Polybasic Region Research Articles

Rac1 Recruits the Adapter Protein CMS/CD2AP to Cell-Cell Contacts

Rac1 is a member of the Rho family of small GTPases, which regulate cell adhesion and migration through their control of the actin cytoskeleton. Rho-GTPases are structurally very similar, with the exception of a hypervariable domain in the C terminus. Using peptide-based pulldown assays in combination with mass spectrometry, we previously showed that the hypervariable domain in Rac1 mediates specific protein-protein interactions. Most recently, we found that the Rac1 C terminus associates to the ubiquitously expressed adapter protein CMS/CD2AP. CD2AP is critical for the formation and maintenance of a specialized cell-cell contact between kidney podocyte foot processes, the slit diaphragm. Here, CD2AP links the cell adhesion protein nephrin to the actin cytoskeleton. In addition, CMS/CD2AP binds actin-regulating proteins, such as CAPZ and cortactin, and has been implicated in the internalization of growth factor receptors. We found that CD2AP specifically interacts with the C-terminal domain of Rac1 but not with that of other Rho family members. Efficient interaction between Rac1 and CD2AP requires both the proline-rich domain and the poly-basic region in the Rac1 C terminus, and at least two of the three N-terminal SH3 domains of CD2AP. CD2AP co-localizes with Rac1 to membrane ruffles, and small interfering RNA-based experiments showed that CD2AP links Rac1 to CAPZ and cortactin. Finally, expression of constitutive active Rac1 recruits CD2AP to cell-cell contacts in epithelial cells, where we found CD2AP to participate in the control of the epithelial barrier function. These data identify CD2AP as a novel Rac1-associated adapter protein that participates in the regulation of epithelial cell-cell contact.

Conquering the complex world of human septins: implications for health and disease

Septins are highly conserved filamentous proteins first characterized in budding yeast and subsequently identified in must eukaryotes. Septins can bind and hydrolyze GTP, which is intrinsically related to their formation of septin hexamers and functional protein interactions. The human septin family is composed of 14 loci, SEPT1-SEPT14, which encode dozens of different septin proteins. Their central GTPase and polybasic domain regions are highly conserved but they diverge in their N-terminus and/or C-terminus. The mechanism by which the different isoforms are generated is not yet well understood, but one can hypothesize that the use of different promoters and/or alternative splicing could give rise to these variants. Septins perform diverse cellular functions according to tissue expression and their interacting partners. Functions identified to date include cell division, chromosome segregation, protein scaffolding, cellular polarity, motility, membrane dynamics, vesicle trafficking, exocytosis, apoptosis, and DNA damage response. Their expression is tightly regulated to maintain proper filament assembly and normal cellular functions. Alterations of these proteins, by mutation or expression changes, have been associated with a variety of cancers and neurological diseases. The association of septins with cancer results from alterations of expression in solid tumors or translocations in leukemias [mixed lineage leukemia (MLL)]. Expression changes in septins have also been associated with neurological conditions such as Alzheimer's and Parkinson's disease, as well as retinopathies, hepatitis C, spermatogenesis and Listeria infection. Pathogenic mutations of SEPT9 were identified in the autosomal dominant neurological disorder hereditary neuralgic amyotrophy (HNA). Human septin research over the past decade has established their importance in cell biology and human disease. Further functional characterization of septins is crucial to our understanding of their possible diagnostic, prognostic, and therapeutic applications.

Nox4 B-loop Creates an Interface between the Transmembrane and Dehydrogenase Domains

By targeting redox-sensitive amino acids in signaling proteins, the NADPH oxidase (Nox) family of enzymes link reactive oxygen species to physiological processes. We previously analyzed the sequences of 107 Nox enzymes and identified conserved regions that are predicted to have important functions in Nox structure or activation. One such region is the cytosolic B-loop, which in Nox1-4 contains a conserved polybasic region. Previous studies of Nox2 showed that certain basic residues in the B-loop are important for activity and translocation of p47(phox)/p67(phox), suggesting this region participates in subunit assembly. However, conservation of this region in Nox4, which does not require p47(phox)/p67(phox), suggested an additional role for the B-loop in Nox function. Here, we show by mutation of Nox4 B-loop residues that this region is important for Nox4 activity. Fluorescence polarization detected binding between Nox4 B-loop peptide and dehydrogenase domain (K(d) = 58 +/- 12 nm). This interaction was weakened with Nox4 R96E B-loop corresponding to a mutation that also markedly decreases the activity of holo-Nox4. Truncations of the dehydrogenase domain localize the B-loop-binding site to the N-terminal half of the NADPH-binding subdomain. Similarly, the Nox2 B-loop bound to the Nox2 dehydrogenase domain, and both the Nox2 and Nox4 interactions were dependent on the polybasic region of the B-loop. These data indicate that the B-loop is critical for Nox4 function; we propose that the B-loop, by binding to the dehydrogenase domain, provides the interface between the transmembrane and dehydrogenase domains of Nox enzymes.

