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PubMedDate not supplied

Development of a 3-D artificial extracellular matrix. Design concept and artificial vascular media.

A photodimerizable monomer was synthesized and subsequently co-polymerized with a hydrophilic monomer to obtain a photocrosslinkable, water soluble polymer. This polymer attained a highly hydrated, gel-like phase on irradiation with ultraviolet (UV) light. Furthermore, incorporation of the well known cell adhesive ligand Arg-Gly-Asp-Ser (RGDS) peptidyl moiety into the above co-polymer resulted, on UV light irradiation, in a three-dimensional hydrophilic gel matrix containing cell adhesive ligands. The UV irradiation on a mixture of photocrosslinkable and cell adhesive polymer and smooth muscle cells in a medium trapped the cells throughout the gel matrix, indicating that the cell-entrapped gel and the designed synthetic polymer biomimic the media of the vascular wall and extracellular matrix, respectively.

Cell Differentiation↗

PubMed1999

Divalent cation interactions with light-dependent K channels. Kinetics of voltage-dependent block and requirement for an open pore.

The light-dependent K conductance of hyperpolarizing Pecten photoreceptors exhibits a pronounced outward rectification that is eliminated by removal of extracellular divalent cations. The voltage-dependent block by Ca(2+) and Mg(2+) that underlies such nonlinearity was investigated. Both divalents reduce the photocurrent amplitude, the potency being significantly higher for Ca(2+) than Mg(2+) (K(1/2) approximately 16 and 61 mM, respectively, at V(m) = -30 mV). Neither cation is measurably permeant. Manipulating the concentration of permeant K ions affects the blockade, suggesting that the mechanism entails occlusion of the permeation pathway. The voltage dependency of Ca(2+) block is consistent with a single binding site located at an electrical distance of delta approximately 0.6 from the outside. Resolution of light-dependent single-channel currents under physiological conditions indicates that blockade must be slow, which prompted the use of perturbation/relaxation methods to analyze its kinetics. Voltage steps during illumination produce a distinct relaxation in the photocurrent (tau = 5-20 ms) that disappears on removal of Ca(2+) and Mg(2+) and thus reflects enhancement or relief of blockade, depending on the polarity of the stimulus. The equilibration kinetics are significantly faster with Ca(2+) than with Mg(2+), suggesting that the process is dominated by the "on" rate, perhaps because of a step requiring dehydration of the blocking ion to access the binding site. Complementary strategies were adopted to investigate the interaction between blockade and channel gating: the photocurrent decay accelerates with hyperpolarization, but the effect requires extracellular divalents. Moreover, conditioning voltage steps terminated immediately before light stimulation failed to affect the photocurrent. These observations suggest that equilibration of block at different voltages requires an open pore. Inducing channels to close during a conditioning hyperpolarization resulted in a slight delay in the rising phase of a subsequent light response; this effect can be interpreted as closure of the channel with a divalent ion trapped inside.

Adaptation, Ocular↗

PubMed2004

Fast and slow voltage-dependent dynamics of magnesium block in the NMDA receptor: the asymmetric trapping block model.

The NMDA receptor (NMDAR) produces a long-lasting component of the glutamatergic EPSC in mammalian central neurons. The current through NMDARs is voltage dependent as a result of block by extracellular magnesium, which has recently been shown to give rise to a complex time dependence, with fast and slow components of responses to changes in membrane potential. Here, we studied the dynamics of block and unblock by measuring voltage step responses in conjunction with fast perfusion of agonist in nucleated patches isolated from rat cortical pyramidal neurons. We found that slow unblock shows a progressive onset during synaptic-like responses to brief pulses of agonist. Repolarizing briefly from +40 to -70 mV revealed that slow unblock is reestablished with a time constant of approximately 5 msec at room temperature. Also, the time course of deactivation, in response to a pulse of agonist, slows twofold over the potential range -30 to +40 mV. An asymmetric "trapping block" model in which the voltage-independent closing rate constant of the blocked channel is approximately three times that of the unblocked channel accounts quantitatively for all of these phenomena and for responses to action potential waveform clamp. This model allows much more accurate prediction of NMDAR current in physiological conditions of magnesium concentration and changing membrane potential than previously possible. It suggests a positive allosteric link between occupation of the NMDAR pore by magnesium and closure of the permeation gate.

Animals↗

PubMed1995

Molecular cloning of a gene from Plasmodium falciparum that codes for a protein sharing motifs found in adhesive molecules from mammals and plasmodia.

