A PH Domain in the Arf GTPase-activating Protein (GAP) ARAP1 Binds Phosphatidylinositol 3,4,5-Trisphosphate and Regulates Arf GAP Activity Independently of Recruitment to the Plasma Membranes

ARAP1 is a phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P₃)-dependent Arf GTPase-activating protein (GAP) with five PH domains that regulates endocytic trafficking of the epidermal growth factor receptor (EGFR). Two tandem PH domains are immediately N-terminal of the Arf GAP domain, and one of these fits the consensus sequence for PtdIns(3,4,5)P₃ binding. Here, we tested the hypothesis that PtdIns(3,4,5)P₃-dependent recruitment mediated by the first PH domain of ARAP1 regulates the in vivo and in vitro function of ARAP1. We found that PH1 of ARAP1 specifically bound to PtdIns(3,4,5)P₃, but with relatively low affinity (≈1.6 μm), and the PH domains did not mediate PtdIns(3,4,5)P₃-dependent recruitment to membranes in cells. However, PtdIns(3,4,5)P₃ binding to the PH domain stimulated GAP activity and was required for in vivo function of ARAP1 as a regulator of endocytic trafficking of the EGFR. Based on these results, we propose a variation on the model for the function of phosphoinositide-binding PH domains. In our model, ARAP1 is recruited to membranes independently of PtdIns(3,4,5)P₃, the subsequent production of which triggers enzymatic activity.Pleckstrin homology (PH)² domains are a common structural motif encoded by the human genome (1, 2). Approximately 10% of PH domains bind to phosphoinositides. These PH domains are thought to mediate phosphoinositide-dependent recruitment to membranes (1–3). Most PH domains likely have functions other than or in addition to phosphoinositide binding. For example, PH domains have been found to bind to protein and DNA (4–12). In addition, some PH domains have been found to be structurally and functionally integrated with adjacent domains (13, 14). A small fraction of PH domain-containing proteins (about 9% of the human proteins) have multiple PH domains arranged in tandem, which have been proposed to function as adaptors but have only been examined in one protein (15, 16). Arf GTPase-activating proteins (GAPs) of the ARAP family are phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P₃)-dependent Arf GAPs with tandem PH domains (17, 18). The function of specific PH domains in regulating Arf GAP activity and for biologic activity has not been described.Arf GAPs are proteins that induce the hydrolysis of GTP bound to Arfs (19–23). The Arf proteins are members of the Ras superfamily of GTP-binding proteins (24–27). The six Arf proteins in mammals (five in humans) are divided into three classes based on primary sequence: Arf1, -2, and -3 are class 1, Arf4 and -5 are class 2, and Arf6 is class 3 (23, 24, 27–29). Class 1 and class 3 Arf proteins have been studied more extensively than class 2. They have been found to regulate membrane traffic and the actin cytoskeleton.The Arf GAPs are a family of proteins with diverse domain structures (20, 21, 23, 30). ARAPs, the most structurally complex of the Arf GAPs, contain, in addition to an Arf GAP domain, the sterile α motif (SAM), five PH, Rho GAP, and Ras association domains (17, 18, 31, 32). The first and second and the third and fourth PH domains are tandem (Fig. 1). The first and third PH domains of the ARAPs fit the consensus for PtdIns(3,4,5)P₃ binding (33–35). ARAPs have been found to affect actin and membrane traffic (21, 23). ARAP3 regulates growth factor-induced ruffling of porcine aortic endothelial cells (31, 36, 37). The function is dependent on the Arf GAP and Rho GAP domains. ARAP2 regulates focal adhesions, an actin cytoskeletal structure (17). ARAP2 function requires Arf GAP activity and a Rho GAP domain capable of binding RhoA·GTP. ARAP1 has been found to have a role in membrane traffic (18). The protein associates with pre-early endosomes involved in the attenuation of EGFR signals. The function of the tandem PH domains in the ARAPs has not been examined.Open in a separate windowFIGURE 1.ARAP1 binding to phospholipids. A, schematic of the recombinant proteins used in this study. Domain abbreviations: Ank, ankyrin repeat; PLCδ-PH, PH domain of phospholipase C δ; RA, Ras association motif; RhoGAP, Rho GTPase-activating domain. B, ARAP1 phosphoinositide binding specificity. 500 nm PH1-Ank recombinant protein was incubated with sucrose-loaded LUVs formed by extrusion through a 1-μm pore filter. LUVs contained PtdIns alone or PtdIns with 2.5 μm PtdIns(3,4,5)P₃, 2.5 μm PtdIns(3)P, 2.5 μm PtdIns(4)P, 2.5 μm PtdIns(5)P, 2.5 μm PtdIns(3,4)P₂, 2.5 μm PtdIns(3,5)P₂, or 2.5 μm PtdIns(4,5)P₂ with a total phosphoinositide concentration of 50 μm and a total phospholipid concentration of 500 μm. Vesicles were precipitated by ultracentrifugation, and associated proteins were separated by SDS-PAGE. The amount of precipitated protein was determined by densitometry of the Coomassie Blue-stained gels with standards on each gel. C, PtdIns(3,4,5)P₃-dependent binding of ARAP1 to LUVs. 1 μm PH1-Ank or ArfGAP-Ank recombinant protein was incubated with 1 mm sucrose-loaded LUVs formed by extrusion through a 1-μm pore size filter containing varying concentration of PtdIns(3,4,5)P₃. Precipitation of LUVs and analysis of associated proteins were performed as described in B. The average ± S.E. of three independent experiments is presented.Here we investigated the role of the first two PH domains of ARAP1 for catalysis and in vivo function. The first PH domain fits the consensus sequence for PtdIns(3,4,5)P₃ binding (33–35). The second does not fit a phosphoinositide binding consensus but is immediately N-terminal to the GAP domain. We have previously reported that the PH domain that occurs immediately N-terminal of the Arf GAP domain of ASAP1 is critical for the catalytic function of the protein (38, 39). We tested the hypothesis that the two PH domains of ARAP1 function independently; one recruits ARAP1 to PtdIns(3,4,5)P₃-rich membranes, and the other functions with the catalytic domain. As predicted, PH1 interacted specifically with PtdIns(3,4,5)P₃, and PH2 did not. However, both PH domains contributed to catalysis independently of recruitment to membranes. None of the PH domains in ARAP1 mediated PtdIns(3,4,5)P₃-dependent targeting to plasma membranes (PM). PtdIns(3,4,5)P₃ stimulated GAP activity, and the ability to bind PtdIns(3,4,5)P₃ was required for ARAP1 to regulate membrane traffic. We propose that ARAP1 is recruited independently of PtdIns(3,4,5)P₃ to the PM where PtdIns(3,4,5)P₃ subsequently regulates its GAP activity to control endocytic events.     

