Carboxy-terminal Tail Research Articles

Opening and closing of KCNKO potassium leak channels is tightly regulated.

Potassium-selective leak channels control neuromuscular function through effects on membrane excitability. Nonetheless, their existence as independent molecular entities was established only recently with the cloning of KCNKO from Drosophila melanogaster. Here, the operating mechanism of these 2 P domain leak channels is delineated. Single KCNKO channels switch between two long-lived states (one open and one closed) in a tenaciously regulated fashion. Activation can increase the open probability to approximately 1, and inhibition can reduce it to approximately 0.05. Gating is dictated by a 700-residue carboxy-terminal tail that controls the closed state dwell time but does not form a channel gate; its deletion (to produce a 300-residue subunit with two P domains and four transmembrane segments) yields unregulated leak channels that enter, but do not maintain, the closed state. The tail integrates simultaneous input from multiple regulatory pathways acting via protein kinases C, A, and G.

Identification of further important residues within the Glut4 carboxy-terminal tail which regulate subcellular trafficking

The insulin-responsive glucose transporter, Glut4, exhibits a unique subcellular distribution such that in the absence of insulin >95% of the protein is stored within intracellular membranes. In response to insulin, Glut4 exhibits a large mobilisation to the plasma membrane. Studies of the amino acid motifs which regulate the unique trafficking of Glut4 have identified several key residues within the soluble cytoplasmic N- and C-terminal domains of Glut4. Of particular note is a Leu-498Leu-499 motif within the C-terminal domain that has been proposed to regulate both internalisation from the plasma membrane and sorting to an insulin-sensitive compartment. In this study, we have examined the role of the adjacent amino acids (Glu-491, Gln-492 and Glu-493) by their sequential replacement with Ala. Our results are consistent with the notion that Glu-491 and Glu-493 play an important role in the sub-endosomal trafficking of Glut4, as substitution of these residues with Ala results in increased levels of these proteins at the cell surface, reduced insulin-stimulated translocation and increased susceptibility to endosomal ablation. These residues, together with other identified sequences within the C-terminus of Glut4, are likely to be crucial targeting elements that regulate Glut4 subcellular distribution.

Tunicates have unusual nuclear lamins with a large deletion in the carboxyterminal tail domain

Lamins are essential proteins of metazoa. They give rise to the nuclear lamina lining the nucleoplasmic face of the inner nuclear membrane. Here we report the isolation of complete lamin cDNA clones from three urochordate (tunicate) libraries — adult Ciona intestinalis, the tailbud stage of Styela clava and the gastrula stage of Molgula oculata. Lamins L1 and L2 of adult Ciona are derived from two distinct genes. The sequence of the 3′ part of the Ciona lamin L1 gene shows that the α and β variants of lamin L1 in Ciona and Styela arise by alternative choice of the 5′ splice site at the last intron. Strikingly, all urochordate sequences reveal a 90 residue deletion which removes nearly the entire 105-box. This region is the only long sequence homology segment in the carboxyterminal tail domain of lamins from animals as diverse as Hydra, Drosophila, Priapulus, Caenorhabditis elegans, several echinoderms, the cephalochordate Branchiostoma and various vertebrates. We discuss this unexpected plasticity of lamin sequences as a urochordate specific marker. To increase the database for the chordates we completed the partial sequence of the Branchiostoma lamin by the N-terminal head and central rod domains. The molecular phylogenetic analysis of the metazoan lamin sequences emphasises the monophyletic nature of the chordates in line with the morphological evidence.