Analysis of the Rac/Rop Small GTPase Family in Rice: Expression, Subcellular Localization and Role in Disease Resistance

Plant-specific Rac/Rop small GTPases function as molecular switches for numerous signal transduction events, including defense responses. To understand the function of each of the seven Rac/Rop family members in rice, we studied the tissue-specific expression patterns of Rac/Rop genes by semi-quantitative reverse transcription-PCR (RT-PCR), and also Rac/Rop subcellular localization using green fluorescent protein (GFP) fusion proteins in transient expression systems. We also investigated the roles of these genes in disease resistance by testing single Rac/Rop-RNAi (RNA interference) plants against the rice blast pathogen Magnaporthe grisea. Our studies show that expression of OsRac2, 6 and 7 is very low in leaf blades, and reveal a strong correlation between the number of lysine and/or arginine (KR) residues in the polybasic region of Rac/Rop GTPases and their subcellular distribution in vivo. Infection assays showed that OsRac1 is a positive regulator of blast resistance, confirming previous observations, whereas OsRac4 and OsRac5 are negative regulators of blast resistance. OsRac6 may make minor contributions to disease resistance, while OsRac3 and OsRac7 are probably not involved in defense. Therefore, our study suggests that the rice Rac/Rop family plays multiple roles in diverse cellular activities and has both positive and negative functions in disease resistance.

Activated Rac1 GTPase Translocates Protein Phosphatase 5 to the Cell Membrane and Stimulates Phosphatase Activity in Vitro

Physiological studies of ion channel regulation have implicated the Ser/Thr protein phosphatase 5 (PP5) as an effector of Rac1 GTPase signaling, but direct biochemical evidence for PP5 regulation by Rac1 is lacking. In this study we used immunoprecipitation, in vitro binding, cellular fractionation, and immunofluorescence techniques to show that the tetratricopeptide repeat domain of PP5 interacts specifically and directly with active Rac1. Consequently, activation of Rac1 promoted PP5 translocation to the plasma membrane in intact cells and stimulated PP5 phosphatase activity in vitro. In contrast, neither constitutively active RhoA-V14 nor dominant negative Rac1N17, which preferentially binds GDP and retains an inactive conformation, bound PP5 or stimulated its activity. In addition, Rac1N17 and Rac1(PBRM), a mutant lacking the C-terminal polybasic region required for Rac1 association with the membrane, both failed to cause membrane translocation of PP5. Mutation of predicted contact residues in the PP5 tetratricopeptide repeat domain or within Rac1 also disrupted co-immunoprecipitation of Rac1-PP5 complexes and membrane translocation of PP5. Specific binding of PP5 to activated Rac1 provides a direct mechanism by which PP5 can be stimulated and recruited to participate in Rac1-mediated signaling pathways.

