Robert V. Stahelin

Binding of the PX domain of p47phox to phosphatidylinositol 3,4-bisphosphate and phosphatidic acid is masked by an intramolecular interaction

p47phox is a key cytosolic subunit required for activation of phagocyte NADPH oxidase. The X‐ray structure of the p47phox PX domain revealed two distinct basic pockets on the membrane‐binding surface, each occupied by a sulfate. These two pockets have different specificities: one preferentially binds phosphatidylinositol 3,4‐bisphosphate [PtdIns(3,4)P2] and is analogous to the phophatidylinositol 3‐phosphate (PtdIns3P)‐binding pocket of p40phox, while the other binds anionic phospholipids such as phosphatidic acid (PtdOH) or phosphatidylserine. The preference of this second site for PtdOH may be related to previously observed activation of NADPH oxidase by PtdOH. Simultaneous occupancy of the two phospholipid‐binding pockets radically increases membrane affinity. Strikingly, measurements for full‐length p47phox show that membrane interaction by the PX domain is masked by an intramolecular association with the C‐terminal SH3 domain (C‐SH3). Either a site‐specific mutation in C‐SH3 (W263R) or a mimic of the phosphorylated form of p47phox [Ser(303, 304, 328, 359, 370)Glu] cause a transition from a closed to an open conformation that binds membranes with a greater affinity than the isolated PX domain.

The Mechanism of Membrane Targeting of Human Sphingosine Kinase 1

Sphingosine 1-phosphate is a bioactive sphingolipid that regulates cell growth and suppresses programmed cell death. The biosynthesis of sphingosine 1-phosphate is catalyzed by sphingosine kinase (SK) but the mechanism by which the subcellular localization and activity of SK is regulated in response to various stimuli is not fully understood. To elucidate the origin and structural determinant of the specific subcellular localization of SK, we performed biophysical and cell studies of human SK1 (hSK1) and selected mutants. In vitro measurements showed that hSK1 selectively bound phosphatidylserine over other anionic phospholipids and strongly preferred the plasma membrane-mimicking membrane to other cellular membrane mimetics. Mutational analysis indicates that conserved Thr54 and Asn89 in the putative membrane-binding surface are essential for lipid selectivity and membrane targeting both in vitro and in the cell. Also, phosphorylation of Ser225 enhances the membrane affinity and plasma membrane selectivity of hSK1, presumably by modulating the interaction of Thr54 and Asn89 with the membrane. Collectively, these studies suggest that the specific plasma membrane localization and activation of SK1 is mediated largely by specific lipid-protein interactions.

Sphingosine analogue drug FTY720 targets I2PP2A/SET and mediates lung tumour suppression via activation of PP2A-RIPK1-dependent necroptosis

Mechanisms that alter protein phosphatase 2A (PP2A)‐dependent lung tumour suppression via the I2PP2A/SET oncoprotein are unknown. We show here that the tumour suppressor ceramide binds I2PP2A/SET selectively in the nucleus and including its K209 and Y122 residues as determined by molecular modelling/simulations and site‐directed mutagenesis. Because I2PP2A/SET was found overexpressed, whereas ceramide was downregulated in lung tumours, a sphingolipid analogue drug, FTY720, was identified to mimick ceramide for binding and targeting I2PP2A/SET, leading to PP2A reactivation, lung cancer cell death, and tumour suppression in vivo. Accordingly, while molecular targeting of I2PP2A/SET by stable knockdown prevented further tumour suppression by FTY720, reconstitution of WT‐I2PP2A/SET expression restored this process. Mechanistically, targeting I2PP2A/SET by FTY720 mediated PP2A/RIPK1‐dependent programmed necrosis (necroptosis), but not by apoptosis. The RIPK1 inhibitor necrostatin and knockdown or genetic loss of RIPK1 prevented growth inhibition by FTY720. Expression of WT‐ or death‐domain‐deleted (DDD)‐RIPK1, but not the kinase‐domain‐deleted (KDD)‐RIPK1, restored FTY720‐mediated necroptosis in RIPK1−/− MEFs. Thus, these data suggest that targeting I2PP2A/SET by FTY720 suppresses lung tumour growth, at least in part, via PP2A activation and necroptosis mediated by the kinase domain of RIPK1.