Receptor Isoform-Specific Interaction of Prostaglandin EP3 Receptor with Muskelin

By using the yeast two-hybrid system, muskelin was found to bind with the carboxy-terminal tail of the prostaglandin EP3 receptor α isoform but not with either the β or γ isoform. A direct interaction between the carboxy-terminal tail of the α isoform and muskelin was confirmed in vitro using recombinant fusion proteins. Analysis by confocal microscopy indicated that the isoform and muskelin were distributed at the plasma membrane in transfected cells. When the isoform was stimulated by agonist, the receptor was internalized in the cells expressing the receptor alone, but this internalization was partially inhibited by the cotransfection with muskelin. Furthermore, muskelin enhanced the Gi activity of the isoform. Thus, muskelin appears to be an isoform-specific anchoring protein for the EP3 receptor.

A carboxy-terminal 16-amino-acid region of sigma(38) of Escherichia coli is important for transcription under high-salt conditions and sigma activities in vivo.

sigma(38) (or sigma(S), the rpoS gene product) is a sigma subunit of RNA polymerase in Escherichia coli and directs transcription from a number of stationary-phase promoters as well as osmotically inducible promoters. In this study, we analyzed the function of the carboxy-terminal 16-amino-acid region of sigma(38) (residues 315 to 330), which is well conserved among the rpoS gene products of enteric bacterial species. Truncation of this region was shown to result in the loss of sigma activity in vivo using promoter-lacZ fusion constructs, but the mutant sigma(38) retained the binding activity in vivo to the core enzyme. The in vitro transcription analysis revealed that the transcription activity of sigma(38) holoenzyme under high potassium glutamate concentrations was significantly decreased by the truncation of the carboxy-terminal tail element.

Chloroplast proteases: Possible regulators of gene expression?

A wide range of proteolytic processes in the chloroplast are well recognized. These include processing of precursor proteins, removal of oxidatively damaged proteins, degradation of proteins missing their prosthetic groups or their partner subunit in a protein complex, and adjustment of the quantity of certain chloroplast proteins in response to changing environmental conditions. To date, several chloroplast proteases have been identified and cloned. The chloroplast processing enzyme is responsible for removing the transit peptides of newly imported proteins. The thylakoid processing peptidase removes the thylakoid-transfer domain from proteins translocated into the thylakoid lumen. Within the lumen, Tsp removes the carboxy-terminal tail of the precursor of the PSII D1 protein. In contrast to these processing peptidases which perform a single endo-proteolytic cut, processive proteases that can completely degrade substrate proteins also exist in chloroplasts. The serine ATP-dependent Clp protease, composed of the proteolytic subunit ClpP and the regulatory subunit ClpC, is located in the stroma, and is involved in the degradation of abnormal soluble and membrane-bound proteins. The ATP-dependent metalloprotease FtsH is bound to the thylakoid membrane, facing the stroma. It degrades unassembled proteins and is involved in the degradation of the D1 protein of PSII following photoinhibition. DegP is a serine protease bound to the lumenal side of the thylakoid membrane that might be involved in the chloroplast response to heat. All these peptidases and proteases are homologues of known bacterial enzymes. Since ATP-dependent bacterial proteases and their mitochondrial homologues are also involved in the regulation of gene expression, via their determining the levels of key regulatory proteins, chloroplast proteases are expected to play a similar role.

Assembly of the coronavirus envelope: homotypic interactions between the M proteins.

The viral membrane proteins M and E are the minimal requirements for the budding of coronavirus particles. Since the E protein occurs in particles only in trace amounts, the lateral interactions between the M proteins apparently generate the major driving force for envelope formation. By using coimmunoprecipitation and envelope incorporation assays, we provide extensive evidence for the existence of such M-M interactions. In addition, we determined which domains of the M protein are involved in this homotypic association, using a mutagenetic approach. Mutant M proteins which were not able to assemble into viruslike particles (VLPs) by themselves (C. A. M. de Haan, L. Kuo, P. S. Masters, H. Vennema, and P. J. M. Rottier, J. Virol. 72:6838-6850, 1998) were tested for the ability to associate with other M proteins and to be rescued into VLPs formed by assembly-competent M proteins. We found that M proteins lacking parts of the transmembrane cluster, of the amphipathic domain, or of the hydrophilic carboxy-terminal tail, or M proteins that had their luminal domain replaced by heterologous ectodomains, were still able to associate with assembly-competent M proteins, resulting in their coincorporation into VLPs. Only a mutant M protein in which all three transmembrane domains had been replaced lost this ability. The results indicate that M protein molecules interact with each other through multiple contact sites, particularly at the transmembrane level. Finally, we tested the stringency with which membrane proteins are selected for incorporation into the coronavirus envelope by probing the coassembly of some foreign proteins. The observed efficient exclusion from budding of the vesicular stomatitis virus G protein and the equine arteritis virus M protein indicates that envelope assembly is indeed a highly selective sorting process. The low but detectable incorporation of CD8 molecules, however, demonstrated that this process is not perfect.

