Inhibition of cytosolic phospholipase A(2) by annexin I - Specific interaction model and mapping of the interaction site

Annexins (ANXs) display regulatory functions in diverse cellular processes, including inflammation, immune suppression, and membrane fusion. However, the exact biological functions of ANXs still remain obscure. Inhibition of phospholipase A(2) (PLA(2)) by ANX-I, a 346-amino acid protein, has been ob served in studies with various forms of PLA(2). "Substrate depletion" and " specific interaction" have been proposed for the mechanism of PLA(2) inhibi tion by ANX-I. Previously, we proposed a specific interaction model for inh ibition of a 100-kDa porcine spleen cytosolic form of PLA(2) (cPLA(2)) by A NX-I (Kim, K. M., Kim, D. K., Park, Y. M., and Na, D. S. (1994) FEES Lett. 343, 251-255). Herein, we present an analysis of the inhibition mechanism o f cPLA(2) by ANX-I in detail using ANX-I and its deletion mutants. Deletion mutants were produced in Escherichia coli, and inhibition of cPLA(2) activ ity was determined. The deletion mutant ANX-I-(1-274), containing the N ter minus to amino acid 274, exhibited no cPLA(2) inhibitory activity, whereas the deletion mutant ANX-I-(275-346), containing amino acid 275 to the C ter minus, retained full activity. The protein-protein interaction between cPLA (2) and ANX-I was examined using the deletion mutants by immunoprecipitatio n and mammalian two-hybrid methods. Full-length ANX-I and ANX-I-(275-346) i nteracted with the calcium-dependent lipid-binding domain of cPLA(2). ANX-I -(1-274) did not interact with cPLA(2). Immunoprecipitation of A549 cell ly sate with anti-ANX-I antibody resulted in coprecipitation of cPLA(2). These results are consistent with the specific interaction mechanism rather than the substrate depletion model. ANX-I may function as a negative regulator of cPLA(2) in cellular signal transduction.