Phosphorylation of the Bin,Amphiphysin, and RSV161/167 (BAR) domain of ACAP4 regulates membrane tubulation

ArfGAP With Coiled-Coil, Ankyrin Repeat And PH Domains 4 (ACAP4) is an ADP-ribosylation factor 6 (ARF6) GTPase-activating protein essential for EGF-elicited cell migration. However, how ACAP4 regulates membrane dynamics and curvature in response to EGF stimulation is unknown. Here, we show that phosphorylation of the N-terminal region of ACAP4, named the Bin, Amphiphysin, and RSV161/167 (BAR) domain, at Tyr34 is necessary for EGF-elicited membrane remodeling. Domain structure analysis demonstrates that the BAR domain regulates membrane curvature. EGF stimulation of cells causes phosphorylation of ACAP4 at Tyr34, which subsequently promotes ACAP4 homodimer curvature. The phospho-mimicking mutant of ACAP4 demonstrates lipid-binding activity and tubulation in vitro, and ARF6 enrichment at the membrane is associated with ruffles of EGF-stimulated cells. Expression of the phospho-mimicking ACAP4 mutant promotes ARF6-dependent cell migration. Thus, the results present a previously undefined mechanism by which EGF-elicited phosphorylation of the BAR domain controls ACAP4 molecular plasticity and plasma membrane dynamics during cell migration.Many cellular processes are orchestrated by dynamic changes in the plasma membrane to form membrane projections and endocytic vesicles. Cell migration is necessary for tissue development and wound repair. During cell migration, the coordination of membrane traffic, actin skeleton remodeling, and formation of new adhesion complexes is required for protrusive activities at the leading edges of the migrating cells (1, 2). Some small GTPases, such as ADP-ribosylation factors (ARFs) and Rhos, are involved in coupling actin dynamics to trafficking of vesicular membranes (3, 4) and in control of membrane curvature. In mammals, the six ARF proteins belong to three classes, based on sequence homology: class I (ARF1–3), class II (ARF4 and 5), and class III (ARF6). The sole member of class III, ARF6, functions in plasma membrane dynamics (5) and in promoting endocytic recycling (6). ARF6 resides on endosomal and plasma membranes to regulate membrane trafficking between these compartments (7–10). Activation of ARF6 promotes cortical actin assembly (9) and plasma membrane remodeling (10). Aberrant expression of ARF6 has been implicated in tumor invasion and metastasis (11).Key determinants of ARF6 function are the lifetime and the subcellular locations of the GTP-bound active state, which is orchestrated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) (12). Our proteomic analyses identified ArfGAP With Coiled-Coil, Ankyrin Repeat And PH Domains 4 (ACAP4) as a GAP protein that selectively binds to active ARF6 and catalyzes GTP hydrolysis (13). The ACAP4 gene encodes 903 amino acids and contains a catalytic core of a pleckstrin homology (PH) domain, a GAP motif, and two ankyrin (ANK) repeats. The GAP activity is regulated by phosphatidylinositol 4,5-biphosphate [PI(4,5)P₂] via binding to the PH domain. The Arg469 in the GAP domain is necessary for its activity in GTP hydrolysis (13). The crystal structure of the catalytic core of ACAP4 in a complex with ARF6 reveals the structural determinants underlying ACAP4 selectivity and specificity as an ARF6 GTPase-activating protein (14). ACAP4 also associates with focal adhesions and with cytoplasmic membranes at the circular dorsal ruffles, where the actin skeleton is remodeled (15). Depletion of ACAP4 by RNA interference suppresses cell migration in wound healing (13, 15).Dynamic changes in the plasma membrane and vesicular trafficking are achieved by recruitment of membrane shaping proteins such as those in the Bin, Amphiphysin, and RSV161/167 (BAR) superfamily. The BAR domain is characterized as a crescent-shaped dimer composed mainly of three long, kinked α-helices. Structural studies indicate that all BAR domains have a concave membrane-binding interface that interacts with negatively charged membranes (16–20). The BAR domain, also found in other contexts in a wide variety of proteins (17, 19), is believed to be responsible for generating, stabilizing, or sensing curvature (18). The concave shape of the BAR domain appears to be responsible for the tubulation of membranes in intracellular trafficking processes (18). The BAR domain also contributes to the membrane scission of budding vesicles (21).Several proteins containing the BAR domain regulate actin-based cytoskeleton dynamics and cell migration (22, 23). The I-BAR protein, MIM, also promotes Arp2/3-mediated assembly of actin filaments at adherent junctions (24). Sensing of membrane curvature is coupled with enzymatic activities, directly, as with GAPs and GEFs, and, indirectly, as in the case of synaptojanin bound to endophilin (18). However, the mechanism underlying the membrane association of these proteins has remained undefined.The present report demonstrates that ACAP4, which contains a BAR domain, is associated with membrane binding and bending activity in vitro and in vivo. The BAR domain is necessary for the recruitment of ACAP4 to membrane structures and for its GAP activity. Importantly, Tyr34 in the BAR domain is phosphorylated in response to EGF stimulation. Phosphorylation of Tyr34 promotes the migratory activity of MDA-MB-231 cells. Thus, phosphorylation of Tyr34 in the BAR domain modulates the membrane-binding capacity of ACAP4. Thus, this study revealed a previously undefined role for ACAP4 in linking the ARF6-mediated vesicular membrane dynamics to EGF-elicited cell migration.     

