Phosphatidylinositol-(4,5)-bisphosphate regulates sorting signal recognition by the clathrin-associated adaptor complex AP2

The alpha,beta2,mu2,sigma2 heterotetrameric AP2 complex is recruited exclusively to the phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P(2))-rich plasma membrane where, amongst other roles, it selects motif-containing cargo proteins for incorporation into clathrin-coated vesicles. Unphosphorylated and mu2Thr156-monophosphorylated AP2 mutated in their alphaPtdIns4,5P(2), mu2PtdIns4,5P(2), and mu2Yxxvarphi binding sites were produced, and their interactions with membranes of different phospholipid and cargo composition were measured by surface plasmon resonance. We demonstrate that recognition of Yxxvarphi and acidic dileucine motifs is dependent on corecognition with PtdIns4,5P(2), explaining the selective recruitment of AP2 to the plasma membrane. The interaction of AP2 with PtdIns4,5P(2)/Yxxvarphi-containing membranes is two step: initial recruitment via the alphaPtdIns4,5P(2) site and then stabilization through the binding of mu2Yxxvarphi and mu2PtdIns4,5P(2) sites to their ligands. The second step is facilitated by a conformational change favored by mu2Thr156 phosphorylation. The binding of AP2 to acidic-dileucine motifs occurs at a different site from Yxxvarphi binding and is not enhanced by mu2Thr156 phosphorylation.     

A phosphatidylinositol (4,5)-bisphosphate binding site within mu2-adaptin regulates clathrin-mediated endocytosis

The clathrin adaptor complex AP-2 serves to coordinate clathrin-coated pit assembly with the sorting of transmembrane cargo proteins at the plasmalemma. How precisely AP-2 assembly and cargo protein recognition at sites of endocytosis are regulated has remained unclear, but recent evidence implicates phosphoinositides, in particular phosphatidylinositol (4,5)-bisphosphate (PI[4,5]P2), in these processes. Here we have identified and functionally characterized a conserved binding site for PI(4,5)P2 within mu2-adaptin, the medium chain of the clathrin adaptor complex AP-2. Mutant mu2 lacking a cluster of conserved lysine residues fails to bind PI(4,5)P2 and to compete the recruitment of native clathrin/AP-2 to PI(4,5)P2-containing liposomes or to presynaptic membranes. Moreover, we show that expression of mutant mu2 inhibits receptor-mediated endocytosis in living cells. We suggest that PI(4,5)P2 binding to mu2-adaptin regulates clathrin-mediated endocytosis and thereby may contribute to structurally linking cargo recognition to coat formation.     

Structural and membrane binding analysis of the Phox homology domain of phosphoinositide 3-kinase-C2alpha

Phox homology (PX) domains, which have been identified in a variety of proteins involved in cell signaling and membrane trafficking, have been shown to interact with phosphoinositides (PIs) with different affinities and specificities. To elucidate the structural origin of diverse PI specificities of PX domains, we determined the crystal structure of the PX domain from phosphoinositide 3-kinase C2alpha (PI3K-C2alpha), which binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). To delineate the mechanism by which this PX domain interacts with membranes, we measured the membrane binding of the wild type domain and mutants by surface plasmon resonance and monolayer techniques. This PX domain contains a signature PI-binding site that is optimized for PtdIns(4,5)P(2) binding. The membrane binding of the PX domain is initiated by nonspecific electrostatic interactions followed by the membrane penetration of hydrophobic residues. Membrane penetration is specifically enhanced by PtdIns(4,5)P(2). Furthermore, the PX domain displayed significantly higher PtdIns(4,5)P(2) membrane affinity and specificity when compared with the PI3K-C2alpha C2 domain, demonstrating that high affinity PtdIns(4,5)P(2) binding was facilitated by the PX domain in full-length PI3K-C2alpha. Together, these studies provide new structural insight into the diverse PI specificities of PX domains and elucidate the mechanism by which the PI3K-C2alpha PX domain interacts with PtdIns(4,5)P(2)-containing membranes and thereby mediates the membrane recruitment of PI3K-C2alpha.     

