p47 phox is a key cytosolic subunit required for activation of phagocyte NADPH oxidase. The X-ray structure of the p47 phox PX domain revealed two distinct basic pockets on the membrane-binding surface, each occupied by a sulfate. These two pockets have different speci®cities: one preferentially binds phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P 2 ] and is analogous to the phophatidylinositol 3-phosphate (PtdIns3P)-binding pocket of p40 phox , 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 phospholipidbinding pockets radically increases membrane af®n-ity. Strikingly, measurements for full-length p47 phox 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-speci®c mutation in C-SH3 (W263R) or a mimic of the phosphorylated form of p47 phox [Ser(303, 304, 328, 359, 370)Glu] cause a transition from a closed to an open conformation that binds membranes with a greater af®nity than the isolated PX domain.

No abstract

Controlled distribution of lipids across various cell membranes is crucial for cell homeostasis and regulation. We developed an imaging method that allows simultaneous in situ quantification of cholesterol in two leaflets of the plasma membrane (PM) using tunable orthogonal cholesterol sensors. Our imaging revealed marked transbilayer asymmetry of PM cholesterol (TAPMC) in various mammalian cells, with the concentration in the inner leaflet (IPM) being ~12-fold lower than that in the outer leaflet (OPM). The asymmetry was maintained by active transport of cholesterol from IPM to OPM and its chemical retention at OPM. Furthermore, the increase in the IPM cholesterol level was triggered in a stimulus-specific manner, allowing cholesterol to serve as a signaling lipid. We found excellent correlation between the IPM cholesterol level and cellular Wnt signaling activity, suggesting that TAPMC and stimulus-induced PM cholesterol redistribution are crucial for tight regulation of cellular processes under physiological conditions.

Gangliosides, glycosphingolipids containing one or more sialic acid(s) in the glyco-chain, are involved in various important physiological and pathological processes in the plasma membrane. However, their exact functions are poorly understood, primarily because of the scarcity of suitable fluorescent ganglioside analogs. Here, we developed methods for systematically synthesizing analogs that behave like their native counterparts in regard to partitioning into raft-related membrane domains or preparations. Single-fluorescent-molecule imaging in the live-cell plasma membrane revealed the clear but transient colocalization and codiffusion of fluorescent ganglioside analogs with a fluorescently labeled glycosylphosphatidylinisotol (GPI)-anchored protein, human CD59, with lifetimes of 12 ms for CD59 monomers, 40 ms for CD59's transient homodimer rafts in quiescent cells, and 48 ms for engaged-CD59-cluster rafts, in cholesterol- and GPI-anchoring-dependent manners. The ganglioside molecules were always mobile in quiescent cells. These results show that gangliosides continually and dynamically exchange between raft domains and the bulk domain, indicating that raft domains are dynamic entities.

Group V phospholipase A 2 is a recently discovered secretory phospholipase A 2 (PLA 2 ) that has been shown to be involved in eicosanoid formation in inflammatory cells, such as macrophages and mast cells. We have demonstrated that human group V PLA 2 (hsPLA 2 -V) can bind phosphatidylcholine (PC) membranes and hydrolyze PC substrates much more efficiently than human group IIa PLA 2 , which makes it better suited for acting on the outer plasma membrane (Han, S.-K., Yoon, E. T., and Cho, W. (1998) Biochem. J. 331, 353-357). In this study, we demonstrate that exogenous hsPLA 2 -V has much greater activity than does group IIa PLA 2 to release fatty acids from various mammalian cells and to elicit leukotriene B 4 formation from human neutrophils. To understand the molecular basis of these activities, we mutated two surface tryptophans of hsPLA 2 -V to alanine (W31A and W79A) and measured the effects of these mutations on the kinetic activity toward various substrates, on the binding affinity for vesicles and phospholipid-coated beads, on the penetration into phospholipid monolayers, and on the activity to release fatty acids and elicit eicosanoid formation from various mammalian cells. These studies show that the relatively high ability of hsPLA 2 -V to induce cellular eicosanoid formation derives from its high affinity for PC membranes and that Trp 31 on its putative interfacial binding surface plays an important role in its binding to PC vesicles and to the outer plasma membrane.

Activation of class I phosphatidylinositol 3-kinase (PI3K) leads to formation of phosphatidylinositol-3,4,5-trisphophate (PIP) and phosphatidylinositol-3,4-bisphophate (PI34P), which spatiotemporally coordinate and regulate a myriad of cellular processes. By simultaneous quantitative imaging of PIP and PI34P in live cells, we here show that they have a distinctively different spatiotemporal distribution and history in response to growth factor stimulation, which allows them to selectively induce the membrane recruitment and activation of Akt isoforms. PI34P selectively activates Akt2 at both the plasma membrane and early endosomes, whereas PIP selectively stimulates Akt1 and Akt3 exclusively at the plasma membrane. These spatiotemporally distinct activation patterns of Akt isoforms provide a mechanism for their differential regulation of downstream signaling molecules. Collectively, our studies show that different spatiotemporal dynamics of PIP and PI34P and their ability to selectively activate key signaling proteins allow them to mediate class I PI3K signaling pathways in a spatiotemporally specific manner.

