This application is to study the regulation of beta2-integrin activation, specifically protein kinase C (PKC)-mediated beta2-integrin activation. In the beta2-integrin-dependent signal transduction pathways, the activation of PKC is required for the activation of integrin. Data suggested that MacMARCKS, a major PKC substrate in leukocytes, plays an essential role in the beta2-integrin-dependent signal transduction pathways. Thus, this proposal is focused on how MacMARCKS regulates the activation of the beta2-integrin family. This proposal will test the hypothesis that MacMARCKS is one of the molecules which transduces PKC-mediated phosphorylation signal to activate beta2-integrin-dependent signal transduction pathways. The phosphorylation-abrogated and pseudo-phosphorylated mutants of MacMARCKS will be generated and their effects on the beta2-integrin-dependent cellular functions will be determined. Preliminary studies suggested that MacMARCKS associates in vivo with paxillin, a focal adhesion protein that is part of the beta2-integrin- dependent signal transduction pathways. The MacMARCKS-paxillin association will be characterized and the effect of PKC-mediated MacMARCKS phosphorylation on the tyrosine phosphorylation of paxillin will be examined. The hypothesis will be tested that MacMARCKS-paxillin association is part of the regulation of the beta2-integrin-dependent pathways. Finally, the cloned novel 47 kDa MacMARCKS binding protein will be characterized. The interaction between MacMARCKS and the 47 kDa protein will also be examined and their regulation will be investigated. The proposed work will investigate the potential role of the 47 kDa protein in integrating MacMARCKS and other focal adhesion proteins, including beta2- integrin itself, into the beta2-integrin-dependent signal transduction pathways. The proposed studies would lead to the development of new strategies to control the leukocyte activation and to limit cancer cell metastasis by modulating their integrin avidity.
