Protein C inhibitor und JFC1/synaptotagmin-like protein 1

Protein C inhibitor (PCI) is a secreted serine protease inhibitor (serpin) with very broad protease reactivity. PCI is present in most human body fluids and seems to be important in reproduction, since male homozygous PCI- knockout mice are infertile. PCI belongs to the heparin-binding serpins, and heparin can modify its activity and target enzyme specificity. Recently we have shown that PCI also binds anionic and oxidized phospholipids, which also regulate its activity. Additionally we have shown that PCI can be internalized by cells via a non-conventional mechanism involving the phospholipid phosphatidylethanolamine. Internalized PCI can translocate to the nucleus. Using different methodological approaches we were able to identify several intracellular proteins interacting with PCI. The interaction of one out of these proteins, JFC1 (synaptotagmin-like protein 1), with PCI was analyzed in more detail. JFC1 has initially been described as an interaction partner of p67phox, a protein of the NADPH complex. JFC1 binds to phospholipids and is an effector molecule of the small GTPase Rab27a, which is involved in vesicular transport processes in the cell. We have shown that endogenous PCI and endogenous JFC1 are co- localized in cells and can be co-immunoprecipitated from cell extracts. These data suggest direct binding of PCI to JFC1. However, so far we have not shown direct binding in purified systems. Therefore it is also not clear, which domains of the molecules are involved in binding and if other factors may influence binding. Also the biological relevance of the interaction between JFC1 and PCI is completely unclear. It is therefore the aim of this project to analyze the biochemistry of the PCI-JFC1 interaction. We want to determine which domains of the two molecules are involved in binding and which factors (proteins, phospholipids) influence binding. Furthermore we want to analyze the biological relevance of the binding of PCI to JFC1. This will be analyzed on the molecular level, by studying known functions of the two proteins in the absence and presence of the reaction partner. Since the Rab27/JFC-1 system plays a role in intracellular transport and regulated secretion we want to determine if JFC1 is necessary for the secretion of PCI and/or for its internalization and nuclear translocation.

Within this project we identified molecular properties, which enable an extracellular protein to penetrate through the phospholipid layer of the cell membrane and to translocate to the nucleus. We also determined how this internalized protein can change the intracellular localization of another protein. We analyzed the interaction of the intracellular protein JFC1 (synaptotagmin-like protein 1) with a secreted, extracellular protein (protein C inhibitor, PCI, serpinA5), which can be internalized by cells via a non-canonical mechanism and which is then transferred to the nucleus. We identified those domains of the PCI molecule, which mediate cell membrane permeation and nuclear translocation, respectively. PCI is a serine protease inhibitor (serpin), which inactivates many proteases and which in the human system is synthesized by many tissues and is present in most body fluids and secretions. In the mouse system PCI is required for male fertility. In the human system PCI seem to have protective effects against cancer and metastasis formation. In this project we could identify the domain of the PCI molecule, which mediates internalization by cells. Proteolytic cleavage by a membrane-anchored protease can release this PCI domain. This may represent one of the mechanisms regulating PCI internalization. We furthermore identified the PCI domain responsible for its translocation to the nucleus (=nuclear localization signal). In a previous project using a molecular biological screening technique we have shown that PCI most likely binds JFC1. JFC1 is a protein, which binds to certain phospholipids present in the inner layer of the cell membrane and which might be involved in cellular secretion. In vitro these JFC1-binding phospholipids also bind PCI. JFC1 can be phosphorylated by AKT/proteinkinase B and is then released from the cell membrane to the cytosol. We could show that in fact PCI and JFC1 interact in several cells/cell lines. In prostate cells the presence of PCI causes translocation of JFC1 from the cell membrane to the nucleus. We could also show that overexpression of PCI as well as adding PCI to cells resulted in AKT/proteinkinase B-activation and that this AKT activation was a requirement for the translocation of JFC1 to the nucleus.