The Molecular Basis of Differential Subcellular Localization of C2 Domains of Protein Kinase C-α and Group IVa Cytosolic Phospholipase A2

The C2 domain is a Ca2+-dependent membrane-targeting module found in many cellular proteins involved in signal transduction or membrane trafficking. C2 domains are unique among membrane targeting domains in that they show a wide range of lipid selectivity for the major components of cell membranes, including phosphatidylserine and phosphatidylcholine. To understand how C2 domains show diverse lipid selectivity and how this functional diversity affects their subcellular targeting behaviors, we measured the binding of the C2 domains of group IVa cytosolic phospholipase A2 (cPLA2) and protein kinase C-α (PKC-α) to vesicles that model cell membranes they are targeted to, and we monitored their subcellular targeting in living cells. The surface plasmon resonance analysis indicates that the PKC-α C2 domain strongly prefers the cytoplasmic plasma membrane mimic to the nuclear membrane mimic due to high phosphatidylserine content in the former and that Asn189 plays a key role in this specificity. In contrast, the cPLA2 C2 domain has specificity for the nuclear membrane mimic over the cytoplasmic plasma membrane mimic due to high phosphatidylcholine content in the former and aromatic and hydrophobic residues in the calcium binding loops of the cPLA2 C2 domain are important for its lipid specificity. The subcellular localization of enhanced green fluorescent protein-tagged C2 domains and mutants transfected into HEK293 cells showed that the subcellular localization of the C2 domains is consistent with their lipid specificity and could be tailored by altering their in vitro lipid specificity. The relative cell membrane translocation rate of selected C2 domains was also consistent with their relative affinity for model membranes. Together, these results suggest that biophysical principles that govern thein vitro membrane binding of C2 domains can account for most of their subcellular targeting properties.

Ceramide-1-phosphate binds group IVA cytosolic phospholipase a2 via a novel site in the C2 domain.

Previously, ceramide-1-phosphate (C1P) was demonstrated to be a potent and specific activator of group IV cytosolic phospholipase A2α (cPLA2α) via interaction with the C2 domain. In this study, we hypothesized that the specific interaction site for C1P was localized to the cationic β-groove (Arg57, Lys58, Arg59) of the C2 domain of cPLA2α. In this regard, mutants of this region of cPLA2α were generated (R57A/K58A/R59A, R57A/R59A, K58A/R59A, R57A/K58A, R57A, K58A, and R59A) and examined for C1P affinity by surface plasmon resonance. The triple mutants (R57A/K58A/R59A), the double mutants (R57A/R59A, K58A/R59A, and R57A/K58A), and the single mutant (R59A) demonstrated significantly reduced affinity for C1P-containing vesicles as compared with wild-type cPLA2α. Examining these mutants for enzymatic activity demonstrated that these five mutants of cPLA2α also showed a significant reduction in the ability of C1P to: 1) increase the Vmax of the reaction; and 2) significantly decrease the dissociation constant (K As) of the reaction as compared with the wild-type enzyme. The mutational effect was specific for C1P as all of the cationic mutants of cPLA2α demonstrated normal basal activity as well as normal affinities for phosphatidylcholine and phosphatidylinositol-4,5-bisphosphate as compared with wild-type cPLA2α. This study, for the first time, demonstrates a novel C1P interaction site mapped to the cationic β-groove of the C2 domain of cPLA2α.

Lipid binding domains: more than simple lipid effectors

The spatial and temporal regulation of lipid molecules in cell membranes is a hallmark of cellular signaling and membrane trafficking events. Lipid-mediated targeting provides for strict control and versatility, because cell membranes harbor a large number of lipid molecules with variation in head group and acyl chain structures. Signaling and trafficking proteins contain a large number of modular domains that exhibit specific lipid binding properties and play a critical role in their localization and function. Nearly 20 years of research including structural, computational, biochemical and biophysical studies have demonstrated how these lipid-binding domains recognize their target lipid and achieve subcellular localization. The integration of this individual lipid-binding domain data in the context of the full-length proteins, macromolecular signaling complexes, and the lipidome is only beginning to be unraveled and represents a target of therapeutic development. This review brings together recent findings and classical concepts to concisely summarize the lipid-binding domain field while illustrating where the field is headed and how the gaps may be filled in with new technologies.

Ceramide kinase uses ceramide provided by ceramide transport protein: localization to organelles of eicosanoid synthesis

Ceramide kinase (CERK) is a critical mediator of eicosanoid synthesis, and its product, ceramide-1-phosphate (C1P), is required for the production of prostaglandins in response to several inflammatory agonists. In this study, mass spectrometry analysis disclosed that the main forms of C1P in cells were C16:0 C1P and C18:0 C1P, suggesting that CERK uses ceramide transported to the trans-Golgi apparatus by ceramide transport protein (CERT). To this end, downregulation of CERT by RNA interference technology dramatically reduced the levels of newly synthesized C1P (kinase-derived) as well as significantly reduced the total mass levels of C1P in cells. Confocal microscopy, subcellular fractionation, and surface plasmon resonance analysis were used to further localize CERK to the trans-Golgi network, placing the generation of C1P in the proper intracellular location for the recruitment of cytosolic phospholipase A2α. In conclusion, these results demonstrate that CERK localizes to areas of eicosanoid synthesis and uses a ceramide “pool” transported in an active manner via CERT.