Control of Strex BK-Channel Palmitoylation Via a Polybasic Domain

Large conductance voltage and calcium sensitive potassium channels (BK) are widely expressed throughout the body and encoded by a single gene (KCNMA1). The splice insertion of the STREX exon at splice site C2, generates a channel phenotype with increased calcium sensitivity and differing regulation by phosphorylation. In mammals, splicing of the STREX exon is dynamically controlled by cellular excitability as well as circulating stress and sex hormones. With STREX insertion, a palmitoylation site and a polybasic region are introduced to the channel. The interaction of the polybasic region with the plasma membrane and palmitoylation of cysteine residues in the STREX-linker between RCK1 and RCK2 may serve as a membrane targeting motif that alters the phenotype of the BK-STREX channel.A GFP-tagged carboxyl terminal construct spanning from the S6 transmembrane domain to the COOH end region of the intracellular carboxyl terminus, localised at the plasma membrane. Membrane localisation was abolished when the STREX insert was excluded. To test whether the polybasic region is important for plasma membrane targeting, site directed mutagenesis was used to mutate positive residues in the polybasic domain into negative (E) or neutral (A) residues. These mutations abolished membrane targeting of S6-COOH-STX to the plasma membrane. Full-length channels with mutations in the polybasic region were studied in patch-clamp electrophysiology to determine calcium and voltage sensitivity, with disruption of the polybasic domain shifting apparent calcium sensitivity towards the zero (insertless) BK channel phenotype. The importance of the polybasic domain for palmitoylation of the BK-STREX channel was further confirmed by assaying 3H-palmitate incorporation into carboxyl terminal constructs.These data suggest that the polybasic region generated by inclusion of the STREX insert is an important determinant of BK-STREX channel palmitoylation and thus contributes to the altered channel properties upon STREX inclusion.

A Polybasic Region in the C-terminus of M3 Muscarinic Acetylcholine Receptors Mediates an Interaction with Gq Heterotrimers

G protein-coupled receptors (GPCRs) form stable complexes with heterotrimeric G proteins when the former are activated and when the latter are not bound to guanine nucleotides. In addition to these active-state ternary (ligand-receptor-G protein) complexes some GPCRs have been suggested to form preassembled or precoupled complexes with G proteins prior to activation. We have previously reported that immobile M3 muscarinic receptors (M3Rs) decrease the mobility of heterotrimers that contain Gαq, consistent with an M3R-Gq complex. This interaction is unaffected by receptor ligands in intact cells, and is specific for M3Rs and Gq, as immobile M4Rs do not decrease the mobility of Gq heterotrimers, and immobile M3Rs do not decrease the mobility of GoA heterotrimers. In order to determine the structural basis of this interaction, we constructed a series of CFP-labeled M3R/M4R chimeras and tested their ability to decrease the mobility of venus-labeled Gq (Gq-V) using fluorescence recovery after photobleaching (FRAP). A chimera consisting of the M3R with the c-terminus of the M4R (M3M4ct) did not decrease Gq-V mobility. A polybasic region (565KKKRRK570) immediately following helix 8 in the c-terminus of M3R was found to be necessary for the decrease in Gq-V mobility. We tested the hypothesis that an electrostatic interaction was involved in the interaction between M3Rs and Gq by repeating our FRAP experiments in permeabilized cells exposed to buffer solutions with high and low ionic strength. High ionic strength solutions inhibited the decrease in Gq mobility, whereas low ionic strength solutions enhanced this effect. These results suggest that an electrostatic interaction mediates an interaction between the c-terminus of M3Rs and Gq heterotrimers. The functional significance of this interaction is currently under study. Supported by grant GM078319 from the National Institutes of Health.

The Nuclear Import of the Small GTPase Rac1 is Mediated by the Direct Interaction with Karyopherin α2

The small GTPase Rac1 is involved in multiple cytosolic functions but recent data point out that Rac1 also translocates to the nucleus to regulate signalling pathways that control gene expression and progression through the cell cycle. Here, we identify the nuclear import receptor karyopherin alpha2 (KPNA2) as a direct interaction partner of Rac1. The C-terminal polybasic region of Rac1 contains a nuclear localization signal (NLS), whereas Rac2 and Rac3 lack a functional NLS and do not bind to KPNA2. The presence of the NLS in Rac1 determines the specificity of the interaction and is a prerequisite for the nuclear import. Although this interaction is independent of the Rac1 GDP/GTP loading, the induction of the translocation requires Rac1 activation. The activation of Rac1 via the cytotoxic necrotizing factor 1 and the concurrent inhibition of its proteasomal degradation are crucial for the nuclear accumulation of Rac1. Conversely, the reduction of KPNA2 expression inhibits the nuclear import of Rac1. For the first time, our results show a direct interaction between Rac1 and KPNA2 and argue for a KPNA2-dependent nuclear import of Rac1. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis revealed that nuclear Rac1 coimmunoprecipitates with numerous proteins. In the nucleus, Rac1 may participate in a variety of so far uncharacterized processes.