Molecular mechanism of membrane constriction and tubulation mediated by the F-BAR protein Pacsin/Syndapin

Peripheral membrane proteins of the Bin/amphiphysin/Rvs (BAR) and Fer-CIP4 homology-BAR (F-BAR) family participate in cellular membrane trafficking and have been shown to generate membrane tubules. The degree of membrane bending appears to be encoded in the structure and immanent curvature of the particular protein domains, with BAR and F-BAR domains inducing high- and low-curvature tubules, respectively. In addition, oligomerization and the formation of ordered arrays influences tubule stabilization. Here, the F-BAR domain-containing protein Pacsin was found to possess a unique activity, creating small tubules and tubule constrictions, in addition to the wide tubules characteristic for this subfamily. Based on crystal structures of the F-BAR domain of Pacsin and mutagenesis studies, vesiculation could be linked to the presence of unique structural features distinguishing it from other F-BAR proteins. Tubulation was suppressed in the context of the full-length protein, suggesting that Pacsin is autoinhibited in solution. The regulated deformation of membranes and promotion of tubule constrictions by Pacsin suggests a more versatile function of these proteins in vesiculation and endocytosis beyond their role as scaffold proteins.     

Phosphorylation of membrane proteins at a cholinergic synapse.

Endogenous membrane protein kinase activity and protein kinase substrates have been found in membrane fractions enriched in the acetylcholine receptor that were prepared from the electric organ of Torpedo californica. Phosphorylation of four polypeptides is stimulated 9-fold by K+. The specific cholinergic ligand, carbachol, inhibited phosphorylation of these four polypeptides by 72% in the presence of 1mM Na+ and 100 mM K+. The 65,000-dalton component of the acetylcholine receptor in the membrane fraction appears to be phosphorylated by the endogenous protein kinase. These results suggest that protein phosphorylation may play an important role in synaptic events at nicotinic cholinergic synapses.     

Phosphorylation of the voltage-gated potassium channel Kv2.1 by AMP-activated protein kinase regulates membrane excitability

Firing of action potentials in excitable cells accelerates ATP turnover. The voltage-gated potassium channel Kv2.1 regulates action potential frequency in central neurons, whereas the ubiquitous cellular energy sensor AMP-activated protein kinase (AMPK) is activated by ATP depletion and protects cells by switching off energy-consuming processes. We show that treatment of HEK293 cells expressing Kv2.1 with the AMPK activator A-769662 caused hyperpolarizing shifts in the current-voltage relationship for channel activation and inactivation. We identified two sites (S440 and S537) directly phosphorylated on Kv2.1 by AMPK and, using phosphospecific antibodies and quantitative mass spectrometry, show that phosphorylation of both sites increased in A-769662-treated cells. Effects of A-769662 were abolished in cells expressing Kv2.1 with S440A but not with S537A substitutions, suggesting that phosphorylation of S440 was responsible for these effects. Identical shifts in voltage gating were observed after introducing into cells, via the patch pipette, recombinant AMPK rendered active but phosphatase-resistant by thiophosphorylation. Ionomycin caused changes in Kv2.1 gating very similar to those caused by A-769662 but acted via a different mechanism involving Kv2.1 dephosphorylation. In cultured rat hippocampal neurons, A-769662 caused hyperpolarizing shifts in voltage gating similar to those in HEK293 cells, effects that were abolished by intracellular dialysis with Kv2.1 antibodies. When active thiophosphorylated AMPK was introduced into cultured neurons via the patch pipette, a progressive, time-dependent decrease in the frequency of evoked action potentials was observed. Our results suggest that activation of AMPK in neurons during conditions of metabolic stress exerts a protective role by reducing neuronal excitability and thus conserving energy.     

