Browsing by Author "Erlmann, Patrik"
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Item Open Access Regulation of START domain containing proteins through membrane interaction(2009) Erlmann, Patrik; Pfizenmaier, Klaus (Prof. Dr.)In mammals, there are 15 proteins that contain a lipid-binding START domain and those that have been characterized are able to transfer lipids between membranes in vitro. A prerequisite for START domains to act as lipid carriers is the interaction of the protein with cellular membranes. The goal of this thesis was to investigate how membrane targeting of these START domaincontaining proteins is regulated and affects protein function. Three START family members were studied in greater detail: StarD10, CERT/StarD11 and DLC1/StarD12. StarD10 is a minimal START domain protein with no additional targeting sequences, which was previously shown to transfer phosphatidylcholine (PC) and to a lesser extent phosphatidylethanolamine (PE) in vitro. By mass spectrometry, we identified a novel phosphorylation site in StarD10 at serine 284. We were able to show that casein kinase II mediated phosphorylation of this site reduced lipid transfer in vitro and membrane binding in vivo. However, the intracellular route of lipid transfer by StarD10 still remains to be determined. By contrast, the ceramide transfer protein CERT is known to shuttle ceramide from the endoplasmatic reticulum (ER) to the Golgi complex. Targeting to these organelles is mediated via the FFAT motif that binds to the ER-resident protein VAP and the PH domain that binds phosphatidylinositol-(4)-phosphate (PI(4)P) enriched in Golgi membranes. Here we identified CERT as a novel substrate for Protein Kinase D (PKD) at the Golgi. PKD-mediated phosphorylation at serine 132 is shown to reduce the PH domain-dependent interaction of CERT with PI(4)P-containing membranes and ceramide transfer. Thus, membrane binding of both StarD10 and CERT is negatively regulated by phosphorylation and this results in decreased lipid transfer efficiency. The tumor suppressor protein DLC1 (Deleted in Liver Cancer 1) is a Rho GTPase activating protein (GAP), which contains a START domain of unknown specificity and function. Through its GAP domain DLC1 inactivates the small GTPase RhoA to control actin cytoskeletal remodeling and biological processes such as cell migration and proliferation. In this thesis, DLC1 was found to interact with phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2) through a previously unrecognized polybasic region (PBR). Interestingly, PI(4,5)P2-containing membranes enhanced DLC1 RhoGAP activity in vitro. In living cells, a DLC1 mutant lacking an intact PBR inactivated Rho signaling less efficiently and was severely compromised in suppressing cell spreading, directed migration and proliferation. PI(4,5)P2 thus appears to be an important cofactor in DLC1 regulation in vivo. The PBR, however, was not sufficient to recruit DLC1 to membranes and its deletion did not alter DLC1 subcellular localization. Instead, we provide evidence for alternative mechanisms towards the regulation of DLC1 localization. DLC1 interaction with the lipid phosphatase PTEN may contribute to plasma membrane localization, while interaction with 14-3-3 adaptor proteins sequesters the protein in the cytoplasm.