Intracellular Proton-Transfer Mutants in a CLC Cl?/H+ Exchanger

CLC-ec1, a bacterial homologue of the CLC family’s transporter subclass, catalyzes transmembrane exchange of Cl⁻ and H⁺. Mutational analysis based on the known structure reveals several key residues required for coupling H⁺ to the stoichiometric countermovement of Cl⁻. E148 (Glu_ex) transfers protons between extracellular water and the protein interior, and E203 (Glu_in) is thought to function analogously on the intracellular face of the protein. Mutation of either residue eliminates H⁺ transport while preserving Cl⁻ transport. We tested the role of Glu_in by examining structural and functional properties of mutants at this position. Certain dissociable side chains (E, D, H, K, R, but not C and Y) retain H⁺/Cl⁻ exchanger activity to varying degrees, while other mutations (V, I, or C) abolish H⁺ coupling and severely inhibit Cl⁻ flux. Transporters substituted with other nonprotonatable side chains (Q, S, and A) show highly impaired H⁺ transport with substantial Cl⁻ transport. Influence on H⁺ transport of side chain length and acidity was assessed using a single-cysteine mutant to introduce non-natural side chains. Crystal structures of both coupled (E203H) and uncoupled (E203V) mutants are similar to wild type. The results support the idea that Glu_in is the internal proton-transfer residue that delivers protons from intracellular solution to the protein interior, where they couple to Cl⁻ movements to bring about Cl⁻/H⁺ exchange.     

A missense mutation accelerating the gating of the lysosomal Cl?/H+-exchanger ClC-7/Ostm1 causes osteopetrosis with gingival hamartomas in cattle

Chloride-proton exchange by the lysosomal anion transporter ClC-7/Ostm1 is of pivotal importance for the physiology of lysosomes and bone resorption. Mice lacking either ClC-7 or Ostm1 develop a lysosomal storage disease and mutations in either protein have been found to underlie osteopetrosis in mice and humans. Some human disease-causing CLCN7 mutations accelerate the usually slow voltage-dependent gating of ClC-7/Ostm1. However, it has remained unclear whether the fastened kinetics is indeed causative for the disease. Here we identified and characterized a new deleterious ClC-7 mutation in Belgian Blue cattle with a severe symptomatology including perinatal lethality and in most cases gingival hamartomas. By autozygosity mapping and genome-wide sequencing we found a handful of candidate variants, including a cluster of three private SNPs causing the substitution of a conserved tyrosine in the CBS2 domain of ClC-7 by glutamine. The case for ClC-7 was strengthened by subsequent examination of affected calves that revealed severe osteopetrosis. The Y750Q mutation largely preserved the lysosomal localization and assembly of ClC-7/Ostm1, but drastically accelerated its activation by membrane depolarization. These data provide first evidence that accelerated ClC-7/Ostm1 gating per se is deleterious, highlighting a physiological importance of the slow voltage-activation of ClC-7/Ostm1 in lysosomal function and bone resorption.KEY WORDS: CLCN7, Hamartomas, Osteopetrosis, Lysosomal storage, Ion homeostasis, Belgian Blue cattle     

Proton block of the CLC-5 Cl?/H+ exchanger

CLC-5 is a H⁺/Cl⁻ exchanger that is expressed primarily in endosomes but can traffic to the plasma membrane in overexpression systems. Mutations altering the expression or function of CLC-5 lead to Dent’s disease. Currents mediated by this transporter show extreme outward rectification and are inhibited by acidic extracellular pH. The mechanistic origins of both phenomena are currently not well understood. It has been proposed that rectification arises from the voltage dependence of a H⁺ transport step, and that inhibition of CLC-5 currents by low extracellular pH is a result of a reduction in the driving force for exchange caused by a pH gradient. We show here that the pH dependence of CLC-5 currents arises from H⁺ binding to a single site located halfway through the transmembrane electric field and driving the transport cycle in a less permissive direction, rather than a reduction in the driving force. We propose that protons bind to the extracellular gating glutamate E211 in CLC-5. It has been shown that CLC-5 becomes severely uncoupled when SCN⁻ is the main charge carrier: H⁺ transport is drastically reduced while the rate of anion movement is increased. We found that in these conditions, rectification and pH dependence are unaltered. This implies that H⁺ translocation is not the main cause of rectification. We propose a simple transport cycle model that qualitatively accounts for these findings.     