Phosphorylation of a UDP-glucuronosyltransferase regulates substrate specificity

UDP-glucuronosyltransferase (UGT) isozymes catalyze detoxification of numerous chemical toxins present in our daily diet and environment by conjugation to glucuronic acid. The special properties and enzymatic mechanism(s) that enable endoplasmic reticulum-bound UGT isozymes to convert innumerable structurally diverse lipophiles to excretable glucuronides are unknown. Inhibition of cellular UGT1A7 and UGT1A10 activities and of [33P]orthophosphate incorporation into immunoprecipitable proteins after exposure to curcumin or calphostin-C indicated that the isozymes are phosphorylated. Furthermore, inhibition of UGT phosphorylation and activity by treatment with PKCepsilon-specific inhibitor peptide supported PKC involvement. Co-immunoprecipitation, colocalization by means of immunofluorescence, and cross-linking studies of PKCepsilon and UGT1A7His revealed that the proteins reside within 11.4 angstroms of each other. Moreover, mutation of three PKC sites in each UGT isozyme demonstrated that T73A/G and T202A/G caused null activity, whereas S432G-UGT1A7 caused a major shift of its pH-8.5 optimum to 6.4 with new substrate selections, including 17beta-estradiol. S432G-UGT1A10 exhibited a minor pH shift without substrate alterations. PKCepsilon involvement was confirmed by the demonstration that PKCepsilon overexpression enhanced activity of UGT1A7 but not of its S432 mutant and the conversion of 17beta-[14C]estradiol by S432G-UGT1A7 but not by UGT1A7. Consistent with these observations, treatment of UGT1A7-transfected cells with PKCepsilon-specific inhibitor peptide or general PKC inhibitors increased 17beta-estradiol catalysis between 5- and 11-fold, with parallel decreases in phosphoserine-432. Here, we report a mechanism involving PKC-mediated phosphorylation of UGT such that phosphoserine/threonine regulates substrate specificity in response to chemical exposures, which possibly confers survival benefit.     

Phosphorylation of murine p53 at ser-18 regulates the p53 responses to DNA damage

Ser-15 of human p53 (corresponding to Ser-18 of mouse p53) is phosphorylated by ataxia-telangiectasia mutated (ATM) family kinases in response to ionizing radiation (IR) and UV light. To determine the effects of phosphorylation of endogenous murine p53 at Ser-18 on biological responses to DNA damage, we introduced a missense mutation (Ser-18 to Ala) by homologous recombination into both alleles of the endogenous p53 gene in mouse embryonic stem (ES) cells. Our analyses showed that phosphorylation of murine p53 at Ser-18 in response to IR or UV radiation was required for a full p53-mediated response to these DNA damage-inducing agents. In contrast, phosphorylation of p53 at Ser-18 was not required for ATM-dependent cellular resistance after exposure to IR. Additionally, efficient acetylation of the C terminus of p53 in response to DNA damage did not require phosphorylation of murine p53 at Ser-18.     

Phosphorylation of TPP1 regulates cell cycle-dependent telomerase recruitment

Telomere maintenance is essential for organisms with linear chromosomes and is carried out by telomerase during cell cycle. The precise mechanism by which cell cycle controls telomeric access of telomerase and telomere elongation in mammals remains largely unknown. Previous work has established oligonucleotide/oligosaccharide binding (OB) fold-containing telomeric protein TPP1, formerly known as TINT1, PTOP, and PIP1, as a key factor that regulates telomerase recruitment and activity. However, the role of TPP1 in cell cycle-dependent telomerase recruitment is unclear. Here, we report that human TPP1 is phosphorylated at multiple sites during cell cycle progression and associates with higher telomerase activity at late S/G₂/M. Phosphorylation of Ser111 (S111) within the TPP1 OB fold appears important for cell cycle-dependent telomerase recruitment. Structural analysis indicates that phosphorylated S111 resides in the telomerase-interacting domain within the TPP1 OB fold. Mutations that disrupt S111 phosphorylation led to decreased telomerase activity in the TPP1 complex and telomere shortening. Our findings provide insight into the regulatory pathways and structural basis that control cell cycle-dependent telomerase recruitment and telomere elongation through phosphorylation of TPP1.     