Full-contact domain labeling: identification of a novel phosphoinositide binding site on gelsolin that requires the complete protein

Gelsolin, an actin and phosphoinositide binding protein, was photoaffinity labeled using a variety of benzophenone-containing phosphoinositide polyphosphate analogues. The N-terminal half and the C-terminal half of gelsolin showed synergy in the binding of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. Competitive displacement experiments with dibutyryl, dioctanoyl, or dipalmitoyl derivatives of PtdIns(4,5)P(2) suggested that, in addition to the inositol headgroup, a diacylglyceryl moiety was important for binding; these analogues also inhibited the gelsolin-severing activity of F-actin. In addition to the previously identified PtdIns(4,5)P2 binding site in the N-terminal half of gelsolin, a new binding site was identified in the C-terminal half by mapping the photocovalently modified peptide fragments. Moreover, increasing concentrations of Ca(2+) decreased the binding of the photolabile analogues to the C-terminal phosphoinositide binding site on gelsolin. A molecular model of the binding of PtdIns(4,5)P2 within two folded repeats of gelsolin has been calculated using these data.     

Myosin VI targeting to clathrin-coated structures and dimerization is mediated by binding to Disabled-2 and PtdIns(4,5)P2

Vesicle transport is essential for the movement of proteins, lipids and other molecules between membrane compartments within the cell. The role of the class VI myosins in vesicular transport is particularly intriguing because they are the only class that has been shown to move 'backwards' towards the minus end of actin filaments. Myosin VI is found in distinct intracellular locations and implicated in processes such as endocytosis, exocytosis, maintenance of Golgi morphology and cell movement. We have shown that the carboxy-terminal tail is the key targeting region and have identified three binding sites: a WWY motif for Disabled-2 (Dab2) binding, a RRL motif for glucose-transporter binding protein (GIPC) and optineurin binding and a site that binds specifically and with high affinity (Kd = 0.3 microM) to PtdIns(4,5)P2-containing liposomes. This is the first demonstration that myosin VI binds lipid membranes. Lipid binding induces a large structural change in the myosin VI tail (31% increase in helicity) and when associated with lipid vesicles, it can dimerize. In vivo targeting and recruitment of myosin VI to clathrin-coated structures (CCSs) at the plasma membrane is mediated by Dab2 and PtdIns(4,5)P2 binding.     

GAP1IP4BP contains a novel group I pleckstrin homology domain that directs constitutive plasma membrane association

The group I family of pleckstrin homology (PH) domains are characterized by their inherent ability to specifically bind phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and its corresponding inositol head-group inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)). In vivo this interaction results in the regulated plasma membrane recruitment of cytosolic group I PH domain-containing proteins following agonist-stimulated PtdIns(3,4,5)P(3) production. Among group I PH domain-containing proteins, the Ras GTPase-activating protein GAP1(IP4BP) is unique in being constitutively associated with the plasma membrane. Here we show that, although the GAP1(IP4BP) PH domain interacts with PtdIns(3,4, 5)P(3), it also binds, with a comparable affinity, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) (K(d) values of 0.5 +/- 0.2 and 0.8 +/- 0.5 microm, respectively). Intriguingly, whereas this binding site overlaps with that for Ins(1,3,4,5)P(4), consistent with the constitutive plasma membrane association of GAP1(IP4BP) resulting from its PH domain-binding PtdIns(4,5)P(2), we show that in vivo depletion of PtdIns(4,5)P(2), but not PtdIns(3,4,5)P(3), results in dissociation of GAP1(IP4BP) from this membrane. Thus, the Ins(1,3,4,5)P(4)-binding PH domain from GAP1(IP4BP) defines a novel class of group I PH domains that constitutively targets the protein to the plasma membrane and may allow GAP1(IP4BP) to be regulated in vivo by Ins(1,3,4,5)P(4) rather than PtdIns(3,4,5)P(3).     