Multiple Roles of the SO42?/Cl?/OH? Exchanger Protein Slc26a2 in Chondrocyte Functions

Mutations in the SO₄²⁻/Cl⁻/OH⁻ exchanger Slc26a2 cause the disease diastrophic dysplasia (DTD), resulting in aberrant bone development and, therefore, skeletal deformities. DTD is commonly attributed to a lack of chondrocyte SO₄²⁻ uptake and proteoglycan sulfation. However, the skeletal phenotype of patients with DTD is typified by reduction in cartilage and osteoporosis of the long bones. Chondrocytes of patients with DTD are irregular in size and have a reduced capacity for proliferation and terminal differentiation. This raises the possibility of additional roles for Slc26a2 in chondrocyte function. Here, we examined the roles of Slc26a2 in chondrocyte biology using two distinct systems: mouse progenitor mesenchymal cells differentiated to chondrocytes and freshly isolated mouse articular chondrocytes differentiated into hypertrophic chondrocytes. Slc26a2 expression was manipulated acutely by delivery of Slc26a2 or shSlc26a2 with lentiviral vectors. We demonstrate that slc26a2 is essential for chondrocyte proliferation and differentiation and for proteoglycan synthesis. Slc26a2 also regulates the terminal stage of chondrocyte cell size expansion. These findings reveal multiple roles for Slc26a2 in chondrocyte biology and emphasize the importance of Slc26a2-mediated protein sulfation in cell signaling, which may account for the complex phenotype of DTD.     

Na/H Exchanger Regulatory Factors Control Parathyroid Hormone Receptor Signaling by Facilitating Differential Activation of Gα Protein Subunits

The Na/H exchanger regulatory factors, NHERF1 and NHERF2, are adapter proteins involved in targeting and assembly of protein complexes. The parathyroid hormone receptor (PTHR) interacts with both NHERF1 and NHERF2. The NHERF proteins toggle PTHR signaling from predominantly activation of adenylyl cyclase in the absence of NHERF to principally stimulation of phospholipase C when the NHERF proteins are expressed. We hypothesized that this signaling switch occurs at the level of the G protein. We measured G protein activation by [³⁵S]GTPγS binding and Gα subtype-specific immunoprecipitation using three different cellular models of PTHR signaling. These studies revealed that PTHR interactions with NHERF1 enhance receptor-mediated stimulation of Gα_q but have no effect on stimulation of Gα_i or Gα_s. In contrast, PTHR associations with NHERF2 enhance receptor-mediated stimulation of both Gα_q and Gα_i but decrease stimulation of Gα_s. Consistent with these functional data, NHERF2 formed cellular complexes with both Gα_q and Gα_i, whereas NHERF1 was found to interact only with Gα_q. These findings demonstrate that NHERF interactions regulate PTHR signaling at the level of G proteins and that NHERF1 and NHERF2 exhibit isotype-specific effects on G protein activation.     

The G215R Mutation in the Cl?/H+-Antiporter ClC-7 Found in ADO II Osteopetrosis Does Not Abolish Function but Causes a Severe Trafficking Defect

Background

ClC-7 is a ubiquitous transporter which is broadly expressed in mammalian tissues. It is implied in the pathogenesis of lysosomal storage disease and osteopetrosis. Because of its endosomal/lysosomal localization it is still poorly characterized.