Phosphorylation of lens fiber cell membrane proteins.

Two intrinsic membrane proteins of calf lens fiber cells can be phosphorylated by a soluble bovine lens cAMP-dependent protein kinase and rabbit muscle cAMP-dependent protein kinase. After electrophoresis of the phosphorylated membranes, 32P comigrates with the lens main intrinsic protein at 26-27 kDa and with a minor band of protein that migrates at 19-20 kDa. 32P is also found with proteins that, based on the molecular sizes, are likely multimers of the 19-kDa and 26-kDa proteins. Upon boiling in NaDodSO4, all the radioactivity is found at the top of the gel, suggesting that both phosphoproteins are intrinsic membrane proteins. Serine is the only phospho amino acid detected in both proteins regardless of the source of protein kinase. The phosphorylation sites of both proteins are lost upon cleavage with trypsin and chymotrypsin. The smaller phosphoprotein is likely not a crystallin, because antibodies directed against alpha-, beta-, or gamma-crystallins do not cross-react with the 19-kDa protein. The 19-kDa 32P-labeled protein does not migrate coincident with calf alpha-crystallin.     

Phosphorylation of the Par-1 polarity kinase by protein kinase D regulates 14-3-3 binding and membrane association

The Par-1 protein kinases are conserved from yeast to humans, where they function as key polarity determinants. The mammalian Par-1 family is comprised of 4 members (Par-1a, -b, -c, and -d). Previously, we demonstrated that atypical protein kinase C (aPKC) phosphorylates the Par-1 kinases on a conserved threonine residue (T595) to regulate localization and kinase activity. Here, we demonstrate that Par-1b is also regulated by another arm of the PKC pathway, one that involves novel PKCs (nPKC) and protein kinase D. Treatment of cells with the PKC activator phorbol-12-myristate-13-acetate (PMA) potently stimulated phosphorylation of Par-1b on serine 400 (S400), a residue that is conserved in all 4 mammalian Par-1 kinases as well as the fly ortholog. We demonstrate that PMA stimulates nPKC to activate PKD, which in turn directly phosphorylates Par-1b on S400 to positively regulate 14-3-3 binding and to negatively regulate membrane association. Thus, 2 arms of the PKC pathway regulate interactions between Par-1b and 14-3-3 proteins: one involving aPKC and the other nPKC/PKD.     

Phosphorylation of the DNA-binding domain of nonhistone high-mobility group I protein by cdc2 kinase: reduction of binding affinity.

Mammalian high-mobility group I nonhistone protein (HMG-I) is a DNA-binding chromatin protein that has been demonstrated both in vitro and in vivo to be localized to the A + T-rich sequences of DNA. Recently an unusual binding domain peptide, "the A.T-hook" motif, that mediates specific interaction of HMG-I with the minor groove of DNA in vitro has been described. Inspection of the A.T-hook region of the binding domain showed that it matches the consensus sequence for phosphorylation by cdc2 kinase. Here we demonstrate that HMG-I is a substrate for phosphorylation by purified mammalian cdc2 kinase in vitro. The site of phosphorylation by this enzyme is a threonine residue at the amino-terminal end of the principal binding-domain region of the protein. Labeling of mitotically blocked mouse cells with [32P]phosphate demonstrates that this same threonine residue in HMG-I is also preferentially phosphorylated in vivo. Competition binding studies show that cdc2 phosphorylation of a synthetic binding-domain peptide significantly weakens its interaction with A + T-rich DNA in vitro, and a similar weakening of DNA binding has been observed for intact murine HMG-I protein phosphorylated by the kinase in vitro. These findings indicate that cdc2 phosphorylation may significantly alter the DNA-binding properties of the HMG-I proteins. Because many cdc2 substrates are DNA-binding proteins, these results further suggest that alteration of the DNA-binding affinity of a variety of proteins is an important general component of the mechanism by which cdc2 kinase regulates cell cycle progression.     

