfer. The same blot was probed with anti-IL-1R1. (B) Analysis of IRAK and IRAK2 mRNAs. Total RNA made from 293-TK/Zeo cells (WT) and mutant I1A was analyzed by the Northern procedure with IRAK or IRAK2 cDNA as the probe.

FIG. 6

Analysis of I1A cells complemented with IRAK or IRAK-K239A. (A) Cells were cotransfected transiently with E-selectin–Luc and increasing amounts of a TK promoter-driven IRAK expression vector or TK promoter-driven IRAK-K239A expression vector. Thirty-six hours later, the cells were either left untreated or stimulated for 4 h more with IL-1 (100 U/ml) before harvest. Luciferase activities were normalized to β-galactosidase. Data are presented as fold induction of luciferase activity in the treated cells. The experiments were repeated four times. Shown are the data from a typical experiment. (B) Western analysis with anti-IRAK of extracts from I1A cells transiently transfected with increasing amounts of IRAK or IRAK-K239A. (C) Western analysis of extracts from 293-TK/Zeo (WT [wild type]), I1A, or I1A cells stably transfected with IRAK or IRAK-K239A.

FIG. 7

IL-1-induced activation of Jun kinase in mutant cells. Immunoprecipitates were prepared from cell extracts with anti-Jun kinase, followed by an in vitro kinase assay. (A) Analysis of extracts from 293-TK/Zeo (WT [wild type]) cells and I1A cells, untransfected or stably transfected with IRAK or IRAK-K239A, untreated, stimulated with IL-1, or treated with UV (40 J/m²). (B) Analysis of extracts from clones I1A, I2A, and I3A untreated, stimulated with IL-1, or treated with UV (40 J/m²).

FIG. 8

Constitutive stimulation of signaling by MyD88 or TRAF6 in I1A cells. 293-TK/Zeo (WT [wild type]) cells and I1A cells stably transfected with IRAK or IRAK-K239A were cotransfected transiently with E-selectin–Luc, with a vector control, or with a MyD88 or TRAF6 expression vector. Luciferase activities were normalized to β-galactosidase. Data are presented as fold induction of luciferase in cells transfected with MyD88 (solid bars) or TRAF6. Shown are the averages and standard deviations from three independent experiments.

FIG. 9

IRAK kinase assay. Cell extracts were made from 293-TK/Zeo (WT [wild type]), I1A, and I1A stably transfected with IRAK or IRAK-K239A. Immunoprecipitates were prepared with anti-IRAK, followed by an in vitro kinase assay. Short and long exposures are presented. The immunoprecipitated samples were also analyzed with anti-IRAK (bottom panel).

FIG. 10

Western analysis of IRAK as a function of time after stimulation with IL-1. Shown are results for wild-type 293TK/Zeo (WT) cells and I3A cells (A), I1A cells transfected with IRAK or IRAK-K239A (B), and I1A/I3A heterokaryons (C), either untreated or treated with IL-1. Cell extracts were analyzed by the Western procedure with anti-IRAK. P-IRAK, phosphorylated IRAK; U-IRAK, ubiquitinated IRAK. The top portions of panels A and B are short exposures, and the bottom portions are long exposures. The same transfers were probed with anti-IL-1R1 to control for loading. (D and E) Extracts of I1A cells transfected with IRAK or IRAK-K239A, with or without IL-1 stimulation, were either untreated or treated with calf intestinal phosphatase (CIP).

FIG. 11

Complementation of I1A cells with IRAK2. E-selectin–Luc was cotransfected transiently with control vector, MyD88, or TRAF6 into I1A cells stably transfected with CMV-IRAK2 (I1A-IRAK2 cells). The I1A-IRAK2 cells transfected with E-selectin–Luc and the control vector were treated with IL-1 (100 U/ml, 4 h). Data are presented as the fold induction of luciferase activity in IL-1-treated or untreated cells transfected with MyD88 or TRAF6 compared to cells transfected with the control vector. Shown are the averages and standard deviations from three independent experiments.