Adhesion of Plasmodium to host cells is an important phenomenon in parasite invasion and in malaria-associated pathology. We report here the molecular cloning of a putative adhesive molecule from P. falciparum that shares both sequence and structural similarities with a sporozoite surface molecule from Plasmodium termed the thrombospondin-related anonymous protein (TRAP) and, to a lesser extent, with the circumsporozoite (CS) protein. The gene, which is present on chromosome 3 as a single copy, was termed CTRP for CS protein-TRAP-related protein. The full-length CTRP encodes a protein containing a putative signal sequence followed by a long extracellular region of 1990 amino acids, a transmembrane domain, and a short cytoplasmic segment. The putative extracellular region of CTRP is defined by two separated adhesive domains. The first domain contains six 210-amino acid-long homologous repeats, the sequence of which is related to the A-type domain found in adhesive molecules including the alpha subunits of several integrins and a number of extracellular matrix glycoproteins. The second domain contains seven repeats of 87-60 amino acids in length, which share similarities with the thrombospondin type 1 domain found in a variety of adhesive molecules. Finally, CTRP also contains consensus motifs found in the superfamily of haematopoietin receptors. Interstrain analysis of eight different parasite isolates revealed that CTRP does not show size polymorphism except in repetitive regions flanking potential adhesive domains.

Amino Acid Sequence↗

PubMed2004

Fiber assembly by the chaperone-usher pathway.

Bacterial pathogens utilize the chaperone-usher pathway to assemble extracellular multi-subunit fibers essential for virulence. The periplasmic chaperone facilitates the initial folding of fiber subunits but then traps them in activated folding transition states. Chaperone dissociation releases the folding energy that drives subunit incorporation into the fiber, which grows through a pore formed by the outer-membrane usher.

Bacterial Proteins↗

PubMed2001

Characteristics and mechanisms of azithromycin accumulation and efflux in human polymorphonuclear leukocytes.

Azithromycin achieves prolonged, high tissue concentrations in spite of low serum levels and obviously must be active at tissue sites of infection to be effective. These unique features prompted us to evaluate the interactions of azithromycin and human polymorphonuclear leukocytes (PMN). Uptake of radiolabeled antibiotic by PMN was determined by a velocity-gradient centrifugation technique and expressed as the ratio of cellular to extracellular drug concentration (C/E). Azithromycin was massively accumulated by human PMN (C/E=387.2 at 2 h). Uptake was not influenced by inhibitors of cellular metabolism, but phagocytosis slightly inhibited the entry of azithromycin into PMN. After removal of extracellular drug, the release (efflux) of azithromycin from PMN was extremely slow. Agents which neutralize lysosomal pH, preventing protonation and trapping of azithromycin, markedly increased antibiotic efflux. Active concentration and prolonged retention of azithromycin by phagocytic cells should allow delivery and subsequent release of accumulated drug at sites of infection.

Anti-Bacterial Agents↗

PubMed2000

The odontoclasts of Ambystoma mexicanum.

The resorption of teeth in Ambystoma mexicanum during postembryonal ontogenesis and induced metamorphosis occurs by means of light-microscopic detectable giant-cells. These have morphological and functional characters similar to those of odontoclasts of other vertebrates. The multinucleated odontoclasts resorb not only the pedicel (base), but the stalk of the tooth, too. When active, the cells form a ruffled border and a sealing zone. In this way they are able to demineralize the hard tissues of teeth (dentin and mineral of the pedicel) and to dissolve the extracellular matrix. Resorption of enamel has not been observed. Marks of resorption resemble the Howship's lacunae of other tetrapods. TRAP as a typical enzyme of odontoclasts could not be detected histochemically. Dependence of PTH, which is supposed to be necessary for the formation and activation of odontoclasts as well as of thyroxine can be excluded, although the resorbing cells are functionally and cytologically identical with those of other vertebrates. This demands some other mechanism for the formation and regulation of the odontoclasts in A. mexicanum.

Ambystoma mexicanum↗

PubMed1998

Thrombin promotes platelet-mediated melanoma cell adhesion to endothelial cells under flow conditions: role of platelet glycoproteins P-selectin and GPIIb-IIIA.

