3' Phosphatase activity toward phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] by voltage-sensing phosphatase (VSP)

Voltage-sensing phosphatases (VSPs) consist of a voltage-sensor domain and a cytoplasmic region with remarkable sequence similarity to phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor phosphatase. VSPs dephosphorylate the 5' position of the inositol ring of both phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] upon voltage depolarization. However, it is unclear whether VSPs also have 3' phosphatase activity. To gain insights into this question, we performed in vitro assays of phosphatase activities of Ciona intestinalis VSP (Ci-VSP) and transmembrane phosphatase with tensin homology (TPTE) and PTEN homologous inositol lipid phosphatase (TPIP; one human ortholog of VSP) with radiolabeled PI(3,4,5)P(3). TLC assay showed that the 3' phosphate of PI(3,4,5)P(3) was not dephosphorylated, whereas that of phosphatidylinositol 3,4-bisphosphate [PI(3,4)P(2)] was removed by VSPs. Monitoring of PI(3,4)P(2) levels with the pleckstrin homology (PH) domain from tandem PH domain-containing protein (TAPP1) fused with GFP (PH(TAPP1)-GFP) by confocal microscopy in amphibian oocytes showed an increase of fluorescence intensity during depolarization to 0 mV, consistent with 5' phosphatase activity of VSP toward PI(3,4,5)P(3). However, depolarization to 60 mV showed a transient increase of GFP fluorescence followed by a decrease, indicating that, after PI(3,4,5)P(3) is dephosphorylated at the 5' position, PI(3,4)P(2) is then dephosphorylated at the 3' position. These results suggest that substrate specificity of the VSP changes with membrane potential.     

Activity-dependent PI(3,5)P2 synthesis controls AMPA receptor trafficking during synaptic depression

Dynamic regulation of phosphoinositide lipids (PIPs) is crucial for diverse cellular functions, and, in neurons, PIPs regulate membrane trafficking events that control synapse function. Neurons are particularly sensitive to the levels of the low abundant PIP, phosphatidylinositol 3,5-bisphosphate [PI(3,5)P₂], because mutations in PI(3,5)P₂-related genes are implicated in multiple neurological disorders, including epilepsy, severe neuropathy, and neurodegeneration. Despite the importance of PI(3,5)P₂ for neural function, surprisingly little is known about this signaling lipid in neurons, or any cell type. Notably, the mammalian homolog of yeast vacuole segregation mutant (Vac14), a scaffold for the PI(3,5)P₂ synthesis complex, is concentrated at excitatory synapses, suggesting a potential role for PI(3,5)P₂ in controlling synapse function and/or plasticity. PI(3,5)P₂ is generated from phosphatidylinositol 3-phosphate (PI3P) by the lipid kinase PI3P 5-kinase (PIKfyve). Here, we present methods to measure and control PI(3,5)P₂ synthesis in hippocampal neurons and show that changes in neural activity dynamically regulate the levels of multiple PIPs, with PI(3,5)P₂ being among the most dynamic. The levels of PI(3,5)P₂ in neurons increased during two distinct forms of synaptic depression, and inhibition of PIKfyve activity prevented or reversed induction of synaptic weakening. Moreover, altering neuronal PI(3,5)P₂ levels was sufficient to regulate synaptic strength bidirectionally, with enhanced synaptic function accompanying loss of PI(3,5)P₂ and reduced synaptic strength following increased PI(3,5)P₂ levels. Finally, inhibiting PI(3,5)P₂ synthesis alters endocytosis and recycling of AMPA-type glutamate receptors (AMPARs), implicating PI(3,5)P₂ dynamics in AMPAR trafficking. Together, these data identify PI(3,5)P₂-dependent signaling as a regulatory pathway that is critical for activity-dependent changes in synapse strength.Phosphorylated phosphoinositide lipids (PIPs) regulate diverse cellular processes (reviewed in refs. 1, 2). These seven interconvertible PIP species are synthesized and turned over by highly regulated lipid kinases and phosphatases. PIPs likely assemble complex protein machines on membrane subdomains through binding of specific downstream protein effectors, which provides tight spatial and temporal control of cellular processes. Such precision is likely critical for complex cellular functions, including regulation of synaptic strength in the CNS.Pleiotropic defects are associated with impairments in phosphatidylinositol 3,5-bisphosphate [PI(3,5)P₂] synthesis (reviewed in ref. 3). Mutations in FIG4, the gene that encodes a positive regulator of PI(3,5)P₂ (4–10), are linked to several neurological disorders, including Charcot–Marie–Tooth type 4J (CMT4J) (4, 11), ALS, and primary lateral sclerosis (12), familial epilepsy with polymicrogyria (13) and Yunis–Varón syndrome (14). Little is known about how perturbations in PI(3,5)P₂ synthesis cause disease.Fig4 is a member of a protein complex that includes the phosphatidylinositol 3-phosphate (PI3P) 5-kinase (PIKfyve; Fab1 in yeast) (10, 15–18) and the scaffolding protein Vac14 (8, 9, 19–22) (Fig. S1). PIKfyve provides the sole source of PI(3,5)P₂ (10, 15, 17, 23–28). The pools of PI3P that are converted to PI(3,5)P₂ may derive from the class III PI 3-kinase VPS34 (29) and/or the class II PI 3-kinase C2α (30). In vivo, depletion of PIKfyve affects both PI(3,5)P₂ and PI5P pools (10, 21, 24, 28). Identification of PI(3,5)P₂ and PI5P protein effectors will likely reveal specific roles for each lipid.The ability to control PI(3,5)P₂ levels dynamically in mammalian cells is likely crucial for cellular function. In yeast, hyperosmotic stress transiently increases and decreases PI(3,5)P₂ levels (6, 31). Similarly, in multicellular organisms, diverse external cues, such as hormones, growth factors, or neurotransmitters, may lead to dynamic regulation of PI(3,5)P₂ levels. Indeed, analysis of the CMT4J disease mutation Fig4-I>T in yeast showed an impairment in stimulus-induced PI(3,5)P₂ synthesis without an effect on basal PI(3,5)P₂ levels (4). In cultured cortical neurons, knockdown of PIKfvye impairs the internalization of an AMPA-type glutamate receptor (AMPAR) subunit, HA-tagged GluA2 (32), and loss of Vac14 and/or Fig4 is associated with strengthened synapses (33). Together, these findings suggest that Vac14 and Fig4 regulate synapse strength via positive regulation of PIKfyve.Here, using multiple approaches, we show that PIKfyve kinase activity negatively regulates postsynaptic strength and plays specialized roles during two distinct forms of synaptic weakening. Chronic down-regulation of PIKfyve activity using shRNA increases postsynaptic strength, whereas brief chemical inhibition of PIKfyve blocks NMDA receptor (NMDAR)-dependent long-term depression (LTD) and reverses homeostatic synaptic weakening (downscaling). Notably, we developed methods to measure the activity-dependent changes in each PIP species in cultured hippocampal neurons and identified that two low abundant PIPs, PI(3,4,5)P₃ and PI(3,5)P₂, are highly dynamic during LTD. Moreover, PI(3,5)P₂ levels increase during homeostatic downscaling, and increasing PI(3,5)P₂ via a dominant-active PIKfyve mutant (PIKfyve^KYA) is sufficient to weaken postsynaptic strength. We further show that these effects on synapses derive, in part, from PI(3,5)P₂-dependent trafficking of AMPARs. Together, these findings demonstrate that PIKfyve lipid kinase activity plays a critical role in regulation of synapse strength.     