Methodology/Principal Findings

An electrophysiological characterization of rat ClC-7 using solid-supported membrane-based electrophysiology is presented. The measured currents show the characteristics of ClC-7 and confirm its function as a Cl⁻/H⁺-antiporter. We have used rat ClC-7 in CHO cells as a model system to investigate the functionality and cellular localization of the wt transporter and its variant G213R ClC-7 which is the analogue of human G215R ClC-7 responsible for autosomal dominant osteopetrosis type II. Our study shows that rat G213R ClC-7 is functional but has a localization defect in CHO cells which prevents it from being correctly targeted to the lysosomal membrane. The electrophysiological assay is tested as a tool for drug discovery. The assay is validated with a number of drug candidates. It is shown that ClC-7 is inhibited by DIDS, NPPB and NS5818 at micromolar concentrations.

Conclusions/Significance

It is suggested that the scenario found in the CHO model system also applies to the human transporter and that mislocalization rather than impaired functionality of G215R ClC-7 is the primary cause of the related autosomal dominant osteopetrosis type II. Furthermore, the robust solid-supported membrane-based electrophysiological assay is proposed for rapid screening for potential ClC-7 inhibitors which are discussed for treatment of osteoporosis.     

Cloning,Expression, and Characterization of the Squid Na+–Ca2+ Exchanger (NCX-SQ1)

We have cloned the squid neuronal Na⁺–Ca²⁺ exchanger, NCX-SQ1, expressed it in Xenopus oocytes, and characterized its regulatory and ion transport properties in giant excised membrane patches. The squid exchanger shows 58% identity with the canine Na⁺–Ca²⁺ exchanger (NCX1.1). Regions determined to be of functional importance in NCX1 are well conserved. Unique among exchanger sequences to date, NCX-SQ1 has a potential protein kinase C phosphorylation site (threonine 184) between transmembrane segments 3 and 4 and a tyrosine kinase site in the Ca²⁺ binding region (tyrosine 462). There is a deletion of 47 amino acids in the large intracellular loop of NCX-SQ1 in comparison with NCX1. Similar to NCX1, expression of NCX-SQ1 in Xenopus oocytes induced cytoplasmic Na⁺-dependent ⁴⁵Ca²⁺ uptake; the uptake was inhibited by injection of Ca²⁺ chelators. In giant excised membrane patches, the NCX-SQ1 outward exchange current showed Na⁺-dependent inactivation, secondary activation by cytoplasmic Ca²⁺, and activation by chymotrypsin. The NCX-SQ1 exchange current was strongly stimulated by both ATP and the ATP-thioester, ATPγS, in the presence of F⁻ (0.2 mM) and vanadate (50 μM), and both effects reversed on application of a phosphatidylinositol-4′,5′-bisphosphate antibody. NCX1 current was stimulated by ATP, but not by ATPγS. Like NCX1 current, NCX-SQ1 current was strongly stimulated by phosphatidylinositol-4′,5′-bisphosphate liposomes. In contrast to results in squid axon, NCX-SQ1 was not stimulated by phosphoarginine (5–10 mM). After chymotrypsin treatment, both the outward and inward NCX-SQ1 exchange currents were more strongly voltage dependent than NCX1 currents. Ion concentration jump experiments were performed to estimate the relative electrogenicity of Na⁺ and Ca²⁺ transport reactions. Outward current transients associated with Na⁺ extrusion were much smaller for NCX-SQ1 than NCX1, and inward current transients associated with Ca²⁺ extrusion were much larger. For NCX-SQ1, charge movements of Ca²⁺ transport could be defined in voltage jump experiments with a low cytoplasmic Ca²⁺ (2 μM) in the presence of high extracellular Ca²⁺ (4 mM). The rates of charge movements showed “U”-shaped dependence on voltage, and the slopes of both charge–voltage and rate–voltage relations (1,600 s⁻¹ at 0 mV) indicated an apparent valency of −0.6 charges for the underlying reaction. Evidently, more negative charge moves into the membrane field in NCX-SQ1 than in NCX1 when ions are occluded into binding sites.     