We investigated the role of platelets in human melanoma cell (line 397) interaction with vascular endothelial cells (ECs) under flow conditions. The ability of the tumour cells to adhere to the EC monolayer was significantly reduced by application of flow at a shear rate of 250 s(-1). A 2.2-fold increase in tumour cell adhesion to ECs under flow was observed upon addition of thrombin receptor agonist peptide (TRAP)-activated platelets but not resting platelets. A similar increase (2.5-fold) in tumour cell adhesion to ECs under flow was observed when the tumour cells were incubated with resting platelets on thrombin-treated ECs. However, thrombin treatment of the ECs alone had no effect on tumour cell adhesion in the absence of platelets. The enhancement of tumour cell adhesion to ECs by TRAP-activated platelets was virtually abolished by blockade of the platelet glycoproteins P-selectin and GPIIb-IIIa by monoclonal antibodies. Blockade of P-selectin also inhibited the direct adhesion of TRAP-activated platelets to ECs, but did not affect the interaction of the tumour cells with platelets immobilized on subendothelial extracellular matrix (ECM). Blockade of GPIIb-IIIa inhibited both platelet-EC and platelet-tumor cell interactions. Our results indicate that tumour cell adhesion to the endothelium under flow is enhanced by platelets under conditions that allow platelet adhesion to ECs. Inhibition studies suggest that activated platelet adhesion to ECs is mediated by P-selectin and GPIIb-IIIA, and tumour cell adhesion to EC-bound platelets--mainly by GPIIb-IIIa.

Animals↗

PubMed1998

Effect of transverse-tubular chloride conductance on excitability in skinned skeletal muscle fibres of rat and toad.

1. The influence of the transverse-tubular (T-) system Cl- conductance on membrane excitability in skeletal muscle fibres of toad and rat was examined because of conflicting conclusions of previous studies on Cl- conductance. A mechanically skinned fibre preparation was used that permitted investigation of Ca2+ release via the normal T-system voltage-sensor mechanism after complete removal of the surface membrane, which thereby allowed estimation of the T-system potential from force measurements. 2. When a skinned fibre was bathed in a high-[K+] solution, the sealed T-system became polarized and could be rapidly depolarized by replacing the K+ with Na+, thereby eliciting Ca2+ release from the sarcoplasmic reticulum. In rat skinned fibres, addition of 20 mM Cl- to the 'myoplasm' (i.e. bathing solution) partially depolarized the T-system, inducing Ca2+ release and subsequent voltage-sensor inactivation. These effects were completely abolished with 100 microM of the Cl- channel blocker 9-anthracene carboxylic acid (9-AC). Voltage-sensor inactivation increased in a graded manner over the range 3-20 mM myoplasmic Cl-. 3. In toad fibres, voltage-sensor inactivation was only detectable at > 10 mM myoplasmic Cl-, and 20 mM Cl- was only able to depolarize the T-system sufficiently to trigger Ca2+ release if the myoplasmic [K+] was reduced by 50 %. In toad fibres, 100 microM 9-AC caused little if any block of the T-system Cl- conductance. 4. It was also found that when skinned fibres were obtained from muscles that had been bathed in a zero Cl- extracellular solution, the initial Na+ substitutions were more effective at depolarizing the T-system. This is consistent with Cl- trapped in the sealed T-system exerting a polarizing effect on T-system potential. 5. These results unequivocally demonstrate that there is a large 9-AC-sensitive Cl- conductance in the T-system of rat fibres, and a smaller, though still appreciable, Cl- conductance in the T-system of toad fibres, which is relatively insensitive to 9-AC. The results are important for understanding the basis of the Cl- channel aberration in myotonia.

Animals↗

PubMed1994

Arsenate arrests flagellar rotation in cytoplasm-free envelopes of bacteria.

The effect of arsenate on flagellar rotation in cytoplasm-free flagellated envelopes of Escherichia coli and Salmonella typhimurium was investigated. Flagellar rotation ceased as soon as the envelopes were exposed to arsenate. Inclusion of phosphate intracellularly (but not extracellular) prevented the inhibition by arsenate. In a parallel experiment, the rotation was not affected by inclusion of an ATP trap (hexokinase and glucose) within the envelopes. It is concluded that arsenate affects the motor in a way other than reversible deenergization. This may be an irreversible damage to the cell or direct inhibition of the motor by arsenate. The latter possibility suggests that a process of phosphorylation or phosphate binding is involved in the motor function.

Adenosine Triphosphate↗

PubMed2005

Parathyroid-hormone-related protein induces expression of receptor activator of NF-{kappa}B ligand in human periodontal ligament cells via a cAMP/protein kinase A-independent pathway.