The Nutrigenetics of Hyperhomocysteinemia: QUANTITATIVE PROTEOMICS REVEALS DIFFERENCES IN THE METHIONINE CYCLE ENZYMES OF GENE-INDUCED VERSUS DIET-INDUCED HYPERHOMOCYSTEINEMIA*

Hyperhomocysteinemia has long been associated with atherosclerosis and thrombosis and is an independent risk factor for cardiovascular disease. Its causes include both genetic and environmental factors. Although homocysteine is produced in every cell as an intermediate of the methionine cycle, the liver contributes the major portion found in circulation, and fatty liver is a common finding in homocystinuric patients. To understand the spectrum of proteins and associated pathways affected by hyperhomocysteinemia, we analyzed the mouse liver proteome of gene-induced (cystathionine β-synthase (CBS)) and diet-induced (high methionine) hyperhomocysteinemic mice using two-dimensional difference gel electrophoresis and Ingenuity Pathway Analysis. Nine proteins were identified whose expression was significantly changed by 2-fold (p ≤ 0.05) as a result of genotype, 27 proteins were changed as a result of diet, and 14 proteins were changed in response to genotype and diet. Importantly, three enzymes of the methionine cycle were up-regulated. S-Adenosylhomocysteine hydrolase increased in response to genotype and/or diet, whereas glycine N-methyltransferase and betaine-homocysteine methyltransferase only increased in response to diet. The antioxidant proteins peroxiredoxins 1 and 2 increased in wild-type mice fed the high methionine diet but not in the CBS mutants, suggesting a dysregulation in the antioxidant capacity of those animals. Furthermore, thioredoxin 1 decreased in both wild-type and CBS mutants on the diet but not in the mutants fed a control diet. Several urea cycle proteins increased in both diet groups; however, arginase 1 decreased in the CBS^+/− mice fed the control diet. Pathway analysis identified the retinoid X receptor signaling pathway as the top ranked network associated with the CBS^+/− genotype, whereas xenobiotic metabolism and the NRF2-mediated oxidative stress response were associated with the high methionine diet. Our results show that hyperhomocysteinemia, whether caused by a genetic mutation or diet, alters the abundance of several liver proteins involved in homocysteine/methionine metabolism, the urea cycle, and antioxidant defense.Homocysteine (Hcy)1 is a thiol-containing amino acid that is produced in every cell of the body as an intermediate of the methionine cycle (Fig. 1, Reactions 1–5) (1). Once formed, the catabolism of homocysteine occurs via three enzymatic pathways. 1) Hcy is remethylated back to methionine using vitamin B₁₂-dependent methionine synthase (Fig. 1, Reaction 4) and/or 2) betaine-homocysteine methyltransferase (BHMT) (Fig. 1, Reaction 5), and 3) Hcy is converted to cysteine via the transsulfuration pathway using CBS and γ-cystathionase (Fig. 1, Reactions 6 and 7). Under normal conditions ∼40–50% of the Hcy that is produced in the liver is remethylated, ∼40–50% is converted to cysteine, and a small amount is exported (1–3). However, when Hcy production is increased (i.e. increased dietary methionine/protein intake) or when Hcy catabolism is decreased (i.e. CBS deficiency or B vitamin deficiencies), excess Hcy is exported into the extracellular space, resulting in hyperhomocysteinemia (1–5).Open in a separate windowFig. 1.Homocysteine metabolism in liver and kidney. In classical homocystinuria, the initial enzyme of the transsulfuration pathway, CBS (Reaction 6), is deficient. MTHF, methylenetetrahydrofolate; THF, tetrahydrofolate; DHF, dihydrofolate; MeCbl, methylcobalamin; DMG, dimethylglycine; PLP, pyridoxal 5′-phosphate.Homocystinuria was first described in the 1960s by Carson et al. (6): they observed 10 pediatric patients with severely elevated levels of Hcy in the urine and hypermethioninemia. Normal concentrations of plasma total homocysteine (tHcy) range from 5 to 12 μm (7); however, in homocystinuria, tHcy levels can exceed 100 μm. Homocystinuric patients present with mental retardation, abnormal bone growth, fine hair, malar flush, and dislocation of the lens of the eye, and most die from premature cardiovascular disease (6, 8). Autopsy findings indicate widespread thromboembolism, arteriosclerosis, and fatty livers (6, 8). Mudd et al. (9, 10) identified the cause of homocystinuria as a defect in the enzyme cystathionine β-synthase. A recent study of newborn infants in Denmark estimated the birth prevalence for CBS heterozygosity to be about 1:20,000 (11).Plasma tHcy concentrations are also directly correlated with dietary methionine/protein intake (12, 13). Guttormsen et al. (13) demonstrated that a protein-rich meal affected tHcy for at least 8–24 h. When normal subjects were fed a low protein-containing breakfast (12–15 g), plasma methionine levels increased slightly after 2 h (22.5–27.5 μm), but tHcy levels did not change significantly. However, when these same subjects were fed a high protein meal (52 g), plasma methionine levels peaked after 4 h (38 μm), and tHcy rose steadily until a maximum level was reached 8 h postmeal (7.6 versus 8.5 μm) (13). Thus, the following questions can be raised. How does the hepatic proteome respond to a hyperhomocysteinemic diet, and are the changes that accompany such a diet the same as or different from those that may be observed in gene-induced hyperhomocysteinemia?Because hyperhomocysteinemia is a strong independent risk factor for cardiovascular, cerebrovascular, and peripheral vascular disease, most of the current research has focused on the mechanisms involved in Hcy-induced endothelial dysfunction (14–24). The results of those studies have concluded that Hcy induces intracellular oxidative stress by generating ROS, which in turn lead to decreased bioavailable nitric oxide (NO), altered gene expression, increased endoplasmic reticulum stress, and activation of cholesterol biosynthesis. Also, several studies have examined the association between hyperhomocysteinemia and alcoholic liver disease, but few have looked at the effect of Hcy on the non-alcoholic liver even though fatty liver is a constant finding in homocystinuria (6, 8), and the liver is the major source of circulating Hcy (4, 5, 10). We hypothesize that 1) the liver proteome will respond to hyperhomocysteinemia by altering the expression of proteins involved in methionine/homocysteine metabolism and antioxidant defense and that 2) the set of proteins that are expressed when hyperhomocysteinemia is induced by CBS deficiency will differ from those expressed as a result of a high methionine diet. In the present study, we use a well established mouse model of CBS deficiency to study the early changes in the liver proteome that accompany hyperhomocysteinemia (25).     

