PROTEIN-KINASE-A STIMULATES BINDING OF MULTIPLE PROTEINS TO A U-RICH DOMAIN IN THE 3'-UNTRANSLATED REGION OF LACTATE-DEHYDROGENASE-A MESSENGER-RNA THAT IS REQUIRED FOR THE REGULATION OF MESSENGER-RNA STABILITY

We have explored the molecular basis of the cAMP-induced stabilization of lactate dehydrogenase A (LDH-A) mRNA and identified four cytoplasm ic proteins of 96, 67, 52, and 50 kDa that specifically bind to a 30-n ucleotide uridine rich sequence in the LDH 3'-untranslated region with a predicted stem-loop structure. Mutational analysis revealed that sp ecific protein binding is dependent upon an intact primary nucleotide sequence in the loop as well as integrity of the adjoining double-stra nded stem structure, thus indicating a high degree of primary and seco ndary structure specificity. The critical stem-loop region is located between nucleotides 1473 and 1502 relative to the mRNA cap site and co ntains a previously identified cAMP-stabilizing region (CSR) required for LDH-A mRNA stability regulation by the protein kinase A pathway. T he 3'-untranslated region binding activity of the proteins is upregula ted after protein kinase A activation, whereas protein dephosphorylati on is associated with a loss of binding activity. These results imply a cause and effect relationship between LDH-A mRNA stabilization and C SR-phosphoprotein binding activity. We propose that the U-rich CSR is a recognition signal for CSR-binding proteins and for an mRNA processi ng pathway that specifically stabilizes LDH mRNA in response to activa tion of the protein kinase A signal transduction pathway.