Phosphorylation of the spindle checkpoint protein Mad2 regulates its conformational transition

Regulated conformational changes of proteins are critical for cellular signal transduction. The spindle checkpoint protein Mad2 is an unusual protein with two native folds: the latent open conformer (O-Mad2) and the activated closed conformer (C-Mad2). During mitosis, cytosolic O-Mad2 binds to the Mad1-Mad2 core complex at unattached kinetochores and undergoes conformational activation to become C-Mad2. C-Mad2 binds to and inhibits Cdc20, an activator of APC/C, to prevent precocious anaphase onset. Here, we show that the conformational transition of Mad2 is regulated by phosphorylation of S195 in its C-terminal region. The phospho-mimicking Mad2(S195D) mutant and the phospho-S195 Mad2 protein obtained using intein-mediated semisynthesis do not form C-Mad2 on their own. Mad2(S195D) fails to bind to Cdc20, a low-affinity ligand, but still binds to high-affinity ligands, such as Mad1 and MBP1, forming ligand-bound C-Mad2. Overexpression of Mad2(S195D) in human cells causes checkpoint defects. Our results indicate that Mad2 phosphorylation inhibits its function through differentially regulating its binding to Mad1 and Cdc20 and establish that the conformational change of Mad2 is regulated by posttranslational mechanisms.     

Phosphorylation of Vpr regulates HIV type 1 nuclear import and macrophage infection

Viral protein R (Vpr) of human immunodeficiency virus type 1 (HIV-1) is a small accessory protein that regulates nuclear import of the viral preintegration complex and facilitates infection of nondividing cells, such as macrophages. Studies demonstrated that a fraction of Vpr molecules is phosphorylated in the virions and in HIV-1-infected cells, but the role of phosphorylation in nuclear import activity of Vpr has not been established. We found that Vpr is phosphorylated predominantly on the serine residue in position 79, and mutations affecting Vpr phosphorylation significantly attenuated viral replication in macrophages, but not in activated T lymphocytes or cell lines. The replication defect was mapped by polymerase chain reaction analysis to the step of nuclear import. These results suggest that phosphorylation of Vpr regulates its activity in the nuclear import of the HIV-1 preintegration complex.     

Electrostatics,hydration, and proton transfer dynamics in the membrane domain of respiratory complex I