Pro-interleukin (IL)-1�� Shares a Core Region of Stability as Compared with Mature IL-1�� While Maintaining a Distinctly Different Configurational Landscape: A COMPARATIVE HYDROGEN/DEUTERIUM EXCHANGE MASS SPECTROMETRY STUDY*

Interleukin-1β (IL-1β) is a master cytokine involved in initiating the innate immune response in vertebrates (Dinarello, C. A. (1994) FASEB J. 8, 1314–1325). It is first synthesized as an inactive 269-residue precursor (pro-interleukin-1β or pro-IL-1β). Pro-IL-1β requires processing by caspase-1 to generate the active, mature 153-residue cytokine. In this study, we combined hydrogen/deuterium exchange mass spectrometry, circular dichroism spectroscopy, and enzymatic digestion comparative studies to investigate the configurational landscape of pro-IL-1β and the role the N terminus plays in modulating the landscape. We find that the N terminus keeps pro-IL-1β in a protease-labile state while maintaining a core region of stability in the C-terminal region, the eventual mature protein. In mature IL-1β, this highly protected region maps back to the area protected earliest in the NMR studies characterizing an on-route kinetic refolding intermediate. This protected region also encompasses two important functional loops that participate in the IL-1β/receptor binding interface required for biological activity. We propose that the purpose of the N-terminal precursor region in pro-IL-1β is to suppress the function of the eventual mature region while keeping a structurally and also functionally important core region primed for the final folding into the native, active state of the mature protein. The presence of the self-inhibiting precursor region provides yet another layer of regulation in the life cycle of this important cytokine.Nearly all cell types respond to interleukin (IL)-1β,⁴ in a very sensitive manner, via binding to the interleukin-1 receptor type 1 (IL-1RI) (2). Although essential in the immune response, overproduction of IL-1β can lead to both acute (sepsis) as well as chronic (rheumatoid arthritis, atherosclerosis, obesity, and diabetes) disease states (3). Thus, the expression, activation, and secretion of this cytokine are tightly controlled (4). Although many cell types express IL-1β, it is predominately produced and secreted by monocytes and macrophages (1). The protein is synthesized as a biologically inactive 269-residue precursor molecule, pro-interleukin-1β (pro-IL-1β), and the 153-residue active mature IL-1β is generated from the C-terminal domain. Processing of the proprotein involves the recently discovered NALP-1 and NALP-3 inflammasomes, which are responsible for activating procaspase-1 (5). The inflammasome function is integral in wound repair as well as for combating infection (6–9).In vivo, the 31-kDa pro-IL-1β precursor is processed to the active C-terminal 17-kDa form by the interleukin-1 converting enzyme, caspase-1 (10, 11). Caspase-1 is a cysteine protease that recognizes two cleavage sites in pro-IL-1β, the Asp²⁷↓Gly²⁸ and Asp¹¹⁶↓Ala¹¹⁷ peptide bonds (Fig. 1A). These cleavage sites are conserved across mammals (12–14). The activation pathway is believed to proceed with cleavage first at Asp²⁷↓Gly²⁸ (site 1) followed by Asp¹¹⁶↓Ala¹¹⁷ (site 2). These processing events lead to the generation of the mature, active IL-1β from the C-terminal domain of pro-IL-1β (15). After cleavage, the mature protein is exported via a cell-specific non-classical pathway (16). The events leading from caspase-1 activation to active IL-1β secretion are poorly understood and constitute an area of active research (16–20).Open in a separate windowFIGURE 1.A, a schematic of pro-interleukin-1β processing by caspase-1. The two caspase-1 cleavage sites are labeled by residue/number. The products for the cleavage scenario are represented as smaller blocks, and the final mature protein as the actual three-dimensional structure shown in blue (Protein Data Bank code 6I1B (74)). B, panel i, important features are highlighted on the structure of mature IL-1β. Residues Tyr⁶⁸ (residue 184 in pro-IL-1β) and Trp¹²⁰ (236 in pro-IL-1β) are indicated by red side chain stick representation. The two loops important for binding at the third Ig domain of the receptor are indicated by blue spheres (the basic/hydrophobic 90s loop, which encompasses residues 85–99 in mature and 201–216 in pro-IL-1β) and yellow spheres (the β-bulge, residues 46–53 and 162–169). The numbering corresponds to mature and pro-IL-1β, respectively. Panel ii, after rotating the structure 90°, the individual trefoils are labeled by color (trefoil 1 in orange, trefoil 2 in yellow, and trefoil 3 in blue). The structural features described in panel i maintain the same coloring. Panel iii, the two-dimensional splay diagram of the trefoils labeled by color as in panel ii showing the 3-fold symmetry of the secondary structure elements.The native structure of IL-1β is classified as a β-trefoil. The global protein-fold contains three pseudo-symmetric βββloopβ motifs that coalesce to form a six-stranded barrel with three hairpins that form a six-stranded cap closing one end of the barrel (see Fig. 1B) (21). Mature IL-1β refolds relatively slowly (22), accessing multiple routes including a major route with a detectable intermediate population (23, 24). Recently, this slow folding has been attributed to repacking of a functionally important loop (the β-bulge) in the mature protein (see Fig. 1B, i) (25–27). Although much information is known about the structure, folding, and function of mature IL-1β, there is little information available on pro-IL-1β, despite the central importance of this molecule in mediating critical inflammatory processes (28–30). What is known is that the presence of the N-terminal 116 amino acids results in a highly protease-sensitive protein with no biological activity (31). Folding of mature IL-1β is believed to occur after cleavage of pro-IL-1β in vivo. Therefore, structural analysis of the precursor is essential for a better understanding of the role the precursor region plays in regulating folding events leading to the generation of the eventual mature protein.The crystal structure of pro-IL-1β has not been determined, despite approximately 25 years of intensive efforts directed toward this goal, as a result of the dynamic nature of this molecule (32–34). Therefore, we used structure-sensitive methods to compare pro-IL-1β in reference to the mature protein. Optical methods in combination with hydrogen/deuterium exchange mass spectrometric analysis (DXMS) and enzymatic digestion were used to investigate how the N-terminal precursor region modulates the properties of the C-terminal mature domain. DXMS is a well established technique for characterizing proteins refractory to standard crystallographic or NMR structure determination techniques (35–37). Taken together, our results indicate that the N terminus inhibits folding to the fully active trefoil structure in the C-terminal region, but maintains the protein in a conformation that is primed for efficient folding upon release after caspase-1 cleavage.     