Targeting of Murine Leukemia Virus Gag to the Plasma Membrane Is Mediated by PI(4,5)P 2 /PS and a Polybasic Region in the Matrix

Membrane targeting of the human immunodeficiency virus Gag proteins is dependent on phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)] located in the plasma membrane. In order to determine if evolutionarily distant retroviral Gag proteins are targeted by a similar mechanism, we generated mutants of the matrix (MA) domain of murine leukemia virus (MuLV) Gag, examined their binding to membrane models in vitro, and analyzed their phenotypes in cell culture. In vitro, we showed that MA bound all the phosphatidylinositol phosphates with significant affinity but displayed a strong specificity for PI(4,5)P(2) only if enhanced by phosphatidylserine. Mutations in the polybasic region in MA dramatically reduced this affinity. In cells, virus production was strongly impaired by PI(4,5)P(2) depletion under conditions of 5ptaseIV overexpression, and mutations in the MA polybasic region altered Gag localization, membrane binding, and virion production. Our results suggest that the N-terminal polybasic cluster of MA is essential for Gag targeting to the plasma membrane. The binding of the MA domain to PI(4,5)P(2) appears to be a conserved feature among retroviruses despite the fact that the MuLV-MA domain is structurally different from that of human immunodeficiency virus types 1 and 2 and lacks a readily identifiable PI(4,5)P(2) binding cleft.

Multifaceted Sequence-Dependent and -Independent Roles for Reovirus FAST Protein Cytoplasmic Tails in Fusion Pore Formation and Syncytiogenesis

Fusogenic reoviruses utilize the FAST proteins, a novel family of nonstructural viral membrane fusion proteins, to induce cell-cell fusion and syncytium formation. Unlike the paradigmatic enveloped virus fusion proteins, the FAST proteins position the majority of their mass within and internal to the membrane in which they reside, resulting in extended C-terminal cytoplasmic tails (CTs). Using tail truncations, we demonstrate that the last 8 residues of the 36-residue CT of the avian reovirus p10 FAST protein and the last 20 residues of the 68-residue CT of the reptilian reovirus p14 FAST protein enhance, but are not required for, pore expansion and syncytium formation. Further truncations indicate that the membrane-distal 12 residues of the p10 and 47 residues of the p14 CTs are essential for pore formation and that a residual tail of 21 to 24 residues that includes a conserved, membrane-proximal polybasic region present in all FAST proteins is insufficient to maintain FAST protein fusion activity. Unexpectedly, a reextension of the tail-truncated, nonfusogenic p10 and p14 constructs with scrambled versions of the deleted sequences restored pore formation and syncytiogenesis, while reextensions with heterologous sequences partially restored pore formation but failed to rescue syncytiogenesis. The membrane-distal regions of the FAST protein CTs therefore exert multiple effects on the membrane fusion reaction, serving in both sequence-dependent and sequence-independent manners as positive effectors of pore formation, pore expansion, and syncytiogenesis.

Targeting of MuLV Gag to the plasma membrane is mediated by PI(4,5)P2 and PhosphatidylSerine

Membrane targeting by the modern human immunodeficiency viruses is dependent on the plasma membrane-located phospholipid PI(4,5)P2. In order to determine if evolutionarily distant retroviruses are targeted by a similar mechanism, we generated mutant Gag constructs in the matrix (MA) domain of the Murine Leukemia Virus (MuLV) and examined their binding to membrane models and phenotypes in cell culture. Mutations in the MA polybasic region altered Gag localization, membrane binding and virion production. In addition, we show that MA binds with good affinity to all the phosphatidylinositol phosphates but displays a strong specificity for PI(4,5)P2 only if enhanced by phophatidylserine. Virus production was strongly impaired by PI(4,5)P2 depletion under 5ptaseIV overexpression. Our results suggest that the N-terminal polybasic region of MA is essential for Gag targeting to the plasma membrane and Gag cellular trafficking. The binding of the MA domain to PI(4,5)P2 appears to be a conserved feature among retroviruses, despite the fact that the MuLV-MA domain is structurally different from that of HIV-1 and -2 and lacks a readily identifiable PI(4,5)P2 binding cleft.

