The goal of this proposal is to study the newly identified inhibitor of the Raf-l kinase designated RKJP. Raf-l is a cytoplasmic serine/threonine kinase whose activity plays a pivotal role in the control of the mitogen-activated protein kinase pathway (MAPK). Raf-1 kinase activity is tightly regulated by an interplay of positive and negative regulators. RKIP is a negative regulator of Raf-l and MAPK pathway signaling that was recently identified in a collaborative effort between my lab and the labs of Walter Kolch and David Rose. In this proposal I outline experiments to investigate two aspects of RKJP function.
In Aim One I will investigate in detail the mechanism by which RKJP contributes to the regulation of the MAPK pathway. Specifically, I will investigate the role of phosphorylation in the regulation of the Raf-RKTP interaction. Several lines of evidence indicate that RKJP binds to phosphorylated residues on proteins. I will thus investigate whether the phosphorylation status of Raf- 1 plays a role in regulating the RKIP-Raf- 1 interaction. The second set of experiments is based on the observation that RKIP is a phosphoprotein in vivo and a substrate of several kinases in vitro. Sites of in vitro phosphorylation will be mapped. In vivo phosphorylation sites will be determined using a combination of mass spectrometry and metabolic labeling followed by peptide mapping and sequencing. Once in vivo sites are ascertained, phosphopeptide-specific antibodies will be raised. The in vivo phosphorylation status of RKIP will be examined under a variety of physiological conditions known to stimulate MAPK activity.
In Aim Two I will investigate the role of RKIP in the NF-KB signaling cascade. I have obtained experimental evidence that modulation of RKIP expression levels affects NF-kB activation, and I have shown that RKIP physically interacts with two components of the NF-kB cascade, NIK and TAK 1. I will employ a combination of genetic and biochemical methods to study the effects of RKIP on NF-kB signaling. The primary goal of this line of investigation will be to define the targets of RKIP in the NF-kB signaling pathway, and to initiate studies into how they are regulated. In summary, I believe that the experiments in this proposal will provide a new handle for understanding Raf- 1 signaling, and will initiate studies to expose the full spectrum of RKIP functions.