Involvement of Protein Tyrosine Kinase in the InsP3-Induced Activation of Ca2+-Dependent Cl− Currents in Cultured Cells of the Rat Retinal Pigment Epithelium

This combined study of patch-clamp and intracellular Ca²⁺ ([Ca²⁺] _i ) measurement was undertaken in order to identify signaling pathways that lead to activation of Ca²⁺-dependent Cl⁻ channels in cultured rat retinal pigment epithelial (RPE) cells. Intracellular application of InsP3 (10 μm) led to an increase in [Ca²⁺] _i and activation of Cl⁻ currents. In contrast, intracellular application of Ca²⁺ (10 μm) only induced transient activation of Cl⁻ currents. After full activation by InsP3, currents were insensitive to removal of extracellular Ca²⁺ and to the blocker of I _CRAC, La³⁺ (10 μm), despite the fact that both maneuvers led to a decline in [Ca²⁺] _i . The InsP3-induced rise in Cl⁻ conductance could be prevented either by thapsigargin-induced (1 μm) depletion of intracellular Ca²⁺ stores or by removal of Ca²⁺ prior to the experiment. The effect of InsP3 could be mimicked by intracellular application of the Ca²⁺-chelator BAPTA (10 mm). Block of PKC (chelerythrine, 1 μm) had no effect. Inhibition of Ca²⁺/calmodulin kinase (KN-63, KN-92; 5 μm) reduced Cl⁻-conductance in 50% of the cells investigated without affecting [Ca²⁺] _i . Inhibition of protein tyrosine kinase (50 μm tyrphostin 51, 5 μm genistein, 5 μm lavendustin) reduced an increase in [Ca²⁺] _i and Cl⁻ conductance. In summary, elevation of [Ca] _i by InsP3 leads to activation of Cl⁻ channels involving cytosolic Ca²⁺ stores and Ca²⁺ influx from extracellular space. Tyrosine kinases are essential for the Ca²⁺-independent maintenance of this conductance. Received: 15 October 1998/Revised: 3 March 1999     

β-Arrestin Interacts with the Beta/Gamma Subunits of Trimeric G-Proteins and Dishevelled in the Wnt/Ca2+ Pathway in Xenopus Gastrulation

β-Catenin independent, non-canonical Wnt signaling pathways play a major role in the regulation of morphogenetic movements in vertebrates. The term non-canonical Wnt signaling comprises multiple, intracellularly divergent, Wnt-activated and β-Catenin independent signaling cascades including the Wnt/Planar Cell Polarity and the Wnt/Ca²⁺ cascades. Wnt/Planar Cell Polarity and Wnt/Ca²⁺ pathways share common effector proteins, including the Wnt ligand, Frizzled receptors and Dishevelled, with each other and with additional branches of Wnt signaling. Along with the aforementioned proteins, β-Arrestin has been identified as an essential effector protein in the Wnt/β-Catenin and the Wnt/Planar Cell Polarity pathway. Our results demonstrate that β-Arrestin is required in the Wnt/Ca²⁺ signaling cascade upstream of Protein Kinase C (PKC) and Ca²⁺/Calmodulin-dependent Protein Kinase II (CamKII). We have further characterized the role of β-Arrestin in this branch of non-canonical Wnt signaling by knock-down and rescue experiments in Xenopus embryo explants and analyzed protein-protein interactions in 293T cells. Functional interaction of β-Arrestin, the β subunit of trimeric G-proteins and Dishevelled is required to induce PKC activation and membrane translocation. In Xenopus gastrulation, β-Arrestin function in Wnt/Ca²⁺ signaling is essential for convergent extension movements. We further show that β-Arrestin physically interacts with the β subunit of trimeric G-proteins and Dishevelled, and that the interaction between β-Arrestin and Dishevelled is promoted by the beta/gamma subunits of trimeric G-proteins, indicating the formation of a multiprotein signaling complex.     

