The lactate dehydrogenase-A (LDH-A) gene, whose product plays a pivotal role in anaerobic glycolysis and whose expression is frequently increased in human cancers, is highly regulated by second messengers at the transcriptional as well as post-transcriptional levels. The application's long-term objectives are to further elucidate the molecular basis responsible for the complex regulation of LDH-A mRNA stability/instability by the cAMP-activated signal transduction pathway involving protein kinase A (PKA). Based on our recent studies, this proposal aims to further delineate (a) post-transcriptional mechanisms controlling LDH-A mRNA stability/instability involving a cAMP-stabilizing region (CSR) which we recently identified in the 3'-untranslated region (3'-UTR) of LDH-A mRNA, and (b) mechanisms whereby recently identified CSR-binding proteins (CSR-BP) are phosphorylated and functionally modified by the interaction of PKA, and (c) elucidate a potential functional interaction between CSR and CSR-BPs. The experimental system in which the studies will be conducted is the rat C6 glioma cell line in which activators of the PKA signal transduction pathway cause a marked stabilization of relatively rapidly decaying LDH-A mRNA leading to significant increase of LDH-A mRNA half-life.
The Specific Aims i nclude: (a) functional analysis of a cis-regulatory module in the 3'-UTR (the CSR) that controls mRNA stability; (b) cloning followed by a structural and functional analysis of trans- regulatory CSR-binding proteins which might be instrumental in controlling LDH-A mRNA half-life; (c) structure/function analysis of the CSR-BPs; and (d) analysis of the role of PKA in modulating the functional activity of CSR-BPs. The studies will contribute to our understanding of gene regulation by cAMP and of regulation of intermediary metabolism.