UNLABELLED: Periodontal ligament (PDL) cells play important roles in root resorption of human deciduous teeth by odontoclasts (osteoclast-like cells). However, it is unclear how PDL cells regulate osteoclastogenesis. We examined the effects of PTHrP, TGF-beta, and EGF, which are all secreted by the tooth germ, on tartrate-resistant acid-phosphatase-positive (TRAP+) cell formation using co-cultures of human PDL cells and mouse spleen cells. Only PTHrP promoted TRAP+ cell formation in co-cultures. PTHrP induced receptor activator of NF-kappaB ligand (RANKL) mRNA expression and slightly reduced osteoprotegerin (OPG) expression in PDL cells. The cAMP/PKA inhibitors Rp-cAMP, H89, and PKI did not affect PTHrP-induced TRAP+ cell formation. The PKC inhibitor, Ro-32-0432, suppressed RANKL expression in PDL cells and PTHrP-induced TRAP+ cell formation. However, this inhibitor directly modulated the number of osteoclast precursors. Thus, PTHrP induces osteoclastogenesis by increasing the relative expression level of RANKL vs. OPG in PDL cells via a cAMP/PKA-independent pathway. ABBREVIATIONS: PTHrP, parathyroid-hormone-related protein; TGF-beta, transforming growth factor-beta; EGF, epidermal growth factor; RANKL, receptor activator of NF-kappaB ligand; OPG, osteoprotegerin; PDL, periodontal ligament; TRAP, tartrate-resistant acid phosphatase; PKA, protein kinase A; PKC, protein kinase C; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; cAMP, cyclic Adenosine 3'5'-Monophosphate.

Acid Phosphatase↗

PubMedDate not supplied

Drug-induced transmembrane lipid scrambling in erythrocytes and in liposomes requires the presence of polyanionic phospholipids.

The asymmetric transmembrane distribution of phospholipids between the two bilayer halves of erythrocyte can be modified upon addition of cationic amphiphilic drugs, such as chlorpromazine or verapamil. We studied this phenomenon in erythrocytes and in lipid vesicles using spin-labelled analogues of the endogenous phospholipids. The extent of the rapid disappearance of the analogues from the erythrocyte outer leaflet depended on the concentration of the drug. Up to 40% of spin-labelled sphingomyelin moved to the inner erythrocyte leaflet in 10 min in the presence of 1.5 mm chlorpromazine. Verapamil or vinblastine gave similar results. On the other hand, the inside-outside movement of the aminophospholipid analogues was less evident, and did not exceed 10%. This apparent discrepancy between inward and outward movements could result from the formation of an endovesicle which is known to occur upon drug addition at high concentration. A fraction of lipids would be trapped in the intravesicular leaflet, corresponding to the cell outer leaflet, and be inaccessible both from the cytoplasm and the extracellular medium. In cells submitted to a metabolic depletion of cellular ATP the intensity of the scrambling induced by the amphipaths was drastically lowered. We attribute this effect to the important reduction of the membrane content in phosphatidylinositol-4,5-bisphosphate (PIP2). The involvement of the latter lipid in triggering scrambling was partly confirmed by experiments carried out with artificial membranes. Indeed, in large unilamellar vesicles PIP2 is required in order to obtain a rapid redistribution of phospholipids between the two leaflets upon addition of drugs. However, the extent of phospholipid redistribution was limited to 15-20%. This redistribution was also induced when the vesicle membrane contained di-anionic phospholipids (phosphatidylinositol-4-monophosphate or diphosphatidylglycerol), but did not occur when it contained mono-anionic phospholipids (phosphatidylserine or phosphatidylinositol). Some drugs such as methochlorpromazine, active in artificial membranes, were ineffective in erythrocyte membranes, probably because they could not cross the membrane and reach PIP2 molecules at the cytoplasmic leaflet.

Adenosine Triphosphate↗

PubMed2002

[Mechanisms of azithromycin accumulation and efflux in human polymorphonuclear leukocytes].