Different membrane targeting of prostaglandin EP3 receptor isoforms dependent on their carboxy-terminal tail structures

Mouse prostaglandin EP3 receptor consists of three isoforms, EP3α, β and γ, with different carboxy-terminal tails. To assess the role of their carboxy-terminal tails in membrane targeting, we examined subcellular localization of myc-tagged EP3 isoforms expressed in MDCK cells. Two isoforms, EP3α and EP3β, were localized in the intracellular compartment but not in the plasma membrane, while the EP3γ isoform was found in the lateral plasma membrane and in part in the intracellular compartment. Mutant EP3 receptor lacking the carboxy-terminal tail was localized in the intracellular compartment but not in the plasma membrane. Thus, EP3 isoforms differ in subcellular targeting, and the carboxy-terminal tails play an important role in determination of the membrane targeting of EP3 receptor.

Transmembrane phosphoprotein Cbp regulates the activities of Src-family tyrosine kinases

The Src family of protein tyrosine kinases (Src-PTKs) is important in the regulation of growth and differentiation of eukaryotic cells. The activity of Src-PTKs in cells of different types is negatively controlled by Csk, which specifically phosphorylates a conserved regulatory tyrosine residue at the carboxy-terminal tail of the Src-PTKs. Csk is mainly cytoplasmic and Src-PTKs are predominantly membrane-associated. This raises a question about the mechanism of interaction between these enzymes. Here we present Cbp--a transmembrane phosphoprotein that is ubiquitously expressed and binds specifically to the SH2 domain of Csk. Cbp is involved in the membrane localization of Csk and in the Csk-mediated inhibition of c-Src. In the plasma membrane Cbp is exclusively localized in the GM1 ganglioside-enriched detergent-insoluble membrane domain, which is important in receptor-mediated signalling. These findings reveal Cbp as a new component of the regulatory mechanism controlling the activity of membrane-associated Src-PTKs.

A novel positive feedback loop mediated by the docking protein Gab1 and phosphatidylinositol 3-kinase in epidermal growth factor receptor signaling.

The Gab1 protein is tyrosine phosphorylated in response to various growth factors and serves as a docking protein that recruits a number of downstream signaling proteins, including phosphatidylinositol 3-kinase (PI-3 kinase). To determine the role of Gab1 in signaling via the epidermal growth factor (EGF) receptor (EGFR) we tested the ability of Gab1 to associate with and modulate signaling by this receptor. We show that Gab1 associates with the EGFR in vivo and in vitro via pTyr sites 1068 and 1086 in the carboxy-terminal tail of the receptor and that overexpression of Gab1 potentiates EGF-induced activation of the mitogen-activated protein kinase and Jun kinase signaling pathways. A mutant of Gab1 unable to bind the p85 subunit of PI-3 kinase is defective in potentiating EGFR signaling, confirming a role for PI-3 kinase as a downstream effector of Gab1. Inhibition of PI-3 kinase by a dominant-interfering mutant of p85 or by Wortmannin treatment similarly impairs Gab1-induced enhancement of signaling via the EGFR. The PH domain of Gab1 was shown to bind specifically to phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3], a product of PI-3 kinase, and is required for activation of Gab1-mediated enhancement of EGFR signaling. Moreover, the PH domain mediates Gab1 translocation to the plasma membrane in response to EGF and is required for efficient tyrosine phosphorylation of Gab1 upon EGF stimulation. In addition, overexpression of Gab1 PH domain blocks Gab1 potentiation of EGFR signaling. Finally, expression of the gene for the lipid phosphatase PTEN, which dephosphorylates PtdIns(3,4, 5)P3, inhibits EGF signaling and translocation of Gab1 to the plasma membrane. These results reveal a novel positive feedback loop, modulated by PTEN, in which PI-3 kinase functions as both an upstream regulator and a downstream effector of Gab1 in signaling via the EGFR.