Analysis of Phosphorylation-dependent Protein Interactions of Adhesion and Degranulation Promoting Adaptor Protein (ADAP) Reveals Novel Interaction Partners Required for Chemokine-directed T cell Migration

Stimulation of T cells leads to distinct changes of their adhesive and migratory properties. Signal propagation from activated receptors to integrins depends on scaffolding proteins such as the adhesion and degranulation promoting adaptor protein (ADAP)¹. Here we have comprehensively investigated the phosphotyrosine interactome of ADAP in T cells and define known and novel interaction partners of functional relevance. While most phosphosites reside in unstructured regions of the protein, thereby defining classical SH2 domain interaction sites for master regulators of T cell signaling such as SLP76, Fyn-kinase, and NCK, other binding events depend on structural context. Interaction proteomics using different ADAP constructs comprising most of the known phosphotyrosine motifs as well as the structured domains confirm that a distinct set of proteins is attracted by pY571 of ADAP, including the ζ-chain-associated protein kinase of 70 kDa (ZAP70). The interaction of ADAP and ZAP70 is inducible upon stimulation either of the T cell receptor (TCR) or by chemokine. NMR spectroscopy reveals that the N-terminal SH2 domains within a ZAP70-tandem-SH2 construct is the major site of interaction with phosphorylated ADAP-hSH3^N and microscale thermophoresis (MST) indicates an intermediate binding affinity (K_d = 2.3 μm). Interestingly, although T cell receptor dependent events such as T cell/antigen presenting cell (APC) conjugate formation and adhesion are not affected by mutation of Y571, migration of T cells along a chemokine gradient is compromised. Thus, although most phospho-sites in ADAP are linked to T cell receptor related functions we have identified a unique phosphotyrosine that is solely required for chemokine induced T cell behavior.T cell migration and the establishment of productive T cell/APC interactions are regulated by the activity of integrins. In resting T cells, integrins are expressed in an inactive state that adopts a conformation with low affinity for their ligands. Members of the intercellular adhesion molecule family (ICAM 1–5) are the physiological ligands of lymphocyte function-associated antigen 1 (LFA-1, αLβ2-integrin) whereas vascular cell adhesion molecule (VCAM) and fibronectin are the ligands for the β1-integrin very late antigen 4 (VLA-4) (1, 2). Triggering of the T cell receptor (TCR) by peptide-major histocompatibility complex (MHC) or stimulation of chemokine receptors (e.g. CCR7 with CCL21 or CXCR4 with CXCL12) induces a conformational change of the integrins that increases their ligand binding (affinity regulation) and subsequently mediates clustering of integrins at the cell surface (avidity regulation). The intracellular events leading to integrin activation have collectively been termed inside-out signaling. Conversely, ligand-bound integrins transmit a signal to the T cell and thereby promote adhesion, activation, proliferation, and migration of T cells (outside-in signaling) (1, 2).In both inside-out and outside-in signaling pathways tyrosine phosphorylation of adaptor proteins, either present as transmembrane scaffolds or as transiently membrane-anchored proteins, is a crucial primary event in signal transmission to integrins.An essential functional module operating at the integrin-membrane-cytoskeleton interface contains the cytosolic adaptor protein ADAP at its core. Ablation of ADAP in mice leads to dysfunctional integrin clustering and activity, thus compromising the adhesive and migratory properties of these cells. In addition to its instantaneous effects on cellular motility, ADAP was shown to act as a regulator of NFκB p65 nuclear translocation (3), a function that might well contribute to the observed modulation of cytokine production, like interleukin-2 (4, 5). A contribution of ADAP to mast cell degranulation has been postulated (6), and its complex formation with cytoskeletal regulators during early phases of phagocytosis in macrophages has been recognized early on (7). ADAP is also critical for normal platelet adhesion (8) and mutations in the human protein have recently been suggested to form an underlying genetic cause for autosomal recessive thrombocytopenia (9).ADAP interacts with several effectors of T cell function, either constitutively or phosphorylation-dependent. The SH3 domain of SKAP55 (Src-kinase associated phosphoprotein of 55 kDa) interacts with a proline-rich sequence (PRS) stretch in ADAP (Fig. 1A) (10, 11), whereas another PRS (FPPPP) is responsible for the interaction with the actin regulator Ena/VASP-like protein (EVL) (12). Membrane binding of the ADAP-SKAP55 complex is conferred by the PH domain of SKAP55 and to a lower extend by the C-terminal hSH3 domain (hSH3^C) of ADAP (13–16). Moreover, ADAP is strongly tyrosine-phosphorylated upon TCR stimulation and thereby serves as a hub for SH2 domain-containing proteins such as SLP76, FYN and NCK (Fig. 1A) (17–21). Beside these well characterized interactions, several other SH2-domain containing binders were identified by pull-down approaches using phosphorylated peptide baits (Fig. 1A) (22, 23). Most of the so far characterized SH2-pTyr interactions in ADAP are mapped to unstructured regions. An exception is Y571, which is located in close proximity to the folded hSH3^N domain of ADAP. Phosphoproteomic profiling of activated T cells has identified Y571 as a major phosphorylation site in ADAP (18, 24–30). Our rationale for the experimental approach chosen here was that the constraints imposed by the folding of the hSH3 domains impact the choice of SH2 domains that bind to such motifs in ADAP.Open in a separate windowFig. 1.Phosphotyrosine sites of ADAP and interaction partners. A, Schematic overview of the ADAP primary structure indicating interaction partners of different phosphotyrosine sites identified by peptide pull-down approaches (22, 23). Black arrows show SH2-pTyr interaction sites. White arrows show interactions dependent on proline rich sequences (PRS). B, Fyn kinase catalyzed in vitro phosphorylation of full-length ADAP. Identification and relative quantification of phosphorylation degrees of individual tyrosine residues was obtained by mass spectrometry. Phosphorylation degrees were estimated by comparing relative MS peak intensities of the corresponding peptide/phosphopeptide pairs as described (33, 34).To meet this challenge, we employed interaction proteomics and immunoprecipitation experiments using different constructs of ADAP that comprise the major phosphotyrosine sites including the two folded hSH3 domains. In particular, Y571, a residue residing at the domain border of the hSH3^N domain, is shown to interact with proteins that are not identified by the peptide pull-down approach. We identify the ZAP70 kinase as the most robust binding partner of pY571 and show that this interaction is maintained in primary T cells. Using NMR spectroscopy we further show that the N-terminal SH2 domain of ZAP70 is responsible for binding and we confirm the direct interaction between the two proteins to be inducible upon T cell receptor or CXCR4 stimulation. Abolishing the interaction by using a Y571F mutation compromises T cell migration along a CXCL12 gradient but does not affect TCR- and CXCR4-mediated adhesion and T cell interaction with APCs. In this way, the Y571F mutant of ADAP provides the striking example of a molecular switch that selectively invokes the migratory, but not the adhesive, signaling pathways in T cells.     

