The Coffin-Lowry syndrome-associated protein RSK2 is implicated in calcium-regulated exocytosis through the regulation of PLD1.

Exocytosis of neurotransmitters and hormones occurs through the fusion of secretory vesicles with the plasma membrane. This highly regulated process involves key proteins, such as SNAREs, and specific lipids at the site of membrane fusion. Phospholipase D (PLD) has recently emerged as a promoter of membrane fusion in various exocytotic events potentially by providing fusogenic cone-shaped phosphatidic acid. We show here that PLD1 is regulated by ribosomal S6 kinase 2 (RSK2)-dependent phosphorylation. RSK2 is activated by a high K(+)-induced rise in cytosolic calcium. Expression of inactive RSK2 mutants or selective knockdown of endogenous RSK2 dramatically affects the different kinetic components of the exocytotic response in chromaffin cells. RSK2 physically interacts with and stimulates PLD activity through the phosphorylation of Thr-147 in the PLD1 amino-terminal phox homology domain. Expression of PLD1 phosphomimetic mutants fully restores secretion in cells depleted of RSK2, suggesting that RSK2 is a critical upstream signaling element in the activation of PLD1 to produce the lipids required for exocytosis. We propose that PLD-related defects in neuronal and endocrine activities could contribute to the effect observed after the loss-of-function mutations in Rsk2 that lead to Coffin-Lowry syndrome, an X-linked form of growth and mental retardation.

[The GIT-PIX protein complex: a hub to ARF and Rac/Cdc42 GTPases]. / Le complexe GIT-PIX: une plate-forme de régulation des GTPases ARF et Rac/Cdc42.

We recently described that the tumor suppressor factor Scribble anchors the PIX exchange factor for Rac/Cdc42 and the ARF-GAP GIT proteins at the plasma membrane. Because it has been postulated that the GIT-PIX proteins dimerize and tightly self-assemble to form a high molecular weight complex, this nexus may be capable of linking together important signalling molecules to control cytosqueleton polymerization and membrane dynamics. To date, most studies that have tempted to unravel the function of these proteins have found their implication in a great variety of cellular functions (receptor recycling, endo-exocytosis, cell migration, synapse formation...) but have mostly neglected to consider the multimeric organization of this hub. There is no doubt that our comprehension of physiopathological disorders such as cancers will be improved when the nature of the complex pathways integrated by the GIT-PIX nodule will be understood.

The Coffin-Lowry syndrome-associated protein RSK2 controls neuroendocrine secretion through the regulation of phospholipase D1 at the exocytotic sites.

Together with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, fusogenic cone-shaped lipids, such as phosphatidic acid (PA), have been recently shown to be important actors in membrane fusion during exocytosis. Phospholipase D (PLD) appears to be the main provider of PA at the exocytotic site in neuroendocrine cells. We show here that ribosomal S6 kinase 2 (RSK2) stimulates PLD activity through the phosphorylation of Thr147 in the PLD1 amino-terminal Phox-homology domain. In PC12 cells, depletion of RSK2 dramatically prevents PA synthesis at exocytotic sites and inhibits hormone release. Expression of PLD1 phosphomimetic mutants fully restores secretion in cells depleted of RSK2, suggesting that RSK2 is a critical upstream signaling element in the activation of PLD1 to produce the lipids required for exocytosis.

Phospholipase D1 production of phosphatidic acid at the plasma membrane promotes exocytosis of large dense-core granules at a late stage.

Substantial efforts have recently been made to demonstrate the importance of lipids and lipid-modifying enzymes in various membrane trafficking processes, including calcium-regulated exocytosis of hormones and neurotransmitters. Among bioactive lipids, phosphatidic acid (PA) is an attractive candidate to promote membrane fusion through its ability to change membrane topology. To date, however, the biosynthetic pathway, the dynamic location, and actual function of PA in secretory cells remain unknown. Using a short interference RNA strategy on chromaffin and PC12 cells, we demonstrate here that phospholipase D1 is activated in secretagogue-stimulated cells and that it produces PA at the plasma membrane at the secretory granule docking sites. We show that phospholipase D1 activation and PA production represent key events in the exocytotic progression. Membrane capacitance measurements indicate that reduction of endogenous PA impairs the formation of fusion-competent granules. Finally, we show that the PLD1 short interference RNA-mediated inhibition of exocytosis can be rescued by exogenous provision of a lipid that favors the transition of opposed bi-layer membranes to hemifused membranes having the outer leaflets fused. Our findings demonstrate that PA synthesis is required during exocytosis to facilitate a late event in the granule fusion pathway. We propose that the underlying mechanism is related to the ability of PA to alter membrane curvature and promote hemi-fusion.