DLC1 Activation Requires Lipid Interaction through a Polybasic Region Preceding the RhoGAP Domain

Deleted in Liver Cancer 1 (DLC1) is a GTPase-activating protein (GAP) with specificity for RhoA, RhoB, and RhoC that is frequently deleted in various tumor types. By inactivating these small GTPases, DLC1 controls actin cytoskeletal remodeling and biological processes such as cell migration and proliferation. Here we provide evidence that DLC1 binds to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)) through a previously unrecognized polybasic region (PBR) adjacent to its RhoGAP domain. Importantly, PI(4,5)P(2)-containing membranes are shown to stimulate DLC1 GAP activity in vitro. In living cells, a DLC1 mutant lacking an intact PBR inactivated Rho signaling less efficiently and was severely compromised in suppressing cell spreading, directed migration, and proliferation. We therefore propose that PI(4,5)P(2) is an important cofactor in DLC1 regulation in vivo and that the PBR is essential for the cellular functions of the protein.

Identification of Sequences in the Polysialyltransferases ST8Sia II and ST8Sia IV That Are Required for the Protein-specific Polysialylation of the Neural Cell Adhesion Molecule, NCAM

The polysialyltransferases ST8Sia II and ST8Sia IV polysialylate the glycans of a small subset of mammalian proteins. Their most abundant substrate is the neural cell adhesion molecule (NCAM). An acidic surface patch and a novel alpha-helix in the first fibronectin type III repeat of NCAM are required for the polysialylation of N-glycans on the adjacent immunoglobulin domain. Inspection of ST8Sia IV sequences revealed two conserved polybasic regions that might interact with the NCAM acidic patch or the growing polysialic acid chain. One is the previously identified polysialyltransferase domain (Nakata, D., Zhang, L., and Troy, F. A. (2006) Glycoconj. J. 23, 423-436). The second is a 35-amino acid polybasic region that contains seven basic residues and is equidistant from the large sialyl motif in both polysialyltransferases. We replaced these basic residues to evaluate their role in enzyme autopolysialylation and NCAM-specific polysialylation. We found that replacement of Arg(276)/Arg(277) or Arg(265) in the polysialyltransferase domain of ST8Sia IV decreased both NCAM polysialylation and autopolysialylation in parallel, suggesting that these residues are important for catalytic activity. In contrast, replacing Arg(82)/Arg(93) in ST8Sia IV with alanine substantially decreased NCAM-specific polysialylation while only partially impacting autopolysialylation, suggesting that these residues may be particularly important for NCAM polysialylation. Two conserved negatively charged residues, Glu(92) and Asp(94), surround Arg(93). Replacement of these residues with alanine largely inactivated ST8Sia IV, whereas reversing these residues enhanced enzyme autopolysialylation but significantly reduced NCAM polysialylation. In sum, we have identified selected amino acids in this conserved polysialyltransferase polybasic region that are critical for the protein-specific polysialylation of NCAM.

Molecular Recognition of the Palmitoylation Substrate Vac8 by Its Palmitoyltransferase Pfa3

Palmitoylation of the yeast vacuolar protein Vac8 is important for its role in membrane-mediated events such as vacuole fusion. It has been established both in vivo and in vitro that Vac8 is palmitoylated by the Asp-His-His-Cys (DHHC) protein Pfa3. However, the determinants of Vac8 critical for recognition by Pfa3 have yet to be elucidated. This is of particular importance because of the lack of a consensus sequence for palmitoylation. Here we show that Pfa3 was capable of palmitoylating each of the three N-terminal cysteines of Vac8 and that this reaction was most efficient when Vac8 is N-myristoylated. Additionally, when we analyzed the Src homology 4 (SH4) domain of Vac8 independent of the rest of the protein, palmitoylation by Pfa3 still occurred. However, the specificity of palmitoylation seen for the full-length protein was lost, and the SH4 domain was palmitoylated by all five of the yeast DHHC proteins tested. These data suggested that a region of the protein C-terminal to the SH4 domain was important for conferring specificity of palmitoylation. This was confirmed by use of a chimeric protein in which the SH4 domain of Vac8 was swapped for that of Meh1, another palmitoylated and N-myristoylated protein in yeast. In this case we saw specificity mimic that of wild type Vac8. Competition experiments revealed that the 11th armadillo repeat of Vac8 is an important element for recognition by Pfa3. This demonstrates that regions distant from the palmitoylated cysteines are important for recognition by DHHC proteins.