Adiponectin Activates AMP-activated Protein Kinase in Muscle Cells via APPL1/LKB1-dependent and Phospholipase C/Ca2+/Ca2+/Calmodulin-dependent Protein Kinase Kinase-dependent Pathways

The binding of the adaptor protein APPL1 to adiponectin receptors is necessary for adiponectin-induced AMP-activated protein kinase (AMPK) activation in muscle, yet the underlying molecular mechanism remains unknown. Here we show that in muscle cells adiponectin and metformin induce AMPK activation by promoting APPL1-dependent LKB1 cytosolic translocation. APPL1 mediates adiponectin signaling by directly interacting with adiponectin receptors and enhances LKB1 cytosolic localization by anchoring this kinase in the cytosol. Adiponectin also activates another AMPK upstream kinase Ca²⁺/calmodulin-dependent protein kinase kinase by activating phospholipase C and subsequently inducing Ca²⁺ release from the endoplasmic reticulum, which plays a minor role in AMPK activation. Our results show that in muscle cells adiponectin is able to activate AMPK via two distinct mechanisms as follows: a major pathway (the APPL1/LKB1-dependent pathway) that promotes the cytosolic localization of LKB1 and a minor pathway (the phospholipase C/Ca²⁺/Ca²⁺/calmodulin-dependent protein kinase kinase-dependent pathway) that stimulates Ca²⁺ release from intracellular stores.Adiponectin, an adipokine abundantly expressed in adipose tissue, exhibits anti-diabetic, anti-inflammatory, and anti-atherogenic properties and hence is a potential therapeutic target for various metabolic diseases (1–3). The beneficial effects of adiponectin are mediated through the direct interaction of adiponectin with its cell surface receptors, AdipoR1 and AdipoR2 (4, 5). Adiponectin increases fatty acid oxidation and glucose uptake in muscle cells by activating AMP-activated protein kinase (AMPK)³ (4, 6), which depends on the interaction of AdipoR1 with the adaptor protein APPL1 (Adaptor protein containing Pleckstrin homology domain, Phosphotyrosine binding domain, and Leucine zipper motif) (5). However, the underlying mechanisms by which APPL1 mediates adiponectin signaling to AMPK activation and other downstream targets remain unclear.AMPK is a serine/threonine protein kinase that acts as a master sensor of cellular energy balance in mammalian cells by regulating glucose and lipid metabolism (7, 8). AMPK is composed of a catalytic α subunit and two noncatalytic regulatory subunits, β and γ. The NH₂-terminal catalytic domain of the AMPKα subunit is highly conserved and contains the activating phosphorylation site (Thr¹⁷²) (9). Two AMPK variants, α1 and α2, exist in mammalian cells that show different localization patterns. AMPKα1 subunit is localized in non-nuclear fractions, whereas the AMPKα2 subunit is found in both nucleus and non-nuclear fractions (10). Biochemical regulation of AMPK activation occurs through various mechanisms. An increase in AMP level stimulates the binding of AMP to the γ subunit, which induces a conformational change in the AMPK heterotrimer and results in AMPK activation (11). Studies have shown that the increase in AMPK activity is not solely via AMP-dependent conformational change, rather via phosphorylation by upstream kinases, LKB1 and CaMKK. Dephosphorylation by protein phosphatases is also important in regulating the activity of AMPK (12).LKB1 has been considered as a constitutively active serine/threonine protein kinase that is ubiquitously expressed in all tissues (13, 14). Under conditions of high cellular energy stress, LKB1 acts as the primary AMPK kinase through an AMP-dependent mechanism (15–17). Under normal physiological conditions, LKB1 is predominantly localized in the nucleus. LKB1 is translocated to the cytosol, either by forming a heterotrimeric complex with Ste20-related adaptor protein (STRADα/β) and mouse protein 25 (MO25α/β) or by associating with an LKB1-interacting protein (LIP1), to exert its biological function (18–22). Although LKB1 has been shown to mediate contraction- and adiponectin-induced activation of AMPK in muscle cells, the underlying molecular mechanisms remain elusive (15, 23).CaMKK is another upstream kinase of AMPK, which shows considerable sequence and structural homology with LKB1 (24–26). The two isoforms of CaMKK, CaMKKα and CaMKKβ, encoded by two distinct genes, share ∼70% homology at the amino acid sequence level and exhibit a wide expression in rodent tissues, including skeletal muscle (27–34). Unlike LKB1, AMPK phosphorylation mediated by CaMKKs is independent of AMP and is dependent only on Ca²⁺/calmodulin (35). Hence, it is possible that an LKB1-independent activation of AMPK by CaMKK exists in muscle cells. However, whether and how adiponectin stimulates this pathway in muscle cells are not known.In this study, we demonstrate that in muscle cells adiponectin induces an APPL1-dependent LKB1 translocation from the nucleus to the cytosol, leading to increased AMPK activation. Adiponectin also activates CaMKK by stimulating intracellular Ca²⁺ release via the PLC-dependent mechanism, which plays a minor role in activation of AMPK. Taken together, our results demonstrate that enhanced cytosolic localization of LKB1 and Ca²⁺-induced activation of CaMKK are the mechanisms underlying adiponectin-stimulated AMPK activation in muscle cells.     