β-arrestin–dependent PI(4,5)P2 synthesis boosts GPCR endocytosis

β-arrestins regulate many cellular functions including intracellular signaling and desensitization of G protein–coupled receptors (GPCRs). Previous studies show that β-arrestin signaling and receptor endocytosis are modulated by the plasma membrane phosphoinositide lipid phosphatidylinositol-(4, 5)-bisphosphate (PI(4,5)P₂). We found that β-arrestin also helped promote synthesis of PI(4,5)P₂ and up-regulated GPCR endocytosis. We studied these questions with the G_q-coupled protease-activated receptor 2 (PAR2), which activates phospholipase C, desensitizes quickly, and undergoes extensive endocytosis. Phosphoinositides were monitored and controlled in live cells using lipid-specific fluorescent probes and genetic tools. Applying PAR2 agonist initiated depletion of PI(4,5)P₂, which then recovered during rapid receptor desensitization, giving way to endocytosis. This endocytosis could be reduced by various manipulations that depleted phosphoinositides again right after phosphoinositide recovery: PI(4)P, a precusor of PI(4,5)P_2, could be depleted at either the Golgi or the plasma membrane (PM) using a recruitable lipid 4-phosphatase enzyme and PI(4,5)P₂ could be depleted at the PM using a recruitable 5-phosphatase. Endocytosis required the phosphoinositides. Knock-down of β-arrestin revealed that endogenous β-arrestin normally doubles the rate of PIP5-kinase (PIP5K) after PAR2 desensitization, boosting PI(4,5)P₂-dependent formation of clathrin-coated pits (CCPs) at the PM. Desensitized PAR2 receptors were swiftly immobilized when they encountered CCPs, showing a dwell time of ∼90 s, 100 times longer than for unactivated receptors. PAR2/β-arrestin complexes eventually accumulated around the edges or across the surface of CCPs promoting transient binding of PIP5K-Iγ. Taken together, β-arrestins can coordinate potentiation of PIP5K activity at CCPs to induce local PI(4,5)P₂ generation that promotes recruitment of PI(4,5)P₂-dependent endocytic machinery.