The Return of the INGs, Histone Mark Sensors and Phospholipid Signaling Effectors

Since their discovery, the members of the ING (inhibitor of growth) family of tumor suppressors have emerged as essential and core components of chromatin modifying complexes. Recent work has identified the ING family as histone mark sensors that orchestrate cellular responses to genotoxic insults and regulate chromatin homeostasis. Dysregulation of chromatin homeostasis is implicated in tumorigenesis through mechanisms such as silencing of tumor suppressor genes, inappropriate activation of oncogenes, and genomic instability due to failure to repair DNA damage. This review will concentrate on the chromatin signaling aspects of the ING proteins, focusing on how recognition of histone H3 trimethylated at lysine 4 (H3K4me3) by the PHD (plant homeodomain) finger of ING proteins is critical for regulating cellular functions such as gene expression. We will also discuss how H3K4me3-recognition by ING proteins plays a critical role in their tumor suppressive functions. Finally, we will discuss the relevance of the association between one ING protein (ING2) and the nuclear phosphoinositide, phosphatidylinositol-5-phosphate (PtdIns(5)P). Interestingly, the ING2-PtdIns(5)P interaction involves the PHD finger and an adjacent polybasic region. The level of nuclear PtdIns(5)P sharply increases upon genotoxic stress, and this increase positively regulates ING2-mediated responses. Thus, the PHD finger of ING2 integrates phosphoinositide and chromatin signaling networks to prevent unchecked cell growth.

Dominant roles of the polybasic proline motif and copper in the PrP23-89-mediated stress protection response

Beta-cleavage of the neurodegenerative disease-associated prion protein (PrP) protects cells from death induced by oxidative insults. The beta-cleavage event produces two fragments, designated N2 and C2. We investigated the role of the N2 fragment (residues 23-89) in cellular stress response, determining mechanisms involved and regions important for this reaction. The N2 fragment differentially modulated the reactive oxygen species (ROS) response induced by serum deprivation, with amelioration when copper bound. Amino acid residues 23-50 alone mediated a ROS reduction response. PrP23-50 ROS reduction was not due to copper binding or direct antioxidant activity, but was instead mediated through proteoglycan binding partners localised in or interacting with cholesterol-rich membrane domains. Furthermore, mutational analyses of both PrP23-50 and N2 showed that their protective capacity requires the sterically constraining double proline motif within the N-terminal polybasic region. Our findings show that N2 is a biologically active fragment that is able to modulate stress-induced intracellular ROS through interaction of its structurally defined N-terminal polybasic region with cell-surface proteoglycans.

Structural characterization of the PtdIns(4,5)P2‐PKCα‐C2 domain interaction

Classical PKCs contain two structural modules, the C1 and the C2 domains, in their regulatory region. These domains, in turn, dictate the cofactor‐dependence of the isoenzymes, being the C1 domain the diacylglycerol sensor and the C2 domain the Ca2+ and acidic phospholipids sensor. Here we show that a polybasic region located in the β3‐β4 strands of the C2 domain of classical PKCs, interacts specifically with PtdIns(4,5)P2 rather than with other polyphosphoinositides or phosphatidylserine. We have used X‐ray crystallography to analyze the structure of the C2 domain of PKCα in complex with PtdIns(4,5)P2, which was found at the conserved polybasic cluster. Site‐directed mutagenesis, on the residues from this cluster that interact directly with the phosphoinositide, revealed an important decrease in the ability of PKCα to interact with the plasma membrane of living cells. Furthermore, the mutant proteins were not able to translocate to the plasma membrane when the cells were stimulated with ionomycin and diacylglycerol. Instead, the protein localized in vesicles and Golgi apparatus, suggesting that the residues mutated are involved in recognizing the proper target of the C2 domain in the plasma membrane. Together, these data reveal the importance of the PtdIns(4,5)P2‐C2 domain interaction for the plasma membrane localization of PKCα.