Azithromycin achieves prolonged, high tissue concentrations in spite of low serum levels and obviously must be effective at tissue sites of infection. These unique features prompted us to evaluate the interactions of azithromycin and human polymorphonuclear leukocytes (PMN). Uptake of radiolabeled antibiotic by PMN was determined by a velocity-gradient centrifugation technique and expressed as the ratio of cellular to extracellular drug concentration (C/E). Azithromycin was massively accumulated by human PMN (C/E = 387.2 at 2 h). Uptake was not influenced by inhibitors of cellular metabolism, but phagocytosis slightly inhibited the entry of azithromycin into PMN. After removal of extracellular drug, the release (efflux) of azithromycin from PMN was extremely slow. Agents which neutralize lysosomal pH, preventing protonation and trapping of azithromycin, markedly increased antibiotic efflux. Active concentration and prolonged retention of azithromycin by phagocytic cells should allow delivery and subsequent release of accumulated drug at sites of infection.

Adenosine↗

PubMed1994

Net uptake of orthophosphate in Ehrlich ascites tumor cells in the presence of purine riboside may be rate limiting for the expansion of the pool of ribonucleotides.

Incubation with adenosine or with structural analogs thereof may in several cell types under some conditions result in the cellular accumulation of abundant amounts of the corresponding triphosphates. In the present work we have found that incubation of cells at high concentrations of orthophosphate (Pi) results in increased intracellular levels thereof, although they become not as high as the extracellular concentration. In the presence of purine riboside (nebularine, Pr) and high concentration of Pi the intracellular Pi is, however, kept at a low steady-state level, probably because it immediately upon uptake is being trapped primarily as the triphosphate of purine riboside. The latter compound accumulates at a constant rate for at least 1 h. The rate of accumulation of the sum of phosphate residues present in Pi, adenosine phosphates and purine riboside phosphates appears to be proportional to the extracellular concentration of Pi and to be highly dependent on pH (6.5 and 7.0 being optimal and 7.9 nonpermissible) but it is unaffected by substitution of Na+ by choline.

Animals↗

PubMed2000

Atomic resolution structures of bacteriorhodopsin photocycle intermediates: the role of discrete water molecules in the function of this light-driven ion pump.

High-resolution X-ray crystallographic studies of bacteriorhodopsin have tremendously advanced our understanding of this light-driven ion pump during the last 2 years, and emphasized the crucial role of discrete internal water molecules in the pump cycle. In the extracellular region an extensive three-dimensional hydrogen-bonded network of protein residues and seven water molecules leads from the buried retinal Schiff base via water 402 and the initial proton acceptor Asp85 to the membrane surface. Near Lys216 where the retinal binds, transmembrane helix G contains a pi-bulge that causes a non-proline kink. The bulge is stabilized by hydrogen bonding of the main chain carbonyl groups of Ala215 and Lys216 with two buried water molecules located in the otherwise very hydrophobic region between the Schiff base and the proton donor Asp96 in the cytoplasmic region. The M intermediate trapped in the D96N mutant corresponds to a late M state in the transport cycle, after protonation of Asp85 and release of a proton to the extracellular membrane surface, but before reprotonation of the deprotonated retinal Schiff base. The M intermediate from the E204Q mutant corresponds to an earlier M, as in this mutant the Schiff base deprotonates without proton release. The structures of these two M states reveal progressive displacements of the retinal, main chain and side chains induced by photoisomerization of the retinal to 13-cis,15-anti, and an extensive rearrangement of the three-dimensional network of hydrogen-bonded residues and bound water that accounts for the changed pK(a)s of the Schiff base, Asp85, the proton release group and Asp96. The structure for the M state from E204Q suggests, moreover, that relaxation of the steric conflicts of the distorted 13-cis,15-anti retinal plays a critical role in the reprotonation of the Schiff base by Asp96. Two additional waters now connect Asp96 to the carbonyl of residue 216, in what appears to be the beginning of a hydrogen-bonded chain that would later extend to the retinal Schiff base. Based on the ground state and M intermediate structures, models of the molecular events in the early part of the photocycle are presented, including a novel model which proposes that bacteriorhodopsin pumps hydroxide (OH(-)) ions from the extracellular to the cytoplasmic side.

Bacteriorhodopsins↗

PubMed2005

Spontaneous thermal motion of the GABA(A) receptor M2 channel-lining segments.