Functional interactions between the extracellular domain and the seven-transmembrane domain in Ca2+ receptor activation.

We studied the activity of mutants involving the aminoterminal extracellular, seven-transmembrane (7TM) and carboxy-terminal tail domains of the human Ca2+ receptor to gain insight into the functional interactions between these domains during receptor activation. Missense mutations of highly conserved residues, D190 and E297, in the extracellular domain (ECD), and a mutation within part of the proximal carboxyterminal tail, A877-880E, resulted in receptors with severely reduced response to Ca2+ despite adequate cell surface expression. Coexpression of either D190A or E297K mutants with A877-880E led to significant reconstitution of function. No such reconstitution occurred when D190A or E297K mutants were coexpressed with a truncation mutant possessing an intact amino-terminal extracellular and first transmembrane domain, despite evidence for heterodimerization and cell surface expression of the respective mutant receptors. In addition, no reconstitution of function was observed when D190A was coexpressed with a deletion Ca2+ receptor mutant lacking only a cysteine-rich region located in the ECD of the Ca2+ receptor (Ca-//-Ca). Moreover, coexpression of this Ca-//-Ca with A877-880E did not recover function. The results show that Ca2+ receptor extracellular and 7TM domains are discrete entities that can communicate within the context of a heterodimer composed of complementary mutant receptors. Two intact 7TM domains and two intact cysteine-rich regions appear to be required for such communication to occur. The results are discussed in the context of a speculative model of receptor structure and function.

Folate pathway gene alterations in patients with neural tube defects.

Periconceptional folate supplementation reduces the recurrence and occurrence risk of neural tube defects (NTD) by as much as 70%, yet the protective mechanism remains unknown. Inborn errors of folate and homocysteine metabolism may be involved in the aetiology of NTDs. Previous studies have demonstrated that both homozygosity for the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene, and combined heterozygosity for the C677T and for another mutation in the same gene, the A1298C polymorphism, represent genetic risk factors for NTDs. In an attempt to identify additional folate related genes that contribute to NTD pathogenesis, we performed molecular genetic analysis of folate receptors (FRs). We identified 4 unrelated patients out of 50 with de novo insertions of pseudogene (PS)-specific mutations in exon 7 and 3'UTR of the FRalpha gene, arising by microconversion events. All of the substitutions affect the carboxy-terminal amino acid membrane tail, or the GPI anchor region of the nascent protein. Furthermore, among 150 control individuals, we also identified one infant with a gene conversion event within the FRalpha coding region. This study, though preliminary, provides the first genetic association between molecular variations of the FRalpha gene and NTDs and suggests that this gene can act as a risk factor for human NTD.

600 ps molecular dynamics reveals stable substructures and flexible hinge points in cAMP dependent protein kinase.