The Positive Effect Of STREX On BK Channels

The large conductance voltage and calcium sensitive potassium channel (BK) is encoded by a single gene KCNMA1. The inclusion of a stress regulated exon (STREX) at a splice site (C2) in the intracellular carboxyl (C) terminus of BK confers differing properties to the channel. STREX insertion generates a putative polybasic region. In a number of other channels, polybasic regions are suggested to interact with negatively charged phospholipids, such as phosphoinositides, to control ion channel gating and membrane targeting. We hypothesised that the polybasic region including STREX may serve as a membrane targeting domain of the STREX BK channel C-terminus. To test this, a GFP-tagged carboxyl terminal construct spanning the S6 transmembrane domain to the COOH end region of the intracellular carboxyl terminus was constructed (S6-COOH-STX) and transiently transfected into HEK293cells. The construct localised at the plasma membrane and this was abolished when the STREX insert was deleted. To test whether the polybasic region is important for plasma membrane targeting, two approaches were taken: Firstly, site directed mutagenesis to change selected positive residues into neutral (alanine) residues, also abolished membrane targeting of S6-COOH-STX to the plasma membrane. Secondly, to discern whether the polybasic region may interact with negatively charged phosphoinositides at the plasma membrane, the S6-COOH-STX construct was co-transfected with a 5′phosphatase, IPP. Cells co-expressing IPP displayed significantly reduced plasma membrane targeting of S6-COOH-STX, however a phosphatase null mutant of IPP did not effect plasma membrane expression. These data suggest that the polybasic region generated by inclusion of the STREX insert is an important determinant of STREX domain interaction with the plasma membrane. The functional role of the polybasic region and the interaction of phospholipids on BK channel calcium and voltage sensitivity were elucidated using patch clamp electrophysiology and high throughput dyes.

Effects of Calcium and PIP2 on the Membrane Binding of Synaptotagmin I

Synaptotagmin I (Syt I) appears to act as the Ca2+ sensor in neuronal exocytosis and it is known to interact both with membranes and with SNAREs, which form the conserved core protein machinery for the fusion process. The interactions of Syt I with membranes were examined here with a combination of vesicle sedimentation and site-directed spin labeling (SDSL). Several interesting features of the interaction are revealed. First, Syt I binds to PC/PS bilayers in a Ca2+-independent manner though one of its cytosolic C2 domains, C2B. The interaction is mediated by the polybasic region of C2B domain, which associates in the electrostatic double-layer, but does not penetrate into the bilayer interior. Second, the affinity of C2B is increased approximately 20 fold in the presence of Ca2+ and now interacts through its Ca2+-binding loops. Remarkably, in the presence of Ca2+, C2A, C2B and a tandem fragment containing both C2A and C2B have approximately the same affinity, indicating the free energy of C2 domain interactions in Syt 1 are not additive. This may be due to demixing of the PS in the bilayer or the effects of curvature strain that are induced by the C2 domains. Finally, PI(4,5)P2 is a lipid that is critical to membrane fusion. Our preliminary data indicate that the addition of 1 mol% PI(4,5)P2 has little effect on the Ca2+-dependent binding of C2A; however, the membrane binding of both C2B and the tandem C2A-C2B domains is enhanced by PI(4,5)P2. As seen for other polybasic segments, the C2 domains appear to sequester or alter the lateral distribution of PI(4,5)P2 in the bilayer.

Gravin dynamics regulates the subcellular distribution of PKA

Gravin, a multivalent A-kinase anchoring protein (AKAP), localizes to the cell periphery in several cell types and is postulated to target PKA and other binding partners to the plasma membrane. An N-terminal myristoylation sequence and three regions rich in basic amino acids are proposed to mediate this localization. Reports indicating that phorbol ester affects the distribution of SSeCKS, the rat orthologue of gravin, further suggest that PKC may also regulate the subcellular distribution of gravin, which in turn may affect PKA distribution. In this study, quantitative confocal microscopy of cells expressing full-length and mutant gravin–EGFP constructs lacking the proposed targeting domains revealed that either the N-myristoylation site or the polybasic regions were sufficient to target gravin to the cell periphery. Moreover, phorbol ester treatment induced redistribution of gravin–EGFP from the cell periphery to a juxtanuclear vesicular compartment, but this required the presence of the N-myristoylation site. Confocal microscopy further revealed that not only did gravin–EGFP target a PKA RII-ECFP construct to the cell periphery, but PKC activation resulted in redistribution of the gravin and PKA constructs to the same subcellular site. It is postulated that this dynamic response by gravin to PKC activity may mediate PKC dependent control of PKA activity.