Complex I serves as the primary electron entry point into the mitochondrial and bacterial respiratory chains. It catalyzes the reduction of quinones by electron transfer from NADH, and couples this exergonic reaction to the translocation of protons against an electrochemical proton gradient. The membrane domain of the enzyme extends ∼180 Å from the site of quinone reduction to the most distant proton pathway. To elucidate possible mechanisms of the long-range proton-coupled electron transfer process, we perform large-scale atomistic molecular dynamics simulations of the membrane domain of complex I from Escherichia coli. We observe spontaneous hydration of a putative proton entry channel at the NuoN/K interface, which is sensitive to the protonation state of buried glutamic acid residues. In hybrid quantum mechanics/classical mechanics simulations, we find that the observed water wires support rapid proton transfer from the protein surface to the center of the membrane domain. To explore the functional relevance of the pseudosymmetric inverted-repeat structures of the antiporter-like subunits NuoL/M/N, we constructed a symmetry-related structure of a possible alternate-access state. In molecular dynamics simulations, we find the resulting structural changes to be metastable and reversible at the protein backbone level. However, the increased hydration induced by the conformational change persists, with water molecules establishing enhanced lateral connectivity and pathways for proton transfer between conserved ionizable residues along the center of the membrane domain. Overall, the observed water-gated transitions establish conduits for the unidirectional proton translocation processes, and provide a possible coupling mechanism for the energy transduction in complex I.Complex I, or NADH:ubiquinone oxidoreductase, is an enzyme crucial for biological energy conversion. By transferring electrons from reduced NADH to quinone (Q), it functions as a primary entry point for electrons into the mitochondrial and bacterial respiratory chains (1, 2). Complex I couples the Q reduction to translocation of three to four protons across the mitochondrial or bacterial membrane (3, 4), thus contributing to the electrochemical proton-motive force subsequently used for synthesis of ATP by F_oF₁-ATPase and for active transport of solutes (5).Complex I is a large (550–980 kDa) L-shaped enzyme, which consists of a hydrophilic domain located in the mitochondrial matrix/bacterial cytoplasm, and a membrane domain, embedded in the mitochondrial inner membrane/bacterial cytoplasmic membrane (1, 2). Its 14 core subunits are evolutionarily conserved in bacteria and eukaryotes (1), with over 20 additional subunits in higher organisms (6). The hydrophilic domain provides an electron transfer chain from NADH via a flavine mononucleotide and eight to nine iron-sulfur centers to Q, located at the end of this chain (Fig. 1A) (7, 8). Leakage from this electron transfer pathway is a likely source of mitochondrial reactive oxygen species (9) associated with neurodegenerative diseases and aging.Open in a separate windowFig. 1.Structure and internal symmetry of complex I from E. coli. (A) Membrane (PDB ID: 3RKO) and hydrophilic domains (PDB ID: 2FUG) of complex I embedded in a lipid membrane. The antiporter-like subunits NuoN, NuoM, and NuoL are shown in yellow, blue, and red, respectively. (B) TM helix segments 4–8 (blue) and 9–13 (red) in NuoN/M/L structurally superimposed. TM helices 4–6 and 9–11 have high structural similarity, whereas TM helices 7–8, and 12–13 are in a different conformation. (C) Position of key residues in the membrane domain of complex I. The amphipathic HL helix (red) lies parallel to the membrane plane, and contacts all three antiporter-like subunits.The proton-translocating membrane domain of complex I comprises three antiporter-like subunits, NuoN, NuoM, and NuoL (Escherichia coli naming), which are connected to the hydrophilic domain by the NuoA/J/K/H subunits (10, 11). The antiporter-like subunits are homologous to each other as well as to, for example, Mrp (multiresistance and pH adaptation) Na⁺/H⁺-antiporters and certain hydrogenases (12). The antiporter-like subunits have an intrinsic sequence identity of ∼20%, but an even more evident structural homology: the transmembrane (TM) helices 4–8 and 9–13 can be structurally superimposed (Fig. 1B). These subunits also contain several crucial residues for the proton translocation process (Fig. 1C): a conserved Lys-Glu (or Asp in NuoL) ion pair in TM helices 5/7a, and one or two other conserved lysines, have been confirmed by site-directed mutagenesis experiments to be crucial for the proton translocation process (1, 2) (Table S1). During the completion of this study, a new X-ray structure of the intact complex I from Thermus thermophilus was released (13). The structure reveals that the NuoH subunit (Nqo8 in T. thermophilus) has structural resemblance to the TM helix segment 4–8 of the antiporter-like NuoN/M/L subunits (13), and may thus also be involved in the proton-pumping machinery. A detailed comparison of key regions of the Escherichia coli and T. thermophilus membrane domains is shown in Fig. S1.Interestingly, mutation of conserved residues in the NuoL subunit, ∼180 Å away from the hydrophilic domain, leads to loss of the Q-reductase activity (1, 2) (Table S1). Although expected for a fully reversible proton-coupled electron transfer machine, this tight coupling imposes severe mechanistic demands. Remarkably, after deletion of subunits equivalent to NuoL and NuoM, the apparent pumping stoichiometry was reduced by about one-half (14). The putative proton transfer pathways through the membrane domain are distant from the redox-active groups mediating electron transfer through the hydrophilic domain (10). As a consequence, the proton-coupled electron transfer in complex I is expected to differ mechanistically from that in other systems, such as ribonucleotide reductase, an enzyme involved in synthesis of DNA from RNA, where the proton and the electron are transferred concertedly along a pathway of conserved residues (15, 16). In contrast, in complex I the electron and proton transfers are separated both kinetically and spatially. To explain this long-range coupling, both “direct” (redox-driven) and “indirect” (conformational-driven) mechanisms have been suggested, but the molecular principles of these mechanisms remain elusive (1, 2, 4, 17–22).Complex I has been suggested to undergo conformational changes, which may drive the proton-translocation process (11, 23). Superimposing the TM helix segments of NuoN, NuoM, and NuoL suggests that TM helices 4–6/9–11 are structurally in a similar conformation, whereas the TM helices 7–8/12–13 have a different tilting angle relative to the membrane normal (Fig. 1B). In terms of its evolutionary homology to antiporters, this internal symmetry would suggest that the two segments are in different conformational states with connectivity to different sides of the membrane, in analogy to what has been observed for carrier-type transporters (24).To gain further insight into the long-range coupling mechanism, we study here the dynamics of the membrane domain of complex I from E. coli by large-scale atomistic molecular dynamics (MD) simulations, hybrid quantum mechanics/molecular mechanics (QM/MM) approaches, and continuum electrostatics calculations. Our simulations, both in the state of the crystal structure and in a putative alternate-access state with inverted symmetry established by harmonic restraints, give molecular insight into the structural dynamics and coupling of key residues involved in the proton translocation process. Instead of large-scale conformational changes associated with traditional transporter function, we show here that extensive changes in internal hydration establish the changes in protonic access required for pumping. With these hydration changes found to be strongly coupled to the charge states of conserved titratable residues, and their charge states in turn coupled to each other, internal water thus emerges as the key element in the redox-coupled proton translocation process that connects the extensive network of buried ionizable residues with each other and with the surfaces.     