Molecular dynamics simulations of the catalytic subunit of cAMP dependent protein kinase (cAPK) have been performed in an aqueous environment. The relations among the protein hydrogen-bonding network, secondary structural elements, and the internal motions of rigid domains were examined. The values of fluctuations of protein dihedral angles during dynamics show quite distinct maxima in the regions of loops and minima in the regions of alpha-helices and beta-strands. Analyses of conformation snapshots throughout the run show stable subdomains and indicate that these rigid domains are constrained during the dynamics by a stable network of hydrogen bonds. The most stable subdomain during the dynamics was in the small lobe including part of the carboxy-terminal tail. The most significant flexible region was the highly conserved glycine-rich loop between beta strands 1 and 2 in the small lobe. Many of the main chain dihedral angle changes measured in a comparison of the crystallographic structures of "open" and "closed" conformations of cAPK correspond to the highly flexible residues found during dynamics.

Ubiquitin-Related Proteins Regulate Interaction of Vimentin Intermediate Filaments with the Plasma Membrane

Integrin-associated protein (IAP, CD47) is a plasma membrane receptor for thrombospondins and signal regulatory proteins (SIRPs) that has an essential role in host defense through its association with integrins. The IAP gene encodes alternatively spliced carboxy-terminal cytoplasmic tails that have no previously described function. IAP cytoplasmic tails can bind two related proteins that mediate interaction between IAP and vimentin-containing intermediate filaments, named p roteins l inking I AP with c ytoskeleton (PLICs). Integrins interact with PLICs indirectly, through IAP. Transfection of PLICs induces redistribution of vimentin and cell spreading in IAP-expressing cells. This novel connection between plasma membrane and cytoskeleton is likely to be significant in many adhesion-dependent cell functions.

Multiple in vivo tyrosine phosphorylation sites in EphB receptors.

Autophosphorylation regulates the function of receptor tyrosine kinases. To dissect the mechanism by which Eph receptors transmit signals, we have developed an approach using matrix-assisted laser desorption-ionization (MALDI) mass spectrometry to map systematically their in vivo tyrosine phosphorylation sites. With this approach, phosphorylated peptides from receptors digested with various endoproteinases were selectively isolated on immobilized anti-phosphotyrosine antibodies and analyzed directly by MALDI mass spectrometry. Multiple in vivo tyrosine phosphorylation sites were identified in the juxtamembrane region, kinase domain, and carboxy-terminal tail of EphB2 and EphB5, and found to be remarkably conserved between these EphB receptors. A number of these sites were also identified as in vitro autophosphorylation sites of EphB5 by phosphopeptide mapping using two-dimensional chromatography. Only two in vitro tyrosine phosphorylation sites had previously been directly identified for Eph receptors. Our data further indicate that in vivo EphB2 and EphB5 are also extensively phosphorylated on serine and threonine residues. Because phosphorylation at each site can affect receptor signaling properties, the multiple phosphorylation sites identified here for the EphB receptors suggest a complex regulation of their functions, presumably achieved by autophosphorylation as well as phosphorylation by other kinases. In addition, we show that MALDI mass spectrometry can be used to determine the binding sites for Src homology 2 (SH2) domains by identifying the EphB2 phosphopeptides that bind to the SH2 domain of the Src kinase.

A head-activator binding protein is present in hydra in a soluble and a membrane-anchored form.

The neuropeptide head activator plays an important role for proliferation and determination of stem cells in hydra. By affinity chromatography a 200 kDa head-activator binding protein, HAB, was isolated from the multiheaded mutant of Chlorohydra viridissima. Partial amino acid sequences were used to clone the HAB cDNA which coded for a receptor with a unique alignment of extracellular modules, a transmembrane domain, and a short carboxy-terminal cytoplasmic tail. A mammalian HAB homologue with identical alignment of these modules is expressed early in brain development. Specific antibodies revealed the presence of HAB in hydra as a transmembrane receptor, but also as secreted protein, both capable of binding head activator. Secretion of HAB during regeneration and expression in regions of high determination potential hint at a role for HAB in regulating the concentration and range of action of head activator.

Mapping of the coronavirus membrane protein domains involved in interaction with the spike protein.