Lipid-mediated membrane binding properties of Disabled-2

Disabled-2 (Dab2) is an adaptor protein involved in several biological processes ranging from endocytosis to platelet aggregation. During endocytosis, the Dab2 phosphotyrosine-binding (PTB) domain mediates protein binding to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) at the inner leaflet of the plasma membrane. As a result of platelet activation, Dab2 is released from α-granules and associates with both the αIIbβ3 integrin receptor and sulfatide lipids on the platelet surface through its N-terminal region including the PTB domain (N-PTB), thus, modulating platelet aggregation. Thrombin, a strong platelet agonist, prevents Dab2 function by cleaving N-PTB within the two basic motifs required for sulfatide association, a reaction that is prevented when Dab2 is bound to these sphingolipids. We have characterized the membrane binding properties of Dab2 N-PTB using micelles enriched with Dab2 lipid ligands, sulfatides and PtdIns(4,5)P(2). Remarkably, NMR spectroscopy studies suggested differences in lipid-binding mechanisms. In addition, we experimentally demonstrated that sulfatide- and PtdIns(4,5)P(2)-binding sites overlap in Dab2 N-PTB and that both lipids stabilize the protein against temperature-induced unfolding. We found that whereas sulfatides induced conformational changes and facilitated Dab2 N-PTB penetration into micelles, Dab2 N-PTB bound to PtdIns(4,5)P(2) lacked these properties. These results further support our model that platelet membrane sulfatides, but not PtdIns(4,5)P(2), protect Dab2 N-PTB from thrombin cleavage.     

Yeast Arf3p modulates plasma membrane PtdIns(4,5)P2 levels to facilitate endocytosis

Phosphatidylinositol-(4,5)-bisphosphate [PtdIns(4,5)P2] is a key regulator of endocytosis. PtdIns(4,5)P2 generation at the plasma membrane in yeast is mediated by the kinase Mss4p, but the mechanism underlying the temporal and spatial activation of Mss4p to increase formation of PtdIns(4,5)P2 at appropriate sites is not known. Here, we show that ADP ribosylation factor (Arf)3p, the yeast homologue of mammalian Arf6, is necessary for wild-type levels of PtdIns(4,5)P2 at the plasma membrane. Arf3p localizes to dynamic spots at the membrane, and the behaviour of these is consistent with it functioning in concert with endocytic machinery. Localization of Arf3p is disrupted by deletion of genes encoding an ArfGAP homology protein Gts1p and a guanine nucleotide exchange factor Yel1p. Significantly, deletion of arf3 causes a reduction in PtdIns(4,5)P2 at the plasma membrane, while increased levels of active Arf3p, caused by deletion of the GTPase-activating protein Gts1, increase PtdIns(4,5)P2 levels. Furthermore, elevated Arf3p correlates with an increase in the number of endocytic sites. Our data provide evidence for a mechanism in yeast to positively regulate plasma membrane production of PtdIns(4,5)P2 levels and that these changes impact on endocytosis.     

Interaction of phosphoinositide cycle intermediates with the plasma membrane-associated clathrin assembly protein AP-2.

Several components of the phosphoinositide cycle have been found to interact specifically and at physiological concentrations with the plasma membrane-associated clathrin assembly (adaptor) protein AP-2. These include phosphatidylinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate, which are present at the plasma membrane, as well as other polyphosphoinositols. ATP and other polyphosphate molecules complete with the polyphosphoinositols, however, they are at least 80-fold less potent. Also, the effect of ATP, unlike the polyphosphoinositols, is blocked by physiological concentrations of Mg2+. Photoaffinity labeling of AP-2 by [alpha-32P]8-azidoadenosine 5'-triphosphate and its competition by polyphosphoinositols has been used to identify the alpha subunit of the AP-2 complex as the site of specific interaction with the polyphosphoinositols and to confirm direct ultrafiltration binding experiments. Proteolytic dissection of the labeled AP-2 demonstrated that binding occurred exclusively on the N-terminal portion of the alpha subunit. Interaction of purified AP-2 with sub-microM concentrations of polyphosphoinositols has inhibitory effects on a novel AP-2 self-association described in the accompanying paper (Beck, K. A., and Keen, J. H., J. Biol. Chem. 266, 4437-4441), and at higher concentrations on the binding of AP-2 to dissociated clathrin trimers as well as AP-2-mediated clathrin coat assembly. Review of the literature shows that several physiological stimuli that are known to result in increased coat pit formation in intact cells correlate with increased phosphoinositide turnover. These in vivo correlations and the in vitro observations reported here suggest that coated membrane and phosphoinositide cycles may be interdependent within cells.     

