Mammary carcinogenesis in response to oncogenes is associated with the regulation of multiple signaling kinase cascades linked to cellular mitogenesis. An innovative and effective therapeutic approach would be to target a domain that is common among multiple signaling kinases. One such domain is the cysteine rich domain (CRD), which modulates intermolecular lipid-protein interactions. CRD domains are present in a large variety of proteins including protein kinase C (PKC),which are critical determinants in breast cancer tumorigenicity and metastasis. PKC is a family of phospholipid- regulated, serine/threonine, kinases that transduces oncogenic signals in breast cancer cells. Therefore, the CRD of PKC may be an important pharmacological target for therapeutic intervention in malignant transformation and progression of breast cancer. D-erythro-N,N-dimethyl- sphingosine (DMS) is a naturally derived sphingomyelin metabolite, which has been shown to inhibit total PKC activity; although the exact molecular mechanism is not known. Our preliminary studies demonstrate that DMS directly inhibits multiple isoforms of PKC activity, suggesting a common mechanism by which DMS recognizes and specifically binds to a conserved domain of PKC isoforms. To minimize interpretation problems, we have chosen to investigate PKC zeta (z) , which contains only one CRD. We have investigated inhibition of PKC z through the CRD as a model system for DMS-induced growth inhibition and/or apoptosis in breast cancer cells. It is hypothesized that DMS specifically binds to the CRD and inactivates PKC z, blocking breast cancer cell invasion and growth. This hypothesis will be investigated using combinations of biochemical, biophysical and molecular experimental approaches. We will first determine the DMS-binding site within CRD using site-directed- and deletion-mutants of PKC z. Second, we will identify the molecular determinants of DMS that mediate interactions with the CRD. Using several analogues of DMS, we will determine the critical molecular structure of DMS that directly interacts with the CRD. Establishing CRD as a molecular target for lipid-derived agents or lipomimetics may lead to potential therapies that simultaneously block multiple pro-survival and pro-mitogenic signaling cascades without appreciable immunological and inflammatory side effects.
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