NMR and mutagenesis evidence for an I domain allosteric site that regulates lymphocyte function-associated antigen 1 ligand binding

The leukocyte integrin, lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18), mediates cell adhesion and signaling in inflammatory and immune responses. To support these functions, LFA-1 must convert from a resting to an activated state that avidly binds its ligands such as intercellular adhesion molecule 1 (ICAM-1). Biochemical and x-ray studies of the Mac-1 (CD11b/CD18) I domain suggest that integrin activation could involve a conformational change of the C-terminal alpha-helix. We report the use of NMR spectroscopy to identify CD11a I domain residues whose resonances are affected by binding to ICAM-1. We observed two distinct sites in the CD11a I domain that were affected. As expected from previous mutagenesis studies, a cluster of residues localized around the metal ion-dependent adhesion site (MIDAS) was severely perturbed on ICAM-1 binding. A second cluster of residues distal to the MIDAS that included the C-terminal alpha-helix of the CD11a I domain was also affected. Substitution of residues in the core of this second I domain site resulted in constitutively active LFA-1 binding to ICAM-1. Binding data indicates that none of the 20 substitution mutants we tested at this second site form an essential ICAM-1 binding interface. We also demonstrate that residues in the I domain linker sequences can regulate LFA-1 binding. These results indicate that LFA-1 binding to ICAM-1 is regulated by an I domain allosteric site (IDAS) and that this site is structurally linked to the MIDAS.     

Phosphorylation near nuclear localization signal regulates nuclear import of adenomatous polyposis coli protein

Mutation of the adenomatous polyposis coli (APC) gene is an early step in the development of colorectal carcinomas. APC protein is located in both the cytoplasm and the nucleus. The objective of this study was to define the nuclear localization signals (NLSs) in APC protein. APC contains two potential NLSs comprising amino acids 1767-1772 (NLS1(APC)) and 2048-2053 (NLS2(APC)). Both APC NLSs are well conserved among human, mouse, rat, and fly. NLS1(APC) and NLS2(APC) each were sufficient to target the cytoplasmic protein beta-galactosidase to the nucleus. Mutational analysis of APC demonstrated that both NLSs were necessary for optimal nuclear import of full-length APC protein. Alignment of NLS2(APC) with the simian virus 40 large T antigen NLS (NLS(SV40 T-ag)) revealed sequence similarity extending to adjacent phosphorylation sites. Changing a serine residue (Ser(2054)) to aspartic acid mutated the potential protein kinase A site adjacent to NLS2(APC), resulting in both inhibition of the NLS2(APC)-mediated nuclear import of a chimeric beta-galactosidase fusion protein and a reduction of full-length APC nuclear localization. Our data provide evidence that control of APC's nuclear import through phosphorylation is a potential mechanism for regulating APC's nuclear activity.     

