The goals of this proposal are (1) to identify and characterize cDNAs capable of complementing IL-1 signaling in IL-1-unresponsive mutant cell lines, thereby providing mechanistic information as to how the IL-1 signal is transduced and (2) to provide an improved genetic system to study signaling pathways in mammalian cells. Better understanding of the IL-1 pathway will provide valuable information for developing anti- inflammatory drugs and will also benefit cancer research. Using a double drug selection strategy, we isolated three independent mutant cell lines unresponsive to IL-1. While two mutants (12A and 13A) are defective in components upstream of IRAK (IL-1 receptor-associated kinase), the third mutant (I1A) lacks the IRAK protein and mRNA.
Specific aim 1, Structure-function analysis of IRAK in mutant I1A cells. IRAK, a serine/threonine kinase, is recruited to the receptor complex upon IL-1 stimulation, where it is highly phosphorylated. IRAK then leaves the complex and interacts with downstream components to continue the pathway. IRAK is ubiquitinated and degraded following its phosphorylation. To study the function of IL-1-induced IRAK phosphorylation, we will first identify the phosphorylation sites and then mutate them. The IRAK phosphorylation mutants will then be analyzed in I1A IRAK-null cells. To identify domains of IRAK that interact with the receptor complex and the downstream components, deletion mutants will be generated and their function will be analyzed in I1A cells. We will also create IRAK mutants that can still be phosphorylated but not ubiquitinated or degraded upon IL-1 stimulation. I1A cells expressing these mutant proteins will be examined for their sustained response to IL-1, compared to cells transfected with wild-type IRAK.
Specific aim 2, Further characterization and complementation of I2A and I3A. Preliminary experiments have indicated that these two mutants are defective in components upstream of IRAK, for example, a receptor component or a protein involved in activating IRAK. Further characterization of the mutants will help to distinguish among possibilities. We will examine receptor affinity for IL-1 formation of a receptor complex, and recuitment of IRAK to this complex upon stimulation. If the mutants are not complemented by any known component, they will be complemented with retroviral expression libraries.
Specific aim 3, Isolation of additional IL-1-unresponsive mutants. Activation of NFkappaB and Jun kinase are major IL-1 mediated signaling pathways. The IL-1-unresponsive mutants that have been isolated so far are defective in both pathways. While we will continue to isolate more of this type of mutant, we will also isolate mutants that are defective specifically either in activating NFkappaB or Jun kinase. The mutants will be characterized and complemented as above.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM060020-03
Application #
6387018
Study Section
Allergy and Immunology Study Section (ALY)
Program Officer
Marino, Pamela
Project Start
1999-08-01
Project End
2004-07-31
Budget Start
2001-08-01
Budget End
2002-07-31
Support Year
3
Fiscal Year
2001
Total Cost
$209,431
Indirect Cost
Name
Cleveland Clinic Lerner
Department
Type
DUNS #
017730458
City
Cleveland
State
OH
Country
United States
Zip Code
44195
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