Ceramide-1-phosphate is required for the translocation of group IVA cytosolic phospholipase A2 and prostaglandin synthesis

Little is known about the regulation of eicosanoid synthesis proximal to the activation of cytosolic phospholipase A2α (cPLA2α), the initial rate-limiting step. The current view is that cPLA2α associates with intracellular/phosphatidylcholine-rich membranes strictly via hydrophobic interactions in response to an increase of intracellular calcium. In opposition to this accepted mechanism of two decades, ceramide 1-phosphate (C1P) has been shown to increase the membrane association of cPLA2α in vitro via a novel site in the cationic β-groove of the C2 domain (Stahelin, R. V., Subramanian, P., Vora, M., Cho, W., and Chalfant, C. E. (2007) J. Biol. Chem. 282, 20467–204741). In this study we demonstrate that C1P is a proximal and required bioactive lipid for the translocation of cPLA2α to intracellular membranes in response to inflammatory agonists (e.g. calcium ionophore and ATP). Last, the absolute requirement of the C1P/cPLA2α interaction was demonstrated for the production of eicosanoids using murine embryonic fibroblasts (cPLA2α−/−) coupled to “rescue” studies. Therefore, this study provides a paradigm shift in how cPLA2α is activated during inflammation.

Differential Roles of Phosphatidylserine, PtdIns(4,5)P2, and PtdIns(3,4,5)P3 in Plasma Membrane Targeting of C2 Domains MOLECULAR DYNAMICS SIMULATION, MEMBRANE BINDING, AND CELL TRANSLOCATION STUDIES OF THE PKCα C2 Domain

Many cytosolic proteins are recruited to the plasma membrane (PM) during cell signaling and other cellular processes. Recent reports have indicated that phosphatidylserine (PS), phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) that are present in the PM play important roles for their specific PM recruitment. To systematically analyze how these lipids mediate PM targeting of cellular proteins, we performed biophysical, computational, and cell studies of the Ca2+-dependent C2 domain of protein kinase Cα (PKCα) that is known to bind PS and phosphoinositides. In vitro membrane binding measurements by surface plasmon resonance analysis show that PKCα-C2 nonspecifically binds phosphoinositides, including PtdIns(4,5)P2 and PtdIns(3,4,5)P3, but that PS and Ca2+ binding is prerequisite for productive phosphoinositide binding. PtdIns(4,5)P2 or PtdIns(3,4,5)P3 augments the Ca2+- and PS-dependent membrane binding of PKCα-C2 by slowing its membrane dissociation. Molecular dynamics simulations also support that Ca2+-dependent PS binding is essential for membrane interactions of PKCα-C2. PtdIns(4,5)P2 alone cannot drive the membrane attachment of the domain but further stabilizes the Ca2+- and PS-dependent membrane binding. When the fluorescence protein-tagged PKCα-C2 was expressed in NIH-3T3 cells, mutations of phosphoinositide-binding residues or depletion of PtdIns(4,5)P2 and/or PtdIns(3,4,5)P3 from PM did not significantly affect the PM association of the domain but accelerated its dissociation from PM. Also, local synthesis of PtdIns(4,5)P2 or PtdIns(3,4,5)P3 at the PM slowed membrane dissociation of PKCα-C2. Collectively, these studies show that PtdIns(4,5)P2 and PtdIns(3,4,5)P3 augment the Ca2+- and PS-dependent membrane binding of PKCα-C2 by elongating the membrane residence of the domain but cannot drive the PM recruitment of PKCα-C2. These studies also suggest that effective PM recruitment of many cellular proteins may require synergistic actions of PS and phosphoinositides.

Mechanism of diacylglycerol-induced membrane targeting and activation of protein kinase C

Protein kinase C (PKC) θ is a novel PKC that plays a key role in T lymphocyte activation. PKCθ has been shown to be specifically recruited to the immunological synapse in response to T cell receptor activation. To understand the basis of its unique subcellular localization properties, we investigated the mechanism of in vitro and cellular sn-1,2-diacylglycerol (DAG)-mediated membrane binding of PKCθ. PKCθ showed phosphatidylserine selectivity in membrane binding and kinase action, which contributes to its translocation to the phosphatidylserine-rich plasma membrane in HEK293 cells. Unlike any other PKCs characterized so far, the isolated C1B domain of PKCθ had much higher affinity for DAG-containing membranes than the C1A domain. Also, the mutational analysis indicates that the C1B domain plays a predominant role in the DAG-induced membrane binding and activation of PKCθ. Furthermore, the Ca2+-independent C2 domain of PKCθ has significant affinity for anionic membranes, and the truncation of the C2 domain greatly enhanced the membrane affinity and enzyme activity of PKCθ. In addition, membrane binding properties of Y90E and Y90F mutants indicate that phosphorylation of Tyr90 of the C2 domain enhances the affinity of PKCθ for model and cell membranes. Collectively, these results show that PKCθ has a unique membrane binding and activation mechanism that may account for its subcellular targeting properties.