The gamma/sigma1 and alpha/sigma2 hemicomplexes of clathrin adaptors AP-1 and AP-2 harbor the dileucine recognition site

The clathrin adaptors AP-1 and AP-2 bind cargo proteins via two types of motifs: tyrosine-based Yxx phi and dileucine-based [DE]XXXL[LI]. Although it is well established that Yxx phi motifs bind to the mu subunits of AP-1 or AP-2, dileucine motifs have been reported to bind to either the mu or beta subunits of these adaptors as well as the gamma/sigma1 hemicomplex of AP-1. To clarify this controversy, the various subunits of AP-1 and AP-2 were expressed individually and in hemicomplex form in insect cells, and they were used in glutathione S-transferase pull-down assays to determine their binding properties. We report that the gamma/sigma1 or alpha/sigma2 hemicomplexes bound the dileucine-based motifs of several proteins quite strongly, whereas binding by the beta1/mu1 and beta2/mu2 hemicomplexes, and the individual beta or mu subunits, was extremely weak or undetectable. The gamma/sigma1 and alpha/sigma2 hemicomplexes displayed substantial differences in their preference for particular dileucine-based motifs. Most strikingly, an aspartate at position -4 compromised binding to the gamma/sigma1 hemicomplex, whereas minimally affecting binding to alpha/sigma2. There was an excellent correlation between binding to the alpha/sigma2 hemicomplex and in vivo internalization mediated by the dileucine-based sorting signals. These findings provide new insights into the trafficking mechanisms of D/EXXXL[LI]-mediated sorting signals.     

Nonclassical PITPs activate PLD via the Stt4p PtdIns-4-kinase and modulate function of late stages of exocytosis in vegetative yeast

Phospholipase D (PLD) is a PtdCho-hydrolyzing enzyme that plays central signaling functions in eukaryotic cells. We previously demonstrated that action of a set of four nonclassical and membrane-associated Sec14p-like phosphatidylinositol transfer proteins (PITPs) is required for optimal activation of yeast PLD in vegetative cells. Herein, we focus on mechanisms of Sfh2p and Sfh5p function in this regulatory circuit. We describe several independent lines of in vivo evidence to indicate these SFH PITPs regulate PLD by stimulating PtdIns-4,5-P2 synthesis and that this stimulated PtdIns-4,5-P2 synthesis couples to action of the Stt4p PtdIns 4-kinase. Furthermore, we provide genetic evidence to suggest that specific subunits of the yeast exocyst complex (i.e. a component of the plasma membrane vesicle docking machinery) and the Sec9p plasma membrane t-SNARE are regulated by PtdIns(4,5)P2 and that Sfh5p helps regulate this interface in vivo. The collective in vivo and biochemical data suggest SFH-mediated stimulation of Stt4p activity is indirect, most likely via a substrate delivery mechanism.     

Pleckstrin homology domain 1 of mouse alpha 1-syntrophin binds phosphatidylinositol 4,5-bisphosphate

Mouse alpha 1-syntrophin sequences were produced as chimeric fusion proteins in bacteria and found to bind phosphatidylinositol 4, 5-bisphosphate (PtdIns4,5P2). Half-maximal binding occurred at 1.9 microM PtdIns4,5P2 and when 1.2 PtdIns4,5P2 were added per syntrophin. Binding was specific for PtdIns4,5P2 and did not occur with six other tested lipids including the similar phosphatidylinositol 4-phosphate. Binding was localized to the N-terminal pleckstrin homology domain (PH1); the second, C-terminal PH2 domain did not bind lipids. Key residues in PtdIns4,5P2 binding to a PH domain were found to be conserved in alpha-syntrophins' PH1 domains and absent in PH2 domains, suggesting a molecular basis for binding.     