Membrane phosphatidylinositide lipids (PPIs) are dynamic regulators of diverse cell functions, and their dysregulation underlies numerous human diseases (1). This paper concerns the key involvement of plasma membrane (PM) phosphatidylinositol-(4, 5)-bisphosphate (PI(4,5)P₂) in refining receptor–G protein and receptor–β-arrestin coupling (2, 3) and preparing for the endocytosis of receptors (4). Endocytosis requires clustering of adapter proteins on the PM, nucleation of clathrin-coated membrane pits, capture of receptors with β-arrestin (5–7), and pinching off of pits as intracellular vesicles by dynamin GTPase (4, 8–10). In clathrin-mediated endocytosis, PI(4,5)P₂ is typically needed for the assembly of the adaptor protein complexes, clathrin-coated pits (CCPs), and dynamin complexes (4, 11–14). Hence, receptor internalization should be compromised if PI(4,5)P₂ pools are depleted. This raises the question of how receptors that signal by depleting PI(4,5)P₂ can still be internalized. In this study, we found roles of receptor stimulation and β-arrestin in promoting resynthesis of PI(4,5)P₂, thus enabling endocytosis at the PM.Synthesis of PPIs starts with phosphatidylinositol and families of lipid kinases that generate the mono-, bis-, and tris-phosphorylated inositol ring. PM phosphatidylinositol 4-phosphate (PI(4)P) and PI(4,5)P₂ are produced by several mechanisms potentially involving other membrane compartments. They can be synthesized by lipid 4-kinases acting on PM phosphatidylinositol and by lipid 5-kinases acting on PM PI(4)P; they can be delivered in exchange for other lipids by phosphatidylinositol exchange proteins; and they can be delivered through fusion with other membranes (15–23). Such studies show that the PPI pools in different membranes are interdependent (21). For example, depleting PI(4)P locally in the trans-Golgi using a recruitable PI(4)P 4-phosphatase tool reduces the generation of PI(4,5)P₂ at the PM (24). Conversely, depleting PI(4,5)P₂ at the PM by activating muscarinic or angiotensin II receptors also strongly decreases total cellular PI(4)P (25–27). New evidence is emerging that the PPI composition controls membrane trafficking between organelles. For instance, trafficking of mannose 6-phosphate receptors from the Golgi to the PM can be slowed by reduction of PPI synthesis (28) presumably because PPIs are important for fusion of receptor-containing vesicles with the PM.Here, we study contributions of PPI pools to the endocytosis of the G_q-coupled protease-activated receptor 2 (PAR2). This receptor is involved in inflammatory responses (29), sensation of inflammatory pain (30), and cancer metastasis (31). It has been a target of drug development (32) facilitated by recent crystal structures (33). Stimulation of this receptor activates phospholipase C (PLC) to cleave and deplete PI(4,5)P₂ with accompanying production of diacylglycerol, inositol trisphosphate, and calcium signals (34, 35). Activation of the PAR-receptor family has unique properties. The receptor is activated by cleavage of the N terminus by serine proteases such as thrombin, tryptase, or trypsin (34, 36), which generates a tethered N-terminal ligand. The activation stimulates G_q but is followed quickly by desensitization that terminates G_q signaling (34, 35, 37). Our previous experimental results and mathematical modeling suggest that rapid phosphorylation of PAR2 precedes desensitization and that β-arrrestin clamps the phosphorylated and ligand-bound state of the receptor, protecting it from dephosphorylation by serine/threonine phosphatases (38). Then, the receptor is internalized slowly via a clathrin- and dynamin-dependent pathway (8). This rapidly desensitizing receptor is well suited to address mechanisms involved in PPI lipid–dependent G_qPCR endocytosis.Using genetic and optical tools to manipulate and measure PI(4)P and PI(4,5)P₂ levels acutely at the Golgi or the PM, we now demonstrate that PAR2 internalization can be controlled by PM PI(4,5)P₂ that is replenished using both PM and Golgi pools of PI(4)P. A β-arrestin–dependent activation of PIP5-kinase (PIP5K) at the PM turned out to be critical in the formation of PI(4,5)P₂- and PI(4)P-requiring CCPs and potentially other endocytic machinery for receptor internalization.     

Golgi and plasma membrane pools of PI(4)P contribute to plasma membrane PI(4,5)P2 and maintenance of KCNQ2/3 ion channel current

Plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] regulates the activity of many ion channels and other membrane-associated proteins. To determine precursor sources of the PM PI(4,5)P₂ pool in tsA-201 cells, we monitored KCNQ2/3 channel currents and translocation of PH_PLCδ1 domains as real-time indicators of PM PI(4,5)P₂, and translocation of PH_OSH2×2, and PH_OSH1 domains as indicators of PM and Golgi phosphatidylinositol 4-phosphate [PI(4)P], respectively. We selectively depleted PI(4)P pools at the PM, Golgi, or both using the rapamycin-recruitable lipid 4-phosphatases. Depleting PI(4)P at the PM with a recruitable 4-phosphatase (Sac1) results in a decrease of PI(4,5)P₂ measured by electrical or optical indicators. Depleting PI(4)P at the Golgi with the 4-phosphatase or disrupting membrane-transporting motors induces a decline in PM PI(4,5)P₂. Depleting PI(4)P simultaneously at both the Golgi and the PM induces a larger decrease of PI(4,5)P₂. The decline of PI(4,5)P₂ following 4-phosphatase recruitment takes 1–2 min. Recruiting the endoplasmic reticulum (ER) toward the Golgi membranes mimics the effects of depleting PI(4)P at the Golgi, apparently due to the trans actions of endogenous ER Sac1. Thus, maintenance of the PM pool of PI(4,5)P₂ appears to depend on precursor pools of PI(4)P both in the PM and in the Golgi. The decrease in PM PI(4,5)P₂ when Sac1 is recruited to the Golgi suggests that the Golgi contribution is ongoing and that PI(4,5)P₂ production may be coupled to important cell biological processes such as membrane trafficking or lipid transfer activity.This paper concerns the dynamics of cellular pools of phosphoinositides, a family of phospholipids located on the cytoplasmic leaflet of cellular membranes, that maintain cell structure, cell motility, membrane identity, and membrane trafficking; they also play key roles in signal transduction (1). Phosphatidylinositol (PI) can be phosphorylated at three positions to generate seven additional species. The subcellular localization of each phosphoinositide is tightly governed by the concurrent presence of lipid kinases and lipid phosphatases (2, 3), giving each membrane within the cell a unique and dynamic phosphoinositide signature (4). Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] is localized to the inner leaflet of the plasma membrane (PM) and is the major substrate of phospholipase C (PLC). As a consequence, PI(4,5)P₂ levels are dynamically regulated by G_q-coupled receptors activating PLC. The activity of lipid kinases and phosphatases also can be modulated by signaling; for example, a PI 4-kinase, when associated with neuronal calcium sensor-1, is accelerated in response to elevated calcium that occurs with PI(4,5)P₂ cleavage (5). In addition, transient apposition between organelles can alter phosphoinositide levels by presenting membrane-bound phosphatases in trans. For example, the endoplasmic reticulum (ER) can make contacts with the Golgi, allowing 4-phosphatases of the ER to dephosphorylate Golgi phosphatidylinositol 4-phosphate [PI(4)P] (6–8).PI(4,5)P₂ is a dynamically regulated positive cofactor required for the activity of many plasma membrane ion channels (9). Current in KCNQ2/3 channels (the molecular correlate of neuronal M-current) can be turned off in a few seconds by depletion of PI(4,5)P₂ following activation of PLC through G_q-coupled M₁ muscarinic receptors (M₁Rs) (10–12). Given the importance of PI(4,5)P₂, we wanted to understand better how it is sourced from its precursor PI(4)P within the cell. How do subcellular compartments influence PI(4,5)P₂ abundance at the plasma membrane? PI(4,5)P₂ is derived from PI in two steps: PI 4-kinases make PI(4)P, and PI(4)P 5-kinases make PI(4,5)P₂. Thus, PI(4)P is the immediate precursor of PI(4,5)P₂. Mammalian cells express at least four distinct isoforms of PI 4-kinase that phosphorylate PI on the 4 position to generate PI(4)P and are commonly referred to as PI4K types II (α and β) and III (α and β) (1, 13). PI 4-kinase type IIIα generates PI(4)P at both the Golgi and the PM (14–16). Originally thought to be localized to an ER/Golgi compartment (17, 18), recent experiments show that it is targeted to the plasma membrane by a palmitoylated peripheral membrane protein (16). PI 4-kinase IIIβ is said to be localized to the Golgi and nucleus and contributes to the biosynthesis of Golgi PI(4)P through its association with Arf1 and neuronal calcium sensor 1 (19–21). Inhibition of PI 4-kinase IIIα and -β with micromolar concentrations of wortmannin prevents the replenishment of PM PI(4,5)P₂ following PLC activation (10, 22, 23). Type IIα and IIβ PI4Ks are membrane-bound proteins due to the palmitoylation of a conserved stretch of cysteines in their catalytic domains (24). Immunocytochemical analysis has revealed that they are mostly associated with trans-Golgi, endoplasmic reticulum, and endosomal membranes (25–27). These type IIα and IIβ enzymes are blocked by adenosine and calcium, but are resistant to wortmannin. Therefore, they are not thought to contribute to the recovery of PM PI(4,5)P₂ following G_q-receptor activation (10, 24). Our understanding of the contribution of the PI 4-kinase isoforms is undergoing refinement by accumulating information concerning the unique localization, trafficking, and activity of each PI 4-kinase.Although PI 4-kinase isoforms are present in the membranes of several organelles, the most abundant pools of PI(4)P appear to be those of the PM, Golgi, and secretory vesicles (13–15, 28). A need for Golgi PI(4)P in the maintenance of PM PI(4,5)P₂ was indirectly revealed when plasma membrane PI(4,5)P₂ recovery was slowed following recruitment of a 4-phosphatase to the trans-Golgi network (28). Depletion of PM PI(4)P has been shown to result in small changes to PM PI(4,5)P₂ (15, 22), and knockout of the PM-bound PI 4-kinase IIIα resulted in loss of PI(4)P and a relocation of PI(4,5)P₂ biosensors to intracellular membranes (16). Nevertheless, others have proposed that PM PI(4)P is redundant for the synthesis of PM PI(4,5)P₂ (29–31) and may not serve as its immediate precursor because treatment with the type III PI 4-kinase inhibitor wortmannin or recruiting a 4-phosphatase to the PM had little effect on the PM localization of the PI(4,5)P₂ reporter, the pleckstrin homology (PH) domain from PLCδ1 (PH_PLCδ1).Here, we revisit the relative contributions of PI(4)P pools to PM PI(4,5)P₂. We find that the majority of PM PI(4,5)P₂ needed for maintenance of KCNQ currents comes from two precursor pools of PI(4)P in the cell, one in the PM and the other in the Golgi. The PM pool makes the larger contribution, but the contribution from both locations is significant and ongoing.     

