Membrane trafficking and Annexin A6 group

LINES OF RESEARCH

Annexin A6 and Signalling

Summary

AnxA6 is recruited to the plasma membrane after different stimuli due to a local increment of calcium levels. It has been shown that AnxA6 binds and targets numerous Ras/MAPK signalling pathway proteins such as p120GAP, Raf-1 and PKCα to the vicinity of the plasma membrane. There, AnxA6 works as a scaffolding protein that regulates these signalling pathways. In this project we study the molecular mechanisms that allow AnxA6 to regulate these signalling pathways and its implication in cell proliferation and cancer.

Description

We are interested in the regulation of the localisation and targeting of the Ras/Raf/MAPK signalling pathway. The Ras signalling pathway has attracted considerable attention since in 30% of human cancers, the Ras family of proteins is activated by oncogenic mutations. Ras signalling has been thought to occur exclusively at the plasma membrane, but recently Ras activity has also been identified in intracellular compartments. The localization of the Ras pathway is regulated by targeting proteins. One of these targeting proteins, Annexin A6, belongs to a family member of Ca2+-dependent membrane binding proteins, and regulates membrane traffic at the plasma membrane, endosomes and in caveolae/lipid rafts. Annexin A6 interacts with Ras regulators (GAP) and Ras effectors (Raf-1) and is involved in the regulation of the localisation and activity of the Ras pathway. We aim to identify the molecular mechanism, how Annexin A6 affects Ras/Raf/MAPK localisation and activity. A better understanding of the mechanisms that regulate the localisation of the Ras pathway will add to a better understanding in human cancer.

 

Along this line, in a recent publication we analyzed a panel of breast cancer cells where overexpression of epidermal growth factor receptor (EGFR) is associated with enhanced activation of wild-type (hyperactive) Ras. Little is known about the regulation of Ras inactivation and GTPase-activating proteins (GAPs), such as p120GAP, in cells with hyperactive Ras. Recently, we showed that in EGFR-overexpressing A431 cells, which lack endogenous Annexin A6 (AnxA6), ectopic expression of AnxA6 stimulates membrane recruitment of p120GAP to modulate Ras signaling.

 

We now demonstrate that, AnxA6 is downregulated in a number of EGFR-overexpressing and estrogen receptor (ER)-negative breast cancer cells. In these cells, AnxA6 overexpression promotes Ca2+- and EGF-inducible membrane targeting of p120GAP. In ER-negative MDA-MB-436 cells, overexpression of p120GAP, but not CAPRI or a p120GAP mutant lacking the AnxA6-binding domain inhibits Ras/MAPK activity. AnxA6 knockdown in MDA-MB-436 increases Ras activity and cell proliferation in anchorage-independent growth assays. Furthermore, AnxA6 co-immunoprecipitates with H-Ras in a Ca2+ and EGF-inducible manner and fluorescence resonance energy transfer (FRET) microscopy confirmed that AnxA6 is in close proximity of active (G12V), but not inactive (S17N) H-Ras. Thus, association of AnxA6 with H-Ras-containing protein complexes may contribute to regulate p120GAP/Ras assembly in EGFR-overexpressing and ER-negative breast cancer cells.

 

Epidemiological, clinical and genetic studies have clearly shown a protective role of high density lipoproteins (HDL) against the development of atherosclerosis. These anti-atherosclerotic properties of HDL are predominantly through its removal of cellular cholesterol and induction of reverse cholesterol transport. This requires a direct interaction of HDL with cells to trigger intracellular signalling events. This project examines the potential role of the HDL-induced activation of the Ras/Raf/MAP Kinase pathway in the development of atherosclerosis. Understanding how this pathway influences lipid metabolism and cholesterol transport may enable cholesterol removal from cells to be enhanced, thus ameliorating the effects of atherosclerosis.

Figure 6
Figure 6. Scheme showing the AnxA6/PKCalpha complex in the regulation of EGFR signaling pathway.
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