The gamma-aminobutyric acid type A (GABA(A)) receptor channel opening involves translational and rotational motions of the five channel-lining, M2 transmembrane segments. The M2 segment's extracellular half is loosely packed and undergoes significant thermal motion. To characterize the extent of the M2 segment's motion, we used disulfide trapping experiments between pairs of engineered cysteines. In alpha1beta1 gamma2S receptors the single gamma subunit is flanked by an alpha and beta subunit. The gamma2 M2-14' position is located in the alpha-gamma subunit interface. Gamma2 13' faces the channel lumen. We expressed either the gamma2 14' or the gamma2 13' cysteine substitution mutants with alpha1 cysteine substitution mutants between 12' and 16' and wild-type beta1. Disulfide bonds formed spontaneously between gamma2 14'C and both alpha1 15'C and alpha1 16'C and also between gamma2 13'C and alpha1 13'C. Oxidation by copper phenanthroline induced disulfide bond formation between gamma2 14'C and alpha1 13'C. Disulfide bond formation rates with gamma2 14'C were similar in the presence and absence of GABA, although the rate with alpha1 13'C was slower than with the other two positions. In a homology model based on the acetylcholine receptor structure, alphaM2 would need to rotate in opposite directions by approximately 80 degrees to bring alpha1 13' and alpha1 15' into close proximity with gamma2 14'. Alternatively, translational motion of alphaM2 would reduce the extent of rotational motion necessary to bring these two alpha subunit residues into close proximity with the gamma2 14' position. These experiments demonstrate that in the closed state the M2 segments undergo continuous spontaneous motion in the region near the extracellular end of the channel gate. Opening the gate may involve similar but concerted motions of the M2 segments.

Animals↗

PubMed2003

Biomolecular cytokine therapy.

As for the chronicity of inflammatory-immune diseases, the medication of them needs to be longterm and thus, quite safe with respect to side effects due to drug actions. Therapy of these diseases includes steroid and non steroid anti-inflammatories given in monotherapy or in combination with cytotoxic antimetabolites. Longterm administration of these active substances cumulate in side effects, not to speak of the probability of developing unresponsiveness to the drug in use. In principle, the earlier the intervention, the better the outcome of medication in therapy. In harmony with this principle, biopharmacology focuses on specific targets in early (acute) phase of inflammatory-immune diseases. One of these targets is the proinflammatory cascade of cytokines (IL1beta, IL6, IL8, IL12, TNFalpha). Among them, the overproduction of tumor necrosis factor (TNFalpha) is suggested to orchestrate and escalate the disease phenotype. Hence, targeting of TNFa may restrict or stop the propagation of pathological reactions. TNFalpha in its excess can be captured at transcription, translation, secretion levels as well as in the extracellular soluble form. This latter approach is supported by clinical records emphasizing the use of recombinant antibodies and soluble receptors in trapping extra amounts of TNFalpha. This review serves as an illustration for the efficacy and safety of infliximab (antibody) and etanercept (soluble receptor) in the example of rheumatoid arthritis (RA).

Antibodies, Monoclonal↗

PubMed1999

Extracellular regulated kinases (ERK) 1 and ERK2 are authentic substrates for the dual-specificity protein-tyrosine phosphatase VHR. A novel role in down-regulating the ERK pathway.

The mammalian dual-specificity protein-tyrosine phosphatase VHR (for VH1-related) has been identified as a novel regulator of extracellular regulated kinases (ERKs). To identify potential cellular substrates of VHR, covalently immobilized mutant VHR protein was employed as an affinity trap. A tyrosine-phosphorylated protein(s) of approximately 42 kDa was specifically adsorbed by the affinity column and identified as ERK1 and ERK2. Subsequent kinetic analyses and transfection studies demonstrated that VHR specifically dephosphorylates and inactivates ERK1 and ERK2 in vitro and in vivo. Only the native structure of phosphorylated ERK was recognized by VHR and was inactivated with a second-order rate constant of 40,000 M-1 s-1. VHR was found to dephosphorylate endogenous ERK, but not p38 and JNK. Immunodepletion of endogenous VHR eliminated the dephosphorylation of cellular ERK. Transfection studies in COS-1 cells demonstrated that in vivo phosphorylation of epidermal growth factor-stimulated ERK depended on VHR protein levels. Overexpression above endogenous levels of VHR led to accelerated ERK inactivation, but did not alter the normal activation of ERK. Unique among reported mitogen activated protein kinase phosphatases, VHR is constitutively expressed, localized to the nucleus, and tyrosine-specific. This study is the first to report the identification of authentic substrates of dual-specificity phosphatases utilizing affinity absorbents and is the first to identify a nuclear, constitutively expressed, and tyrosine-specific ERK phosphatase. The data strongly suggest that VHR is responsible for the rapid inactivation of ERK following stimulation and for its repression in quiescent cells.

Animals↗