Plasminogen activator inhibitor type 1 promotes fibrosarcoma cell migration by modifying cellular attachment to vitronectin via alpha(v)beta(5) integrin

Both urokinase plasminogen activator (u-PA) and plasminogen activator inhibitor type 1 (PAI-1) are associated with a poor prognosis in cancer patients. We demonstrate that PAI-1 inhibits human fibrosarcoma cell (HT-1080) adhesion to vitronectin (Vn) via alpha (v)beta (5) integrin, and stimulates cell migration from Vn toward collagen type IV (Col). The cells attached more strongly to Vn and Col than to fibronectin (Fn), whereas PAI-1 interfered with cell attachment to Vn only. An integrin antagonist, RGD peptide, and anti-alpha (v)beta (5) integrin antibodies, which similarly inhibited cell attachment to Vn, also stimulated cell migration from Vn toward Col. u-PA did not modify cell attachment directly, but reversed the PAI-1-mediated inhibitory effect on cell adhesion to Vn, and its stimulatory effect on cell migration from Vn toward Col. Thus HT-1080 cell migration appears to be modified by u-PA and PAI-1, altering cell adhesion to Vn via alpha (v)beta (5) integrin. This may be related to their tumor-promoting effect.     

Syndecan-4 proteoliposomes enhance fibroblast growth factor-2 (FGF-2)-induced proliferation, migration, and neovascularization of ischemic muscle

Ischemia of the myocardium and lower limbs is a common consequence of arterial disease and a major source of morbidity and mortality in modernized countries. Inducing neovascularization for the treatment of ischemia is an appealing therapeutic strategy for patients for whom traditional treatment modalities cannot be performed or are ineffective. In the past, the stimulation of blood vessel growth was pursued using direct delivery of growth factors, angiogenic gene therapy, or cellular therapy. Although therapeutic angiogenesis holds great promise for treating patients with ischemia, current methods have not found success in clinical trials. Fibroblast growth factor-2 (FGF-2) was one of the first growth factors to be tested for use in therapeutic angiogenesis. Here, we present a method for improving the biological activity of FGF-2 by codelivering the growth factor with a liposomally embedded coreceptor, syndecan-4. This technique was shown to increase FGF-2 cellular signaling, uptake, and nuclear localization in comparison with FGF-2 alone. Delivery of syndecan-4 proteoliposomes also increased endothelial proliferation, migration, and angiogenic tube formation in response to FGF-2. Using an animal model of limb ischemia, syndecan-4 proteoliposomes markedly improved the neovascularization following femoral artery ligation and recovery of perfusion of the ischemic limb. Taken together, these results support liposomal delivery of syndecan-4 as an effective means to improving the potential of using growth factors to achieve therapeutic neovascularization of ischemic tissue.     

Profilin1 regulates PI(3,4)P2 and lamellipodin accumulation at the leading edge thus influencing motility of MDA-MB-231 cells

Profilin1, a ubiquitously expressed actin-binding protein, plays a critical role in cell migration through actin cytoskeletal regulation. Given the traditional view of profilin1 as a promigratory molecule, it is difficult to reconcile observations that profilin1 is down-regulated in various invasive adenocarcinomas and that reduced profilin1 expression actually confers increased motility to certain adenocarcinoma cells. In this study, we show that profilin1 negatively regulates lamellipodin targeting to the leading edge in MDA-MB-231 breast cancer cells and normal cells; profilin1 depletion increases lamellipodin concentration at the lamellipodial tip (where it binds Ena/VASP), and this mediates the hypermotility. We report that the molecular mechanism underlying profilin1's modulation of lamellipodin localization relates to phosphoinositide control. Specifically, we show that phosphoinositide binding of profilin1 inhibits the motility of MDA-MB-231 cells by negatively regulating PI(3,4)P(2) at the membrane and thereby limiting recruitment of lamellipodin [a PI(3,4)P(2)-binding protein] and Ena/VASP to the leading edge. In summary, this study uncovers a unique biological consequence of profilin1-phosphoinositide interaction, thus providing direct evidence of profilin1's regulation of cell migration independent of its actin-related activity.     

Function recovery after chemobleaching (FRAC): evidence for activity silent membrane receptors on cell surface

Membrane proteins represent approximately 30% of the proteome of both prokaryotes and eukaryotes. Unique to cell surface receptors is their biogenesis pathway, which involves vesicular trafficking from the endoplasmic reticulum through the Golgi apparatus and to the cell surface. Increasing evidence suggests specific regulation of biogenesis for different membrane receptors, hence affecting their surface expression. We report the development of a pulse-chase assay to monitor function recovery after chemobleaching (FRAC) to probe the transit time of the Kir2.1 K+ channel to reach the cell surface. Our results reveal that the channel activity is contributed by a small fraction of channel protein, providing evidence of activity-silent "sleeping" molecules on the cell surface. This method distinguishes molecular density from functional density, and the assay strategy is generally applicable to other membrane receptors. The ability of the reported method to access the biogenesis pathways in a high-throughput manner facilitates the identification and evaluation of molecules affecting receptor trafficking.     

