p21-activated kinases (PAKs) are serine/threonine kinases that have been identified as targets for the small GTPases Rac and Cdc42. PAKs have been implicated in cytoskeletal regulation, stimulation of mitogen-activated protein kinase cascades, and in control of the phagocyte NADPH oxidase. Membrane targeting of PAK1 induced increased kinase activity in a GTPase-independent manner, suggesting that other mechanisms for PAK regulation exist. We observed concentration- and time-dependent activation of PAK1 by sphingosine and several related long chain sphingoid bases but not by ceramides or a variety of other lipids. Although phospholipids were generally ineffective, phosphatidic acid and phosphatidylinositol also had stimulatory effects on PAK1. Lipid stimulation induced a similar level of PAK1 activity as did stimulation by GTPases, and the patterns of PAK1 autophosphorylation determined after partial tryptic digestion and two-dimensional peptide analysis were similar with each class of activator. Lipid stimulation of PAK1 activity was dependent upon intact PAK kinase activity, as indicated by studies with a kinase-dead PAK1 mutant. Treatment of COS-7 cells expressing wild type PAK1 with sphingosine, fumonisin B, or sphingomyelinase, all of which are able to elevate the levels of free sphingosine, induced increased activity of PAK1 as determined using a p47(phox) peptide substrate. Studies using PAK1 mutants suggest that lipids act at a site overlapping or identical to the GTPase-binding domain on PAK. The inactive sphingosine derivative N,N-dimethylsphingosine was an effective inhibitor of PAK1 activation in response to either sphingosine or Cdc42. Our results demonstrate a novel GTPase-independent mechanism of PAK activation and, additionally, suggest that PAK(s) may be important mediators of the biological effects of sphingolipids.

Recent research has shed light on the cellular and molecular functions of bioactive lipids that go far beyond what was known about their role as dietary lipids. Bioactive lipids regulate inflammation and its resolution as signaling molecules. Genetic studies have identified key factors that can increase the risk of cardiovascular diseases and metabolic syndrome through their effects on lipogenesis. Lipid scientists have explored how these signaling pathways affect lipid metabolism in the liver, adipose tissue, and macrophages by utilizing a variety of techniques in both humans and animal models, including novel lipidomics approaches and molecular dynamics models. Dissecting out these lipid pathways can help identify mechanisms that can be targeted to prevent or treat cardiometabolic conditions. Continued investigation of the multitude of functions mediated by bioactive lipids may reveal additional components of these pathways that can provide a greater understanding of metabolic homeostasis.

Membrane contact sites (MCS), close membrane apposition between organelles, are platforms for interorganellar transfer of lipids including cholesterol, regulation of lipid homeostasis, and co-ordination of endocytic trafficking. Sphingosine kinases (SphKs), two isoenzymes that phosphorylate sphingosine to the bioactive sphingosine-1-phosphate (S1P), have been implicated in endocytic trafficking. However, the physiological functions of SphKs in regulation of membrane dynamics, lipid trafficking and MCS are not known. Here, we report that deletion of SphKs decreased S1P with concomitant increases in its precursors sphingosine and ceramide, and markedly reduced endoplasmic reticulum (ER) contacts with late endocytic organelles. Expression of enzymatically active SphK1, but not catalytically inactive, rescued the deficit of these MCS. Although free cholesterol accumulated in late endocytic organelles in SphK null cells, surprisingly however, cholesterol transport to the ER was not reduced. Importantly, deletion of SphKs promoted recruitment of the ER-resident cholesterol transfer protein Aster-B (also called GRAMD1B) to the plasma membrane (PM), consistent with higher accessible cholesterol and ceramide at the PM, to facilitate cholesterol transfer from the PM to the ER. In addition, ceramide enhanced in vitro binding of the Aster-B GRAM domain to phosphatidylserine and cholesterol liposomes. Our study revealed a previously unknown role for SphKs and sphingolipid metabolites in governing diverse MCS between the ER network and late endocytic organelles versus the PM to control the movement of cholesterol between distinct cell membranes.