The coronavirus membrane (M) protein is the key player in virion assembly. One of its functions is to mediate the incorporation of the spikes into the viral envelope. Heterotypic interactions between M and the spike (S) protein can be demonstrated by coimmunoprecipitation and by immunofluorescence colocalization, after coexpression of their genes in eukaryotic cells. Using these assays in a mutagenetic approach, we have mapped the domains in the M protein that are involved in complex formation between M and S. It appeared that the 25-residue luminally exposed amino-terminal domain of the M protein is not important for M-S interaction. A 15-residue deletion, the insertion of a His tag, and replacement of the ectodomain by that of another coronavirus M protein did not affect the ability of the M protein to associate with the S protein. However, complex formation was sensitive to changes in the transmembrane domains of this triple-spanning protein. Deletion of either the first two or the last two transmembrane domains, known not to affect the topology of the protein, led to a considerable decrease in complex formation, but association was not completely abrogated. Various effects of changes in the part of the M protein that is located at the cytoplasmic face of the membrane were observed. Deletions of the extreme carboxy-terminal tail appeared not to interfere with M-S complex formation. However, deletions in the amphipathic domain severely affected M-S interaction. Interestingly, changes in the amino-terminal and extreme carboxy-terminal domains of M, which did not disrupt the interaction with S, are known to be fatal to the ability of the protein to engage in virus particle formation (C. A. M. de Haan, L. Kuo, P. S. Masters, H. Vennema, and P. J. M. Rottier, J. Virol. 72:6838-6850, 1998). Apparently, the structural requirements of the M protein for virus particle assembly differ from the requirements for the formation of M-S complexes.

Ubiquitin-dependent degradation of active Src

Signaling by members of the Src family of protein tyrosine kinases, such as Src and Fyn, is important in many biological responses, including gene transcription, cell-cycle progression, and cell adhesion and spreading [1,2]. Unregulated Src kinase activity has been implicated in the progression of colon cancer and transformation of cultured cells [3–6]. Thus, precise regulation of Src activity is critical for normal cell growth. Src kinase activity is downregulated by the carboxy-terminal Src kinase (Csk), a tyrosine kinase that phosphorylates a conserved tyrosine residue in the carboxy-terminal tail of Src [7,8]. When phosphorylated, this tyrosine residue mediates an intramolecular interaction that results in a ‘closed’ or inactive conformation [1,2,9,10]. Here, we report that loss of csk resulted in a reduction in the abundance of the Src and Fyn proteins, which could be restored by reintroducing catalytically active Csk. The effect of Csk on Src expression was not due to an increase in Src message, but to stabilization of the Src protein. Inhibition of proteasome activity also increased the level of Src protein in csk-deficient cells. Src was found to be ubiquitinated, and activation of Src increased the extent of polyubiquitination. Thus, ubiquitin–proteasome-dependent degradation represents an additional mechanism by which active Src can be downregulated.

Single-molecule analysis of kinesin motility reveals regulation by the cargo-binding tail domain.

Conventional kinesin transports membranes along microtubules in vivo, but the majority of cellular kinesin is unattached to cargo. The motility of non-cargo-bound, soluble kinesin may be repressed by an interaction between the amino-terminal motor and carboxy-terminal cargo-binding tail domains, but neither bead nor microtubule-gliding assays have shown such inhibition. Here we use a single-molecule assay that measures the motility of kinesin unattached to a surface. We show that full-length kinesin binds microtubules and moves about ten times less frequently and exhibits discontinuous motion compared with a truncated kinesin lacking a tail. Mutation of either the stalk hinge or neck coiled-coil domain activates motility of full-length kinesin, indicating that these regions are important for tail-mediated repression. Our results suggest that the motility of soluble kinesin in the cell is inhibited and that the motor becomes activated by cargo binding.

Pseudohypoparathyroidism: from bedside to bench and back.

Pseudohypoparathyroidism: from bedside to bench and back.