Printor, a Novel TorsinA-interacting Protein Implicated in Dystonia Pathogenesis

Early onset generalized dystonia (DYT1) is an autosomal dominant neurological disorder caused by deletion of a single glutamate residue (torsinA ΔE) in the C-terminal region of the AAA⁺ (ATPases associated with a variety of cellular activities) protein torsinA. The pathogenic mechanism by which torsinA ΔE mutation leads to dystonia remains unknown. Here we report the identification and characterization of a 628-amino acid novel protein, printor, that interacts with torsinA. Printor co-distributes with torsinA in multiple brain regions and co-localizes with torsinA in the endoplasmic reticulum. Interestingly, printor selectively binds to the ATP-free form but not to the ATP-bound form of torsinA, supporting a role for printor as a cofactor rather than a substrate of torsinA. The interaction of printor with torsinA is completely abolished by the dystonia-associated torsinA ΔE mutation. Our findings suggest that printor is a new component of the DYT1 pathogenic pathway and provide a potential molecular target for therapeutic intervention in dystonia.Early onset generalized torsion dystonia (DYT1) is the most common and severe form of hereditary dystonia, a movement disorder characterized by involuntary movements and sustained muscle spasms (1). This autosomal dominant disease has childhood onset and its dystonic symptoms are thought to result from neuronal dysfunction rather than neurodegeneration (2, 3). Most DYT1 cases are caused by deletion of a single glutamate residue at positions 302 or 303 (torsinA ΔE) of the 332-amino acid protein torsinA (4). In addition, a different torsinA mutation that deletes amino acids Phe³²³–Tyr³²⁸ (torsinA Δ323–328) was identified in a single family with dystonia (5), although the pathogenic significance of this torsinA mutation is unclear because these patients contain a concomitant mutation in another dystonia-related protein, ϵ-sarcoglycan (6). Recently, genetic association studies have implicated polymorphisms in the torsinA gene as a genetic risk factor in the development of adult-onset idiopathic dystonia (7, 8).TorsinA contains an N-terminal endoplasmic reticulum (ER)³ signal sequence and a 20-amino acid hydrophobic region followed by a conserved AAA⁺ (ATPases associated with a variety of cellular activities) domain (9, 10). Because members of the AAA⁺ family are known to facilitate conformational changes in target proteins (11, 12), it has been proposed that torsinA may function as a molecular chaperone (13, 14). TorsinA is widely expressed in brain and multiple other tissues (15) and is primarily associated with the ER and nuclear envelope (NE) compartments in cells (16–20). TorsinA is believed to mainly reside in the lumen of the ER and NE (17–19) and has been shown to bind lamina-associated polypeptide 1 (LAP1) (21), lumenal domain-like LAP1 (LULL1) (21), and nesprins (22). In addition, recent evidence indicates that a significant pool of torsinA exhibits a topology in which the AAA⁺ domain faces the cytoplasm (20). In support of this topology, torsinA is found in the cytoplasm, neuronal processes, and synaptic terminals (2, 3, 15, 23–26) and has been shown to bind cytosolic proteins snapin (27) and kinesin light chain 1 (20). TorsinA has been proposed to play a role in several cellular processes, including dopaminergic neurotransmission (28–31), NE organization and dynamics (17, 22, 32), and protein trafficking (27, 33). However, the precise biological function of torsinA and its regulation remain unknown.To gain insights into torsinA function, we performed yeast two-hybrid screens to search for torsinA-interacting proteins in the brain. We report here the isolation and characterization of a novel protein named printor (protein interactor of torsinA) that interacts selectively with wild-type (WT) torsinA but not the dystonia-associated torsinA ΔE mutant. Our data suggest that printor may serve as a cofactor of torsinA and provide a new molecular target for understanding and treating dystonia.     

Magnitude of a Conformational Change in the Glycine Receptor ��1-��2 Loop Is Correlated with Agonist Efficacy

The efficacy of agonists at Cys-loop ion channel receptors is determined by the rate they isomerize receptors to a pre-open flip state. Once the flip state is reached, the shut-open reaction is similar for low and high efficacy agonists. The present study sought to identify a conformational change associated with the closed-flip transition in the α1-glycine receptor. We employed voltage-clamp fluorometry to compare ligand-binding domain conformational changes induced by the following agonists, listed from highest to lowest affinity and efficacy: glycine > β-alanine > taurine. Voltage-clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. Agonist affinity and efficacy correlated inversely with maximum fluorescence magnitudes at labeled residues in ligand-binding domain loops D and E, suggesting that large conformational changes in this region preclude efficacious gating. However, agonist affinity and efficacy correlated directly with maximum fluorescence magnitudes from a label attached to A52C in loop 2, near the transmembrane domain interface. Because glycine experiences the largest affinity increase between closed and flip states, we propose that the magnitude of this fluorescence signal is directly proportional to the agonist affinity increase. In contrast, labeled residues in loops C, F, and the pre-M1 domain yielded agonist-independent fluorescence responses. Our results support the conclusion that a closed-flip conformation change, with a magnitude proportional to the agonist affinity increase from closed to flip states, occurs in the microenvironment of Ala-52.Glycine receptors (GlyRs)³ are pentameric chloride-selective ion channels that mediate fast inhibitory neurotransmission (1). They are members of the Cys-loop receptor family that includes the prototypical nicotinic acetylcholine receptor (nAChR), the γ-aminobutyric acid type-A receptors (GABA_ARs), and serotonin type-3 receptors (5-HT₃Rs). Recent structural studies have provided a wealth of information on the structure and function of this receptor family (2–6). In Cys-loop receptors, the ligand-binding domain (LBD) preceding the four transmembrane helices consists of two twisted β-sheets. The inner (vestibule facing) β-sheet comprises seven β-strands, while the outer β-sheet is formed by three β-strands (3). The ligand binding site is located at the interface of adjacent subunits and is lined by six domains: three loops from the principal and the complementary sides, termed A-C and D-F, respectively (3).GlyRs are activated by endogenous amino acid agonists in the following order of efficacy: glycine > β-alanine > taurine (7, 8). As these amino acids share considerable structural similarity (Fig. 1A), they are likely to compete for the same binding site (9–11). A recent ground-breaking study on an intermediate pre-open state, the so-called “flip” state (12), has provided new insights into the mechanism of partial agonism in Cys-loop receptors (13). This study suggested that agonist efficacy depends on the ability of the agonist to convert the inert agonist-bound receptor to the pre-open flip state. Once the flip state is reached, the shut-open reaction is similar for high and low efficacy agonists. To date there is, however, very little information concerning the structural basis for the lower efficacies of partial agonists. To address this, the present study employed the voltage-clamp fluorometry (VCF) technique (14) to compare the conformational changes induced by glycine, β-alanine, and taurine at various positions in the GlyR LBD.Open in a separate windowFIGURE 1.A, structures of glycine, β-alanine, and taurine. B, model of the LBD, based on carbomylcholine-bound AChBP (PDB code 1uv6). The inner β-sheet is displayed in red, the outer β-sheet in blue. Connecting loops are shown in gray. Colored balls represent approximate locations of selected residues labeled in regions flanking the outer β-sheet (black, G181C in loop F; N203C in loop C; Q219C in the pre-M1 domain) and in the inner β-sheet (yellow, L127C in loop E; Q67C in loop D; A52C in loop 2).VCF involves tethering of an environmentally sensitive fluorophore to a cysteine engineered into a domain of interest. If ligand-binding and/or channel opening leads to a changed dielectric environment surrounding the fluorophore, a change in quantum yield or emission spectrum can be detected. VCF was first employed on voltage-gated potassium channels (15) and has since provided a wealth of information on Cys-loop receptor structure and function (16–23). Here we employ VCF to identify an agonist-specific conformational change that may control or reflect the rate at which the GlyR isomerizes to the flip state.     