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 PKCalpha 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)P(2)), and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) 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 Ca(2+)-dependent C2 domain of protein kinase Calpha (PKCalpha) that is known to bind PS and phosphoinositides. In vitro membrane binding measurements by surface plasmon resonance analysis show that PKCalpha-C2 nonspecifically binds phosphoinositides, including PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3), but that PS and Ca(2+) binding is prerequisite for productive phosphoinositide binding. PtdIns(4,5)P(2) or PtdIns(3,4,5)P(3) augments the Ca(2+)- and PS-dependent membrane binding of PKCalpha-C2 by slowing its membrane dissociation. Molecular dynamics simulations also support that Ca(2+)-dependent PS binding is essential for membrane interactions of PKCalpha-C2. PtdIns(4,5)P(2) alone cannot drive the membrane attachment of the domain but further stabilizes the Ca(2+)- and PS-dependent membrane binding. When the fluorescence protein-tagged PKCalpha-C2 was expressed in NIH-3T3 cells, mutations of phosphoinositide-binding residues or depletion of PtdIns(4,5)P(2) and/or PtdIns(3,4,5)P(3) 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)P(2) or PtdIns(3,4,5)P(3) at the PM slowed membrane dissociation of PKCalpha-C2. Collectively, these studies show that PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3) augment the Ca(2+)- and PS-dependent membrane binding of PKCalpha-C2 by elongating the membrane residence of the domain but cannot drive the PM recruitment of PKCalpha-C2. These studies also suggest that effective PM recruitment of many cellular proteins may require synergistic actions of PS and phosphoinositides.     

The pleckstrin homology domain proteins Slm1 and Slm2 are required for actin cytoskeleton organization in yeast and bind phosphatidylinositol-4,5-bisphosphate and TORC2

Phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P(2)] is a key second messenger that regulates actin and membrane dynamics, as well as other cellular processes. Many of the effects of PtdIns(4,5)P(2) are mediated by binding to effector proteins that contain a pleckstrin homology (PH) domain. Here, we identify two novel effectors of PtdIns(4,5)P(2) in the budding yeast Saccharomyces cerevisiae: the PH domain containing protein Slm1 and its homolog Slm2. Slm1 and Slm2 serve redundant roles essential for cell growth and actin cytoskeleton polarization. Slm1 and Slm2 bind PtdIns(4,5)P(2) through their PH domains. In addition, Slm1 and Slm2 physically interact with Avo2 and Bit61, two components of the TORC2 signaling complex, which mediates Tor2 signaling to the actin cytoskeleton. Together, these interactions coordinately regulate Slm1 targeting to the plasma membrane. Our results thus identify two novel effectors of PtdIns(4,5)P(2) regulating cell growth and actin organization and suggest that Slm1 and Slm2 integrate inputs from the PtdIns(4,5)P(2) and TORC2 to modulate polarized actin assembly and growth.     

The C2 domains of classical PKCs are specific PtdIns(4,5)P2-sensing domains with different affinities for membrane binding

C2 domains are conserved protein modules in many eukaryotic signaling proteins, including the protein kinase (PKCs). The C2 domains of classical PKCs bind to membranes in a Ca(2+)-dependent manner and thereby act as cellular Ca(2+) effectors. Recent findings suggest that the C2 domain of PKCalpha interacts specifically with phosphatidylinositols 4,5-bisphosphate (PtdIns(4,5)P(2)) through its lysine rich cluster, for which it shows higher affinity than for POPS. In this work, we compared the three C2 domains of classical PKCs. Isothermal titration calorimetry revealed that the C2 domains of PKCalpha and beta display a greater capacity to bind to PtdIns(4,5)P(2)-containing vesicles than the C2 domain of PKCgamma. Comparative studies using lipid vesicles containing both POPS and PtdIns(4,5)P(2) as ligands revealed that the domains behave as PtdIns(4,5)P(2)-binding modules rather than as POPS-binding modules, suggesting that the presence of the phosphoinositide in membranes increases the affinity of each domain. When the magnitude of PtdIns(4,5)P(2) binding was compared with that of other polyphosphate phosphatidylinositols, it was seen to be greater in both PKCbeta- and PKCgamma-C2 domains. The concentration of Ca(2+) required to bind to membranes was seen to be lower in the presence of PtdIns(4,5)P(2) for all C2 domains, especially PKCalpha. In vivo experiments using differentiated PC12 cells transfected with each C2 domain fused to ECFP and stimulated with ATP demonstrated that, at limiting intracellular concentration of Ca(2+), the three C2 domains translocate to the plasma membrane at very similar rates. However, the plasma membrane dissociation event differed in each case, PKCalpha persisting for the longest time in the plasma membrane, followed by PKCgamma and, finally, PKCbeta, which probably reflects the different levels of Ca(2+) needed by each domain and their different affinities for PtdIns(4,5)P(2).     