Functional interaction with filamin A and intracellular Ca2+ enhance the surface membrane expression of a small-conductance Ca2+-activated K+ (SK2) channel

For an excitable cell to function properly, a precise number of ion channel proteins need to be trafficked to distinct locations on the cell surface membrane, through a network and anchoring activity of cytoskeletal proteins. Not surprisingly, mutations in anchoring proteins have profound effects on membrane excitability. Ca²⁺-activated K⁺ channels (K_Ca2 or SK) have been shown to play critical roles in shaping the cardiac atrial action potential profile. Here, we demonstrate that filamin A, a cytoskeletal protein, augments the trafficking of SK2 channels in cardiac myocytes. The trafficking of SK2 channel is Ca²⁺-dependent. Further, the Ca²⁺ dependence relies on another channel-interacting protein, α-actinin2, revealing a tight, yet intriguing, assembly of cytoskeletal proteins that orchestrate membrane expression of SK2 channels in cardiac myocytes. We assert that changes in SK channel trafficking would significantly alter atrial action potential and consequently atrial excitability. Identification of therapeutic targets to manipulate the subcellular localization of SK channels is likely to be clinically efficacious. The findings here may transcend the area of SK2 channel studies and may have implications not only in cardiac myocytes but in other types of excitable cells.Small-conductance Ca²⁺-activated K⁺ (SK or K_Ca2) channels are highly unique in that they are gated solely by changes in intracellular Ca²⁺ (Ca²⁺_i) concentration. Hence, the channels function to integrate changes in Ca²⁺ concentration with changes in membrane potentials. SK channels have been shown to be expressed in a wide variety of cells (1–3) and mediate afterhyperpolarizations following action potentials in neurons (1, 4, 5). We have previously documented the expression of several isoforms of SK channels in human and mouse atrial myocytes that mediate the repolarization phase of the atrial action potentials (6, 7). We further demonstrated that SK2 (K_Ca2.2) channel knockout mice are prone to the development of atrial arrhythmias and atrial fibrillation (AF) (8). Conversely, a recent study by Diness et al. suggests that inhibition of SK channels may prevent AF (9). Together, these studies underpin the importance of the precise control for the expression of these ion channels in atria and their potential to serve as a future therapeutic target for AF.Current antiarrhythmic agents target the permeation and gating properties of ion channel proteins; however, increasing evidence suggests that membrane localization of ion channels may also be pharmacologically altered (10). Furthermore, a number of disorders have been associated with mistrafficking of ion channel proteins (11, 12). We have previously demonstrated the critical role of α-actinin2, a cytoskeletal protein, in the surface membrane localization of cardiac SK2 channels (13, 14). Specifically, we demonstrated that cardiac SK2 channel interacts with α-actinin2 cytoskeletal protein via the EF hand motifs in α-actinin2 protein and the helical core region of the calmodulin (CaM) binding domain (CaMBD) in the C terminus of SK2 channel. Moreover, direct interactions between SK2 channel and α-actinin2 are required for the increase in cell surface localization of SK2 channel.Here, to further define the functional interactome of SK2 channel in the heart, we demonstrate the role of filamin A (FLNA), another cytoskeletal protein, in SK2 channel surface membrane localization. In contrast to α-actinin2 protein, FLNA interacts not with the C terminus, but with the N terminus of the cardiac SK2 channel. FLNA is a scaffolding cytoskeletal protein with two calponin homology domains that has been shown to be critical for the trafficking of a number of membrane proteins (15–19). Our data demonstrate that FLNA functions to enhance membrane localization of SK2 channels. Moreover, using live-cell imaging, we demonstrate the critical roles of Ca²⁺_i on the membrane localization of SK2 channel when the channels are coexpressed with α-actinin2, but not FLNA. A decrease in Ca²⁺_i results in a significant decrease in SK2 channel membrane localization. Our findings may have important clinical implications. A rise in Ca²⁺_i—for example, during rapid pacing or atrial tachyarrhythmias—is predicted to increase the membrane localization of SK2 channel and result in the abbreviation of the atrial action potentials and maintenance of the arrhythmias.     

Autophagosome targeting and membrane curvature sensing by Barkor/Atg14(L)

The class III phosphatidylinositol 3-kinase (PI3KC3) is crucial for autophagosome biogenesis. It has been long speculated to nucleate the autophagosome membrane, but the biochemical mechanism of such nucleation activity remains unsolved. We recently identified Barkor/Atg14(L) as the targeting factor for PI3KC3 to autophagosome membrane. Here, we show that we have characterized the region of Barkor/Atg14(L) required for autophagosome targeting and identified the BATS [Barkor/Atg14(L) autophagosome targeting sequence] domain at the carboxyl terminus of Barkor. Bioinformatics and mutagenesis analyses revealed that the BATS domain binds to autophagosome membrane via the hydrophobic surface of an intrinsic amphipathic alpha helix. BATS puncta overlap with Atg16 and LC3, and partially with DFCP1, in a stress-inducible manner. Ectopically expressed BATS accumulates on highly curved tubules that likely represent intermediate autophagic structures. PI3KC3 recruitment and autophagy stimulation by Barkor/Atg14(L) require the BATS domain. Furthermore, our biochemical analyses indicate that the BATS domain directly binds to the membrane, and it favors membrane composed of phosphatidylinositol 3-phosphate [PtdIns(3)P] and phosphatidylinositol 4,5-biphosphate [PtdIns(4,5)P2]. By binding preferentially to curved membranes incorporated with PtdIns(3)P but not PtdIns(4,5)P2, the BATS domain is capable of sensing membrane curvature. Thus, we propose a novel model of PI3KC3 autophagosome membrane nucleation in which its autophagosome-specific adaptor, Barkor, accumulates on highly curved PtdIns(3)P enriched autophagic membrane via its BATS domain to sense and maintain membrane curvature.     

