| Various roles have been assigned to intracellular annexin II; however, no consensus has been reached on an actual in vivo function. Besides a conserved ability to bind Ca2+-dependently to acidic phospholipids, previous work demonstrated that annexin II also interacts Ca2+-dependently with the actin cytoskeleton, suggesting that actin binding may play a critical role in the intracellular function of annexin II. Initial work in this thesis was aimed at studying the F-actin binding activity of annexin II through two distinct mechanisms. First, inactivation of the Ca2+-binding sites of annexin II resulted in a Ca2+-independent interaction of the heterotetrameric form of the protein with both acidic phospholipid vesicles as well as F-actin. In contrast, the interaction with heparin remained Ca2+-dependent, suggesting that the known Ca2+-binding sites do not directly participate in binding to these biomolecules and that novel Ca2+-binding sites may regulate heparin binding. Secondly, annexin II mutants lacking its carboxyl-terminal 16, 13 or 9 amino acids was unable to bind to F-actin but still retained its ability to interact with both anionic phospholipids and heparin. A mutant annexin II lacking only its carboxyl-terminal 4 amino acids, however, retained its ability to interact with F-actin, establishing that the annexin II carboxyl-terminal amino acid residues, LLYLC, participate in F-actin-binding. The cysteine residue within this novel F-actin-binding domain is shown to be critical for F-actin binding. Finally, in a search for other intracellular binding partners of annexin II, it was established that annexin II acts as an RNA-binding protein in vivo. The binding of annexin II to RNA is activated by Ca 2+ and is highly dependent on the tyrosine phosphorylation state of the protein with only unphosphorylated protein capable of RNA binding. Subsequent work demonstrates that c-myc mRNA is a component of the ribonucleoprotein complex formed by annexin II in vivo. The interaction of annexin II with c-myc RNA results in the repression of c-myc translation. These findings suggest that annexin II may provide a link between the Ca2+ second messenger system, the tyrosine kinase signaling pathways and the translational regulation of mRNA. |