The C2 domain is a Ca 2؉ -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 A 2 (cPLA 2 ) and protein kinase C-␣ (PKC-␣) 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-␣ C2 domain strongly prefers the cytoplasmic plasma membrane mimic to the nuclear membrane mimic due to high phosphatidylserine content in the former and that Asn 189 plays a key role in this specificity. In contrast, the cPLA 2 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 cPLA 2 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 the in vitro membrane binding of C2 domains can account for most of their subcellular targeting properties.The agonist-induced subcellular targeting of protein is an important process in cell signaling and regulation. Recently, the membrane targeting of peripheral proteins (e.g. phospholipases, lipid-dependent protein kinases, lipid kinases, and lipid phosphatases) by Ca 2ϩ and lipid mediators, including phosphoinositides, has received much attention as an important event in cell signaling and membrane trafficking. It has been shown that the subcellular targeting of peripheral proteins is driven by a growing number of membrane targeting domains. These domains include protein kinase C (PKC) 1 conserved 1 (C1) domain, PKC conserved 2 (C2) domain, pleckstrin homology (PH) domain, Fab1, YOTB, Vac 1 and EEA1 (FYVE) domain, band four-point-one, ezrin, radixin and moesin (FERM) domain, epsin amino-terminal homology (ENTH) domain, and phox (PX) domains (1-5).The C2 domain has been identified in many cellular proteins involved in signal transduction or membrane trafficking (5-7). A majority of C2 domains bind the membrane in a Ca 2ϩ -dependent manner and thereby play an important role in Ca 2ϩ -dependent membrane targeting of pe...

The regulatory domains of conventional and novel protein kinases C (PKC) have two C1 domains (C1A and C1B) that have been identified as the interaction site for diacylglycerol (DAG) and phorbol ester. It has been reported that C1A and C1B domains of individual PKC isoforms play different roles in their membrane binding and activation; however, DAG affinity of individual C1 domains has not been quantitatively determined. In this study, we measured the affinity of isolated C1A and C1B domains of two conventional PKCs, PKC␣ and PKC␥, for soluble and membrane-incorporated DAG and phorbol ester by isothermal calorimetry and surface plasmon resonance. The C1A and C1B domains of PKC␣ have opposite affinities for DAG and phorbol ester; i.e. the C1A domain with high affinity for DAG and the C1B domain with high affinity for phorbol ester. In contrast, the C1A and C1b domains of PKC␥ have comparably high affinities for both DAG and phorbol ester. Consistent with these results, mutational studies of full-length proteins showed that the C1A domain is critical for the DAG-induced activation of PKC␣, whereas both C1A and C1B domains are involved in the DAG-induced activation of PKC␥. Further mutational studies in conjunction with in vitro activity assay and monolayer penetration analysis indicated that, unlike the C1A domain of PKC␣, neither the C1A nor the C1B domain of PKC␥ is conformationally restricted. Cell studies with enhanced green fluorescent protein-tagged PKCs showed that PKC␣ did not translocate to the plasma membrane in response to DAG at a basal intracellular calcium concentration due to the inaccessibility of its C1A domain, whereas PKC␥ rapidly translocated to the plasma membrane under the same conditions. These data suggest that differential activation mechanisms of PKC isoforms are determined by the DAG affinity and conformational flexibility of their C1 domains. Protein kinase C (PKC)1 are a family of serine/threonine kinases that mediate numerous cellular processes (1, 2). All PKCs contain an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. Based on structural differences in the regulatory domain, PKCs are generally classified into three groups: conventional PKC (␣, ␤I, ␤II, and ␥ subtypes), novel PKC (␦, ⑀, , and subtypes), and atypical PKC ( and / subtypes). Regulatory domains of both conventional and novel PKCs contain tandem C1 (C1A and C1B) domains and a C2 domain. The C1 domain (ϳ50 residues) is a cysteine-rich compact structure that contains five short ␤ strands, a short helix, and two zinc ions (3-7), whereas the C2 domain (ϳ130 residues) is composed of eight-stranded antiparallel ␤ strands (5, 8 -10) and interconnecting loops some of which are involved in Ca 2ϩ -dependent membrane binding. The C1 domain was first identified as the interaction site for diacylglycerol (DAG) and phorbol ester in PKCs (11), but it was subsequently found in other proteins with diverse functions, including protein kinase D (PKD/PKC), chimaerin, Ras-GRP, DAG kinases, and Raf-1 kinase (4, 6, 7). ...