Annexin A6 (AnxA6) belongs to a family of Ca2+-dependent membrane-binding proteins and is involved in the regulation of endocytic and exocytic pathways. We previously demonstrated that AnxA6 regulates receptor-mediated endocytosis and lysosomal targeting of low-density lipoproteins and translocates to cholesterol-enriched late endosomes (LE). As cholesterol modulates the membrane binding and the cellular location of AnxA6, but also affects the intracellular distribution of caveolin, we investigated the localization and trafficking of caveolin in AnxA6-expressing cells. Here, we show that cells expressing high levels of AnxA6 are characterized by an accumulation of caveolin-1 (cav-1) in the Golgi complex. This is associated with a sequestration of cholesterol in the LE and lower levels of cholesterol in the Golgi and the plasma membrane, both likely contributing to retention of caveolin in the Golgi apparatus and a reduced number of caveolae at the cell surface. Further strengthening these findings, knock down of AnxA6 and the ectopic expression of the Niemann–Pick C1 protein in AnxA6-overexpressing cells restore the cellular distribution of cav-1 and cholesterol, respectively. In summary, this study demonstrates that elevated expression levels of AnxA6 perturb the intracellular distribution of cholesterol, which indirectly inhibits the exit of caveolin from the Golgi complex.

Annexin A6 (AnxA6) belongs to a conserved family of Ca(2+)-dependent membrane-binding proteins. Like other annexins, the function of AnxA6 is linked to its ability to bind phospholipids in cellular membranes in a dynamic and reversible fashion, in particular during the regulation of endocytic and exocytic pathways. High amounts of AnxA6 sequester cholesterol in late endosomes, thereby lowering the levels of cholesterol in the Golgi and the plasma membrane. These AnxA6-dependent redistributions of cellular cholesterol pools give rise to reduced cytoplasmic phospholipase A2 (cPLA(2)) activity, retention of caveolin in the Golgi apparatus and a reduced number of caveolae at the cell surface. In addition to regulating cholesterol and caveolin distribution, AnxA6 acts as a scaffold/targeting protein for several signaling proteins, the best characterized being the Ca(2+)-dependent membrane targeting of p120GAP to downregulate Ras activity. AnxA6 also stimulates the Ca(2+)-inducible involvement of PKC in the regulation of HRas and possibly EGFR signal transduction pathways. The ability of AnxA6 to recruit regulators of the EGFR/Ras pathway is likely potentiated by AnxA6-induced actin remodeling. Accordingly, AnxA6 may function as an organizer of membrane domains (i) to modulate intracellular cholesterol homeostasis, (ii) to create a scaffold for the formation of multifactorial signaling complexes, and (iii) to regulate transient membrane-actin interactions during endocytic and exocytic transport. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Inhibition of cholesterol export from late endosomes causes cellular cholesterol imbalance, including cholesterol depletion in the trans-Golgi network (TGN). Here, using Chinese hamster ovary (CHO) Niemann-Pick type C1 (NPC1) mutant cell lines and human NPC1 mutant fibroblasts, we show that altered cholesterol levels at the TGN/endosome boundaries trigger Syntaxin 6 (Stx6) accumulation into VAMP3, transferrin, and Rab11-positive recycling endosomes (REs). This increases Stx6/VAMP3 interaction and interferes with the recycling of αVβ3 and α5β1 integrins and cell migration, possibly in a Stx6-dependent manner. In NPC1 mutant cells, restoration of cholesterol levels in the TGN, but not inhibition of VAMP3, restores the steady-state localization of Stx6 in the TGN. Furthermore, elevation of RE cholesterol is associated with increased amounts of Stx6 in RE. Hence, the fine-tuning of cholesterol levels at the TGN-RE boundaries together with a subset of cholesterol-sensitive SNARE proteins may play a regulatory role in cell migration and invasion.

Protein kinase Cα (PKCα) can phosphorylate the epidermal growth factor receptor (EGFR) at threonine 654 (T654) to inhibit EGFR tyrosine phosphorylation (pY-EGFR) and the associated activation of downstream effectors. However, upregulation of PKCα in a large variety of cancers is not associated with EGFR inactivation, and factors determining the potential of PKCα to downregulate EGFR are yet unknown. Here, we show that ectopic expression of annexin A6 (AnxA6), a member of the Ca(2+) and phospholipid-binding annexins, strongly reduces pY-EGFR levels while augmenting EGFR T654 phosphorylation in EGFR overexpressing A431, head and neck and breast cancer cell lines. Reduced EGFR activation in AnxA6 expressing A431 cells is associated with reduced EGFR internalization and degradation. RNA interference (RNAi)-mediated PKCα knockdown in AnxA6 expressing A431 cells reduces T654-EGFR phosphorylation, but restores EGFR tyrosine phosphorylation, clonogenic growth and EGFR degradation. These findings correlate with AnxA6 interacting with EGFR, and elevated AnxA6 levels promoting PKCα membrane association and interaction with EGFR. Stable expression of the cytosolic N-terminal mutant AnxA6(1-175), which cannot promote PKCα membrane recruitment, does not increase T654-EGFR phosphorylation or the association of PKCα with EGFR. AnxA6 overexpression does not inhibit tyrosine phosphorylation of the T654A EGFR mutant, which cannot be phosphorylated by PKCα. Most strikingly, stable plasma membrane anchoring of AnxA6 is sufficient to recruit PKCα even in the absence of EGF or Ca(2+). In summary, AnxA6 is a new PKCα scaffold to promote PKCα-mediated EGFR inactivation through increased membrane targeting of PKCα and EGFR/PKCα complex formation.