Identification and Characterization of Post-Translational Modifications in the Raf Kinase Inhibitory Protein: Implication for a Molecular-Based Therapy of Breast Cancer

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This study focuses on elucidating the function of Raf Kinase Inhibitor Protein (RKIP), a novel kinase inhibitor that was identified as a potent metastasis suppressor and the regulation of which constitutes a key element in understanding the signaling pathways involved in cancer progression and metastasis. Mechanistically this protein functions as a negative regulator of both the Raf and NF-KB signaling pathways. Consistent with its inhibitory effects on these two pathways, a significant inverse correlation was observed between the expression of RKIP and the stage of cancer development in tumors. Particularly low levels of RKIP were noted in several cancer metastases including breast, prostate, colon cancers and melanoma. Importantly, restoration of RKIP expression in highly metastatic prostate and breast cancer cell lines sensitized them to apoptosis and inhibited metastasis in mouse models, suggesting this protein as promising candidate for cancer therapy. In order to exploit this potential, the present application proposes to define the molecular mechanisms that regulate RKIP function. In this study experiments are outlined to investigate the mechanism of RKIP regulation by phosphorylation and its relevance in breast cancer progression. RKIP is a phosphoprotein in vivo. PKC phosphorylates RKIP at Ser153. Importantly, RKIP phosphorylation by PKC in response to growth factor stimulation decreases the affinity of RKIP to Raf-1. However, the effect of Ser153 phosphorylation on RKIP activity toward its other targets remains undefined. In addition to PKC, ERK is identified as a putative kinase of RKIP. Thus, the first aim of the project is to explore if ERK is a bonafide kinase of RKIP. ERK phosphorylation sites in RKIP will be mapped in vitro and the intact cells using a combination of site-specific mutagenesis and proteomic/ mass spectrometry. 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 ERK activity. The second aim is to examine the effects of ERK or/ and PKC phosphorylation on the biological activities of RKIP towards its known targets. The effects of phosphorylation on RKIP pro-apoptotic function and RKIP-mediated suppression of breast cancer metastasis using both xenograft and allograft mouse models will also be determined. Finally, the third aim is to demonstrate the clinical relevance using clinically-derived breast cancer tissues to study the relationship between level of phosphorylated RKIP and breast cancer progression and patient survival. Overall, our current data, together with those to be obtained from the above proposed investigation should provide more in-depth understanding of pathways regulating RKIP function in addition to identifying RKIP as a novel target that can be manipulated therapeutically to delay the progression of cancer and improve patient survival.