Crystal structure of the C2 domain of class II phosphatidylinositide 3-kinase C2alpha

Phosphatidylinositide (PtdIns) 3-kinase catalyzes the addition of a phosphate group to the 3'-position of phosphatidyl inositol. Accumulated evidence shows that PtdIns 3-kinase can provide a critical signal for cell proliferation, cell survival, membrane trafficking, glucose transport, and membrane ruffling. Mammalian PtdIns 3-kinases are divided into three classes based on structure and substrate specificity. A unique characteristic of class II PtdIns 3-kinases is the presence of both a phox homolog domain and a C2 domain at the C terminus. The biological function of the C2 domain of the class II PtdIns 3-kinases remains to be determined. We have determined the crystal structure of the mCPK-C2 domain, which is the first three-dimensional structural model of a C2 domain of class II PtdIns 3-kinases. Structural studies reveal that the mCPK-C2 domain has a typical anti-parallel beta-sandwich fold. Scrutiny of the surface of this C2 domain has identified three small, shallow sulfate-binding sites. On the basis of the structural features of these sulfate-binding sites, we have studied the lipid binding properties of the mCPK-C2 domain by site-directed mutagenesis. Our results show that this C2 domain binds specifically to PtdIns(3,4)P(2) and PtdIns(4,5)P(2) and that three lysine residues at SBS I site, Lys-1420, Lys-1432, and Lys-1434, are responsible for the phospholipid binding affinity.     

Amer1/WTX couples Wnt-induced formation of PtdIns(4,5)P2 to LRP6 phosphorylation

Phosphorylation of the Wnt receptor low-density lipoprotein receptor-related protein 6 (LRP6) by glycogen synthase kinase 3β (GSK3β) and casein kinase 1γ (CK1γ) is a key step in Wnt/β-catenin signalling, which requires Wnt-induced formation of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). Here, we show that adenomatous polyposis coli membrane recruitment 1 (Amer1) (also called WTX), a membrane associated PtdIns(4,5)P(2)-binding protein, is essential for the activation of Wnt signalling at the LRP6 receptor level. Knockdown of Amer1 reduces Wnt-induced LRP6 phosphorylation, Axin translocation to the plasma membrane and formation of LRP6 signalosomes. Overexpression of Amer1 promotes LRP6 phosphorylation, which requires interaction of Amer1 with PtdIns(4,5)P(2). Amer1 translocates to the plasma membrane in a PtdIns(4,5)P(2)-dependent manner after Wnt treatment and is required for LRP6 phosphorylation stimulated by application of PtdIns(4,5)P(2). Amer1 binds CK1γ, recruits Axin and GSK3β to the plasma membrane and promotes complex formation between Axin and LRP6. Fusion of Amer1 to the cytoplasmic domain of LRP6 induces LRP6 phosphorylation and stimulates robust Wnt/β-catenin signalling. We propose a mechanism for Wnt receptor activation by which generation of PtdIns(4,5)P(2) leads to recruitment of Amer1 to the plasma membrane, which acts as a scaffold protein to stimulate phosphorylation of LRP6.     

Inactivation of the phosphoinositide phosphatases Sac1p and Inp54p leads to accumulation of phosphatidylinositol 4,5-bisphosphate on vacuole membranes and vacuolar fusion defects