The Vav binding site of the non-receptor tyrosine kinase Syk at Tyr 348 is critical for beta2 integrin (CD11/CD18)-mediated neutrophil migration

Leukocyte adhesion via beta(2) integrins (CD11/CD18) activates the tyrosine kinase Syk. We found that Syk was enriched at the lamellipodium during N-formyl-Met-Leu-Phe-induced migration of neutrophil-like differentiated HL-60 cells. Here, Syk colocalized with Vav, a guanine nucleotide exchange factor for Rac and Cdc42. The enrichment of Syk at the lamellipodium and its colocalization with Vav were absent upon expression of a Syk kinase-dead mutant (Syk K402R) or a Syk mutant lacking the binding site of Vav (Syk Y348F). Live cell imaging revealed that both mutations resulted in excessive lamellipodium formation and severely compromised migration compared with control cells. Similar results were obtained upon down-regulation of Syk by RNA interference (RNAi) technique as well as in Syk(-/-) neutrophils from wild-type mice reconstituted with Syk(-/-) bone marrow. A pivotal role of Syk in vivo was demonstrated in the Arthus reaction, where neutrophil extravasation, edema formation, and hemorrhage were profoundly diminished in Syk(-/-) bone marrow chimeras compared with those in control animals. In the inflamed cremaster muscle, Syk(-/-) neutrophils revealed a defect in adhesion and migration. These findings indicate that Syk is critical for beta(2) integrin-mediated neutrophil migration in vitro and plays a fundamental role in neutrophil recruitment during the inflammatory response in vivo.     

Insulin-mediated upregulation of K(Ca)3.1 channels promotes cell migration and proliferation in rat vascular smooth muscle

The detailed molecular mechanisms underlying pathogenesis of various vascular diseases such as atherosclerosis are not fully understood in type-2 diabetes. The present study was designed to investigate whether insulin regulates K_Ca3.1 channels and participates in vasculopathy in type-2 diabetes. A rat model with experimental insulin-resistant type-2 diabetes was used for detecting pathological changes in the aorta wall, and cultured vascular smooth muscle cells (VSMCs) were employed to investigate the regulation of K_Ca3.1 channels by insulin and roles of K_Ca3.1 channels in cell migration and proliferation using molecular biology and electrophysiology. Early pathological changes were observed and expression of K_Ca3.1 channels increased in the aorta wall of the type 2 diabetic rats. K_Ca3.1 channel mRNA, protein levels and current density were greatly enhanced in cultured VSMCs treated with insulin, and the effects were countered in the cells treated with the ERK1/2 inhibitor PD98059, but not the p38-MAPK inhibitor SB203580. In addition, insulin stimulated cell migration and proliferation in cultured VSMCs, and the effects were fully reversed in the cells treated with the K_Ca3.1 blocker TRAM-34 or PD98059, but not SB203580. These results demonstrate the novel information that insulin increases expression of K_Ca3.1 channels by stimulating ERK1/2 phosphorylation thereby promoting migration and proliferation of VSMCs, which likely play at least a partial role in the development of vasculopathy in type-2 diabetes.     

A novel integrin alpha5beta1 binding domain in module 4 of connective tissue growth factor (CCN2/CTGF) promotes adhesion and migration of activated pancreatic stellate cells

BACKGROUND: Connective tissue growth factor (CCN2) is upregulated in pancreatic fibrosis and desmoplastic pancreatic tumours. CCN2 interacts with integrin alpha5beta1 on pancreatic stellate cells (PSC) in which it stimulates fibrogenesis, adhesion, migration, and proliferation. AIM: To determine the structural domain(s) in CCN2 that interact with integrin alpha5beta1 to regulation PSC functions. METHODS: Primary activated rat PSC were tested for their adherence to isoforms of CCN2 comprising modules 1-4 (CCN2(1-4)), modules 3-4 (CCN2(3-4)), module 3 alone (CCN2(3)), or module 4 alone (CCN2(4)). Adhesion studies were performed in the presence of EDTA, divalent cations, anti-integrin alpha5beta1 antibodies, CCN2 synthetic peptides, or heparin, or after pretreatment of the cells with heparinase, chondroitinase, or sodium chlorate. CCN2 integrin alpha5beta1 binding was analysed in cell free systems. The ability of CCN2(1-4), CCN2(3-4), or CCN2(4) to stimulate PSC migration was evaluated in the presence of anti-integrin alpha5beta1 or heparin. RESULTS: PSC adhesion was stimulated by CCN2(1-4), CCN2(3-4), or CCN2(4) and supported by Mg2+ but not Ca2+. CCN2(4) supported PSC adhesion or migration were blocked by anti-integrin alpha5beta1 antibodies or by treatment of cells with heparinase or sodium chlorate. A direct interaction between CCN2(4) and integrin alpha5beta1 was demonstrated in cell free assays. The sequence GVCTDGR in module 4 mediated the binding between CCN2(4) and integrin alpha5beta1 as well as CCN2(4) mediated PSC adhesion and migration. CONCLUSIONS: A GVCTDGR sequence in module 4 of CCN2 is a novel integrin alpha5beta1 binding site that is essential for CCN2 stimulated functions in PSC and which represents a new therapeutic target in PSC mediated fibrogenesis.     