Inhibition of Human Steroid 5��-Reductase (AKR1D1) by Finasteride and Structure of the Enzyme-Inhibitor Complex

The Δ⁴-3-ketosteroid functionality is present in nearly all steroid hormones apart from estrogens. The first step in functionalization of the A-ring is mediated in humans by steroid 5α- or 5β-reductase. Finasteride is a mechanism-based inactivator of 5α-reductase type 2 with subnanomolar affinity and is widely used as a therapeutic for the treatment of benign prostatic hyperplasia. It is also used for androgen deprivation in hormone-de pend ent prostate carcinoma, and it has been examined as a chemopreventive agent in prostate cancer. The effect of finasteride on steroid 5β-reductase (AKR1D1) has not been previously reported. We show that finasteride competitively inhibits AKR1D1 with low micromolar affinity but does not act as a mechanism-based inactivator. The structure of the AKR1D1·NADP⁺·finasteride complex determined at 1.7 Å resolution shows that it is not possible for NADPH to reduce the Δ^1-2-ene of finasteride because the cofactor and steroid are not proximal to each other. The C3-ketone of finasteride accepts hydrogen bonds from the catalytic residues Tyr-58 and Glu-120 in the active site of AKR1D1, providing an explanation for the competitive inhibition observed. This is the first reported structure of finasteride bound to an enzyme involved in steroid hormone metabolism.The Δ⁴-3-ketosteroid functionality is present in many important steroid hormones, e.g. testosterone, cortisone, and progesterone. An initial step in steroid hormone metabolism is the reduction of the Δ⁴-ene, which in humans is mediated by steroid 5α-reductases (SRD5A1, SRD5A2) or steroid 5β-reductase (AKR1D1)³ to yield the corresponding 5α- or 5β-dihydrosteroids, respectively (1, 2). The products of these reactions are not always inactive. 5α-Reductase is responsible for the conversion of testosterone to 5α-dihydrotestosterone (5α-DHT), which is the most potent natural ligand for the androgen receptor. By contrast, in addition to being involved in bile acid biosynthesis, 5β-reductase is responsible for generating 5β-pregnanes, which are natural ligands for the pregnane-X receptor (PXR) in the liver (3, 4). PXR is involved in the induction of CYP3A4, which is responsible for the metabolism of a large proportion of drugs (5, 6). Thus both 5α-reductase and 5β-reductase are involved in the formation of potent ligands for nuclear receptors.Finasteride is a selective 5α-reductase type 2 inhibitor that reduces plasma 5α-dihydrotestosterone levels and shrinks the size of the prostate (7). It is a widely used therapeutic agent in the treatment of benign prostatic hyperplasia (8, 9), it is used in androgen deprivation therapy to treat prostate cancer (10), and it has been examined as a chemopreventive agent for hormone-dependent prostate cancer (11). Finasteride was originally thought to act as a competitive inhibitor with nanomolar affinity for 5α-reductase type 2 (12). More recently, it was found that finasteride acts as a mechanism-based inactivator of this enzyme (13). Subsequent to inhibitor binding, there is hydride transfer from the NADPH cofactor to the Δ^1-2-ene double bond of finasteride. The intermediate enolate tautomerizes at the enzyme active site to form a bisubstrate analogue in which dihydrofinasteride is covalently bound to NADP⁺ (13). The bisubstrate analogue has subnanomolar affinity for 5α-reductase type 2 (Fig. 1). No structural information exists for 5α-reductase type 1 or type 2; therefore, it is not possible to determine how finasteride would bind to the active site of a human steroid double bond reductase in the absence of an experimentally determined crystal structure.Open in a separate windowFIGURE 1.Mechanism-based inactivation of 5α-reductase type 2 by finasteride. Adapted from Bull et al. (13). R = −C(=O)-NH₂; PADPR = 2′-phosphoadenosine-5″-diphosphoribose.Human steroid 5β-reductase is a member of the aldo-keto reductase (AKR) superfamily and is formally designated (AKR1D1) (14). The AKRs are soluble NADP(H)-dependent oxidoreductases with monomeric molecular masses of 37 kDa. These enzymes are amenable to x-ray crystallography, and during the last year, we and others have reported crystal structures of ternary complexes of AKR1D1 (15–17). The ternary complexes containing steroid substrates include: AKR1D1·NADP⁺·testosterone (PDB: 3BUR), AKR1D1·NADP⁺·progesterone (PDB: 3COT), AKR1D1·NADP⁺·cortisone (PDB: 3CMF), and AKR1D1·NADP⁺·Δ⁴-androstene-3,17-dione (PDB: 3CAS) (17). In addition, ternary complexes containing the products 5β-dihydroprogesterone (PDB: 3CAV) and 5β-dihydrotestosterone (PDB: 3DOP) have also been described (16, 18).As part of an ongoing inhibitor screen of AKR1D1, we now report that finasteride acts as a competitive inhibitor with low micromolar affinity. Additionally, we report the x-ray crystal structure of the AKR1D1·NADP⁺·finasteride complex.     