Phosphorylation of the Cav3.2 T-type calcium channel directly regulates its gating properties

Phosphorylation is a major mechanism regulating the activity of ion channels that remains poorly understood with respect to T-type calcium channels (Cav3). These channels are low voltage-activated calcium channels that play a key role in cellular excitability and various physiological functions. Their dysfunction has been linked to several neurological disorders, including absence epilepsy and neuropathic pain. Recent studies have revealed that T-type channels are modulated by a variety of serine/threonine protein kinase pathways, which indicates the need for a systematic analysis of T-type channel phosphorylation. Here, we immunopurified Cav3.2 channels from rat brain, and we used high-resolution MS to construct the first, to our knowledge, in vivo phosphorylation map of a voltage-gated calcium channel in a mammalian brain. We identified as many as 34 phosphorylation sites, and we show that the vast majority of these sites are also phosphorylated on the human Cav3.2 expressed in HEK293T cells. In patch-clamp studies, treatment of the channel with alkaline phosphatase as well as analysis of dephosphomimetic mutants revealed that phosphorylation regulates important functional properties of Cav3.2 channels, including voltage-dependent activation and inactivation and kinetics. We also identified that the phosphorylation of a locus situated in the loop I-II S442/S445/T446 is crucial for this regulation. Our data show that Cav3.2 channels are highly phosphorylated in the mammalian brain and establish phosphorylation as an important mechanism involved in the dynamic regulation of Cav3.2 channel gating properties.Voltage-gated calcium channels (L-, N-, P/Q-, R-, and T-types) mediate calcium entry in many different cell types in response to membrane depolarization and action potentials. Calcium influx through these channels serves as an important second messenger of electrical signaling, initiating a variety of cellular events and physiological functions (1, 2). Among the family of voltage-gated calcium channels, T-type calcium channels (Cav3 family) have unique electrophysiological properties, because they display low voltage-activated calcium currents with rapid activation/inactivation kinetics. In neurons, small changes in the membrane potential near the resting potential can activate T-type channels, favoring further membrane depolarization and repetitive firing of action potentials (3–5). These unique gating properties of T-type channels make them important in many different processes, including neuronal spontaneous firing and pacemaker activities, rebound burst firing, sleep rhythms, sensory processing, and neuronal differentiation, as well as in pathological conditions, such as epilepsy and neuropathic pain (6).To properly assure this plurality of physiological functions, a tight control of T-type calcium channels is necessary. An important regulatory mechanism is phosphorylation, the fastest and most frequent posttranslational modification for a protein. Ion channels, especially voltage-gated channels, are critically regulated by phosphorylation. Voltage-dependent sodium and potassium channels have been shown to be the target of multiple phosphorylation events, regulating different channel functions and being involved in pathological states, like epilepsy (7–11). Also, several studies have shown the crucial role of the L-type/Cav1 phosphorylation in important physiological functions, like the fight or flight response (12–15).Regarding T-type channels, phosphorylation remains poorly understood. There are three Cav3 pore-forming proteins (Cav3.1, Cav3.2, and Cav3.3 subunits) all displaying typical properties of T-type channels when expressed in heterologous cell systems (3, 16). Among them, the Cav3.2 channel seems to be particularly sensitive to various types of regulation, including phosphorylation. To date, several serine/threonine kinases, like PKA, PKC, or CamKII, have been shown to regulate Cav3.2 activity (reviewed in refs. 17–20); however, in most cases, this regulation is tissue-dependent, and little is still known about its molecular basis. Cav3.2 channels bear more than 100 multiple intracellular serine and threonine residues that are predicted to be phosphorylated by common prediction algorithms, like NetPhos2.0 (21). However, which of these residues are actually phosphorylated and what functional impact this phosphorylation will have remain to be determined.In this study, we investigate the phosphorylation pattern of the Cav3.2 isoform of the T-type channels and its role in Cav3.2 channel properties. Using an MS approach, we have established the first, to our knowledge, phosphorylation map of the Cav3.2 channel in a mammalian brain and a human cell line. Then, by using alkaline phosphatase (AP) and dephosphomimetic mutants in patch-clamp experiments, we reveal the importance of phosphorylation in modulating Cav3.2 gating properties. We have also identified a phosphorylation hot spot situated in the loop connecting domains I and II of the channel that plays a crucial role in this regulation. Altogether, this study provides important insights regarding how phosphorylation regulates Cav3.2 channels.