VEGF(165) promotes survival of leukemic cells by Hsp90-mediated induction of Bcl-2 expression and apoptosis inhibition

Similar to endothelial cells (ECs), vascular endothelial growth factor (VEGF) induces Bcl-2 expression on VEGF receptor-positive (VEGFR(+)) primary leukemias and cell lines, promoting survival. We investigated the molecular pathways activated by VEGF on such leukemias, by performing a gene expression analysis of VEGF-treated and untreated HL-60 leukemic cells. One gene to increase after VEGF stimulation was heat shock protein 90 (Hsp90). This was subsequently confirmed at the protein level, on primary leukemias and leukemic cell lines. VEGF increased the expression of Hsp90 by interacting with KDR and activating the mitogen-activated protein kinase cascade. In turn, Hsp90 modulated Bcl-2 expression, as shown by a complete blockage of VEGF-induced Bcl-2 expression and binding to Hsp90 by the Hsp90-specific inhibitor geldanamycin (GA). GA also blocked the VEGF-induced Hsp90 binding to APAF-1 on leukemic cells, a mechanism shown to inhibit apoptosis. Notably, VEGF blocked the proapoptotic effects of GA, correlating with its effects at the molecular level. Earlier, we showed that in some leukemias, a VEGF/KDR autocrine loop is essential for cell survival, whereas here we identified the molecular correlates for such an effect. We also demonstrate that the generation of a VEGF/VEGFR autocrine loop on VEGFR(+) cells such as ECs, also protected them from apoptosis. Infection of ECs with adenovirus-expressing VEGF resulted in elevated Hsp90 levels, increased Bcl-2 expression, and resistance to serum-free or GA-induced apoptosis. In summary, we demonstrate that Hsp90 mediates antiapoptotic and survival-promoting effects of VEGF, which may contribute to the survival advantage of VEGFR(+) cells such as subsets of leukemias.     

P2X(1)-mediated activation of extracellular signal-regulated kinase 2 contributes to platelet secretion and aggregation induced by collagen

Adenosine triphosphate (ATP) and its stable analog, alpha,beta-methylene ATP, activate the platelet P2X(1) ion channel, causing a rapid Ca(++) influx. Here, we show that, in washed apyrase-treated platelets, alpha,beta-methylene ATP elicits reversible extracellular signal-regulated kinase 2 (ERK2) phosphorylation through a Ca(++)- and protein kinase C-dependent pathway. In contrast, high-performance liquid chromatography-purified adenosine diphosphate (ADP) did not trigger ERK2 phosphorylation. alpha,beta-Methylene ATP also activated the ERK2 pathway in P2X(1)-transfected HEK293 cells but not in cells expressing mutated P2X(1)delL nonfunctional channels. Because ATP released from the dense granules during platelet activation contributes to platelet aggregation elicited by low doses of collagen, and because collagen causes ERK2 phosphorylation, we have investigated the role of P2X(1)-mediated ERK2 activation in these platelet responses. We found that the antagonism of P2X(1) with ADP or desensitization of this ion channel with alpha,beta-methylene ATP both resulted in impaired ERK2 phosphorylation, ATP secretion, and platelet aggregation induced by low concentrations of collagen (< or = 1 microg/mL) without affecting the minor early dense granule release. Selective MEK1/2 inhibition by U-0126 and Ca(++) chelation with EGTA (ethyleneglycoltetraacetic acid) behaved similarly, whereas the PKC inhibitor GF109203-X totally prevented collagen-induced secretion and ERK2 activation. In contrast, when elicited by high collagen concentrations (2 microg/mL), platelet aggregation and secretion no longer depended on P2X(1) or ERK2 activation, as shown by the lack of their inhibition by alpha,beta-methylene ATP or U-0126. We thus conclude that mild platelet stimulation with collagen rapidly releases ATP, which activates the P2X(1)-PKC-ERK2 pathway. This process enhances further degranulation of the collagen-primed granules allowing platelet aggregation to be completed.     

Microfibril-associate glycoprotein-2 (MAGP-2) promotes angiogenic cell sprouting by blocking notch signaling in endothelial cells

Angiogenesis is highly sensitive to the composition of the vascular microenvironment, however, our understanding of the structural and matricellular components of the vascular microenvironment that regulate angiogenesis and the molecular mechanisms by which these molecules function remains incomplete. Our previous results described a novel pro-angiogenic activity for Microfibril-Associated Glycoprotein-2 (MAGP-2), but did not address the molecular mechanism(s) by which this is accomplished. We now demonstrate that MAGP-2 promotes angiogenic cell sprouting by antagonizing Notch signaling pathways in endothelial cells. MAGP-2 decreased basal and Jagged1 induced expression from the Notch sensitive Hes-1 promoter in ECs, and blocked Jagged1 stimulated Notch1 receptor processing in transiently transfected 293T cells. Interestingly, inhibition of Notch signaling by MAGP-2 seems to be restricted to ECs since MAGP-2 increased Hes-1 promoter activity and Notch1 receptor processing in heterologous cell types. Importantly, constitutive activation of the Notch signaling pathway blocked the ability of MAGP-2 to promote angiogenic cell sprouting, as well as morphological changes associated with angiogenesis. Collectively, these observations indicate that MAGP-2 promotes angiogenic cell spouting in vitro by antagonizing Notch signaling pathways in ECs.