Differential Effect of Membrane Cholesterol Removal on ��- and ��-Opioid Receptors: A PARALLEL COMPARISON OF ACUTE AND CHRONIC SIGNALING TO ADENYLYL CYCLASE*

According to the lipid raft theory, the plasma membrane contains small domains enriched in cholesterol and sphingolipid, which may serve as platforms to organize membrane proteins. Using methyl-β-cyclodextrin (MβCD) to deplete membrane cholesterol, many G protein-coupled receptors have been shown to depend on putative lipid rafts for proper signaling. Here we examine the hypothesis that treatment of HEK293 cells stably expressing FLAG-tagged μ-opioid receptors (HEK FLAG-μ) or δ-opioid receptors (HEK FLAG-δ) with MβCD will reduce opioid receptor signaling to adenylyl cyclase. The ability of the μ-opioid agonist [d-Ala²,N-Me-Phe⁴,Gly⁵-ol]enkephalin to acutely inhibit adenylyl cyclase or to cause sensitization of adenylyl cyclase following chronic treatment was attenuated with MβCD. These effects were due to removal of cholesterol, because replenishment of cholesterol restored [d-Ala²,N-Me-Phe⁴,Gly⁵-ol]enkephalin responses back to control values, and were confirmed in SH-SY5Y cells endogenously expressing μ-opioid receptors. The effects of MβCD may be due to uncoupling of the μ receptor from G proteins but were not because of decreases in receptor number and were not mimicked by cytoskeleton disruption. In contrast to the results in HEK FLAG-μ cells, MβCD treatment of HEK FLAG-δ cells had no effect on acute inhibition or sensitization of adenylyl cyclase by δ-opioid agonists. The differential responses of μ- and δ-opioid agonists to cholesterol depletion suggest that μ-opioid receptors are more dependent on cholesterol for efficient signaling than δ receptors and can be partly explained by localization of μ- but not δ-opioid receptors in cholesterol- and caveolin-enriched membrane domains.Membrane cholesterol can alter the function of integral proteins, such as G protein-coupled receptors, through cholesterol-protein interactions and by changes in membrane viscosity (1). In addition, cholesterol interacts with other lipids found in the bilayer, particularly sphingolipids (2), which allows for tight and organized packing that can precipitate the formation of specialized domains within the plasma membrane (3). These domains have become an area of intense research interest and have been termed lipid or membrane rafts (4). The study of membrane rafts in intact cells is controversial, due in part to the limitations of the current methods used to study rafts (5, 6). Regardless, the membrane environment formed in regions of high cholesterol and sphingolipids may be such that certain proteins have an affinity for these regions, especially proteins with a propensity to interact with cholesterol.Many G protein-coupled receptors and signaling proteins have been found to prefer cholesterol-enriched domains leading to the hypothesis that these domains can organize signaling molecules in the membrane to enhance or inhibit specific signaling events (7). This includes μ- (8, 9), δ- (10, 11), and κ-opioid receptors (12). In addition, Gα_i (12–17), Gα_o (16), and adenylyl cyclase isoforms 3 (18), 5/6 (9, 18, 19), and 8 (20) have been found to associate with cholesterol and/or the cholesterol-binding protein caveolin. Activated opioid receptors couple to Gα_i/o proteins and acutely inhibit the activity of adenylyl cyclase. Longer term exposure to opioid agonists causes sensitization of adenylyl cyclase and a rebound overshoot of cAMP production upon withdrawal of the agonist (21). Consequently, we sought to assess the role of cholesterol depletion on the ability of μ- and δ-opioid receptor agonists to inhibit and cause sensitization of adenylyl cyclase.There are conflicting data for the effect of changes in membrane cholesterol on opioid signaling. For example, an increase in plasma membrane microviscosity by addition of cholesteryl hemisuccinate to SH-SY5Y cell membranes increased μ-opioid receptor coupling to G proteins (22). Conversely, removal of membrane cholesterol from Chinese hamster ovary cells has been shown to either decrease (23) or increase (24) the coupling of μ-opioid receptors to G proteins, as measured by [³⁵S]GTPγS³ binding stimulated by the μ-opioid agonist DAMGO. Furthermore, the effect of cholesterol removal on δ-opioid agonist-stimulated [³⁵S]GTPγS binding varies by cell type (10, 25). In these previous studies, the variety of cell types utilized and the conflicting results make comparisons between opioid receptor types difficult. The objective of this study was to directly compare the role of membrane cholesterol in modulating acute and chronic μ- and δ-opioid signaling in the same cell systems using identical methods, including the following: 1) depletion of cholesterol by the cholesterol-sequestering agent methyl-β-cyclodextrin (MβCD); 2) separation of cholesterol-enriched membranes by sucrose gradient ultracentrifugation; and 3) clustering of lipid raft patches in whole cells with cholera toxin B subunit.In initial experiments using human embryonic kidney (HEK) cells heterologously expressing μ- or δ-opioid receptors, we found that δ-opioid receptors were located in caveolin-poor fractions following 1% Triton X-100 homogenization and sucrose gradient ultracentrifugation. This differs from studies using a detergent-free method to identify lipid raft fractions (10, 11). In contrast, we found that the μ-opioid receptor was found in both caveolin-poor and caveolin-rich fractions, in accordance with previous literature (8, 9). This differential localization of opioid receptors led us to test the hypothesis that, in contrast to the μ-opioid receptor, the δ-opioid receptor would not be dependent on cholesterol for signaling. The results show that μ- but not δ-opioid receptors have a dependence on cholesterol for signaling to adenylyl cyclase and that this effect is much more pronounced following chronic exposure to opioids.