Phosphoinositides direct membrane trafficking, facilitating the recruitment of effectors to specific membranes. In yeast phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) isproposed to regulate vacuolar fusion; however, in intact cells this phosphoinositide can only be detected at the plasma membrane. In Saccharomyces cerevisiae the 5-phosphatase, Inp54p, dephosphorylates PtdIns(4,5)P2 forming PtdIns(4)P, a substrate for the phosphatase Sac1p, which hydrolyzes (PtdIns(4)P). We investigated the role these phosphatases in regulating PtdIns(4,5)P2 subcellular distribution. PtdIns(4,5)P2 bioprobes exhibited loss of plasma membrane localization and instead labeled a subset of fragmented vacuoles in Deltasac1 Deltainp54 and sac1ts Deltainp54 mutants. Furthermore, sac1ts Deltainp54 mutants exhibited vacuolar fusion defects, which were rescued by latrunculin A treatment, or by inactivation of Mss4p, a PtdIns(4)P 5-kinase that synthesizes plasma membrane PtdIns(4,5)P2. Under these conditions PtdIns(4,5)P2 was not detected on vacuole membranes, and vacuole morphology was normal, indicating vacuolar PtdIns(4,5)P2 derives from Mss4p-generated plasma membrane PtdIns(4,5)P2. Deltasac1 Deltainp54 mutants exhibited delayed carboxypeptidase Y sorting, cargo-selective secretion defects, and defects in vacuole function. These studies reveal PtdIns(4,5)P2 hydrolysis by lipid phosphatases governs its spatial distribution, and loss of phosphatase activity may result in PtdIns(4,5)P2 accumulation on vacuole membranes leading to vacuolar fragmentation/fusion defects.     

Characterization of a protein phosphatase 2A holoenzyme that dephosphorylates the clathrin adaptors AP-1 and AP-2

The AP-2 complex is a key factor in the formation of endocytic clathrin-coated vesicles (CCVs). AP-2 sorts and packages cargo membrane proteins into CCVs, binds the coat protein clathrin, and recruits numerous other factors to the site of vesicle formation. Structural information on the AP-2 complex and biochemical work have allowed understanding its function on the molecular level, and recent studies showed that cycles of phosphorylation are key steps in the regulation of AP-2 function. The complex is phosphorylated on both large subunits (alpha- and beta2-adaptins) as well as at a single threonine residue (Thr-156) of the medium subunit mu2. Phosphorylation of mu2 is necessary for efficient cargo recruitment, whereas the functional context of the large subunit phosphorylation is unknown. Here, we show that the subunit phosphorylation of AP-2 exhibits striking differences, with calculated half-lives of <1 min for mu2, approximately 25 min for beta2, and approximately 70 min for alpha. We were also able to purify a phosphatase that dephosphorylates the mu2 subunit. The enzyme is a member of the protein phosphatase 2A family and composed of a catalytic Cbeta subunit, a scaffolding Abeta subunit, and a regulatory Balpha subunit. RNA interference knock down of the latter subunit in HeLa cells resulted in increased levels of phosphorylated adaptors and altered endocytosis, showing that a specific PP2A holoenzyme is an important regulatory enzyme in CCV-mediated transport.     

Regulation of CK2 activity by phosphatidylinositol phosphates

The process of clathrin-coated vesicle (CCV) formation/disassembly involves numerous proteins that act cooperatively. Phosphorylation is an important regulatory mechanism governing protein interactions in CCVs, and many of the core and accessory proteins of the CCV machinery are reversibly phosphorylated in vivo. CK2 is highly enriched in CCVs and is capable of phosphorylating a number of peripheral membrane proteins involved in the process of clathrin-mediated endocytosis. At least some of these phosphorylation events have been shown to be inhibitory for CCV assembly, and CK2 has been shown to be inactive when associated with intact CCVs. Here we show that CCV membranes inhibit CK2 activity even after incubation in trypsin, indicating that a component of the lipid bilayer may be the inhibitory factor. Consistent with this, we showed that liposomes containing phosphatidylinositol phosphates inhibit the activity of CK2 and that CK2 binds to those liposomes. Notably, liposomes containing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)), a component of CCVs, bind CK2 and inhibit its activity. Furthermore, we showed that the binding of CK2 to PtdIns(4,5)P(2)-containing liposomes is via the active site of CK2, thus providing a molecular explanation for the inhibition of CK2 activity when it is bound to PtdIns(4,5)P(2)-containing liposomes. Thus CK2 is inactive in CCVs because of the fact that it is bound to the CCV membrane via an interaction between PtdIns(4,5)P(2) in the CCV membrane and the active site in CK2.