Osmosensitivity of the 2H+−K+-exchange and the H+-ATPase complex F0F1 in anaerobically grownEscherichia coli

Escherichia coli grown anaerobically for osmotic studies upon increased osmolarity in alkaline medium carried out H⁺–K⁺-exchange in two steps, the first of which was DCCD¹ sensitive and osmo-dependent and had the 2H⁺/K⁺ stoichiometry. H⁺-efflux in the presence of protonophore (CCCP) upon increase of osmolarity was shown to be high and inhibited by DCCD, whereas H⁺-efflux induced by a decrease of osmolarity was small and not inhibited by DCCD. The 2H⁺/K⁺-exchange was absent intrkA anduncA mutants. InuncB mutant 2H⁺/K⁺-exchange was not DCCD-and osmosensitive. Competition between DCCD and osmoshock on inhibition of 2H⁺/K⁺-exchange was found. Osmosensitivity of this exchange disappeared in spheroplasts. Osmosensitivity of both 2H⁺/K⁺-exchange and the F₀F₁ and osmoregulation of the F₀F₁ via F₀ and a periplasmic space are postulated.Abbreviations F₀F₁ H⁺-ATPase complex - F₀ H⁺-channel, proteolipid - F₁ H⁺-ATPase - Trk constitutive system for K⁺ uptake - PV periplasmic protein valve - DCCD N,N-dicyclohexylcarbodiimide - CCCP carbonylcyanide-m-chlorophenylhydrazone - _H or _K transmembrane electrochemical gradient for H⁺ or K⁺ respectively - membrane potential - upshock or downshock increase or decrease of medium osmolarity, respectively - CGSC E. coli Genetic Stock Center, Yale University, USA     

SORLA/SORL1 Functionally Interacts with SPAK To Control Renal Activation of Na+-K+-Cl? Cotransporter 2

Proper control of NaCl excretion in the kidney is central to bodily functions, yet many mechanisms that regulate reabsorption of sodium and chloride in the kidney remain incompletely understood. Here, we identify an important role played by the intracellular sorting receptor SORLA (sorting protein-related receptor with A-type repeats) in functional activation of renal ion transporters. We demonstrate that SORLA is expressed in epithelial cells of the thick ascending limb (TAL) of Henle''s loop and that lack of receptor expression in this cell type in SORLA-deficient mice results in an inability to properly reabsorb sodium and chloride during osmotic stress. The underlying cellular defect was correlated with an inability of the TAL to phosphorylate Na⁺-K⁺-Cl⁻ cotransporter 2 (NKCC2), the major sodium transporter in the distal nephron. SORLA functionally interacts with Ste-20-related proline-alanine-rich kinase (SPAK), an activator of NKCC2, and receptor deficiency is associated with missorting of SPAK. Our data suggest a novel regulatory pathway whereby intracellular trafficking of SPAK by the sorting receptor SORLA is crucial for proper NKCC2 activation and for maintenance of renal ion balance.Sorting-protein-related receptor with A-type repeats (SORLA; also known as SORL1 or LR11) is a member of the VPS10P domain receptor gene family, a group of sorting receptors structurally related to the yeast vacuolar protein sorting 10 protein (VPS10P) (11, 30). VPS10P is a receptor that transports carboxypeptidase Y into the vacuole (the yeast lysosome) (12). VPS10P domain receptors, including SORLA, have been shown to shuttle between the cell surface, early endosomes, and the trans-Golgi network (TGN) in various cell types (16, 26). Based on this distinct trafficking pattern and on their homology to VPS10P, a role for SORLA and other family members as intracellular sorting receptors has been suggested (reviewed in reference 29).Recently, a role for SORLA in protein trafficking was confirmed in neurons in the brain, where it acts as a receptor for the amyloid precursor protein (APP), the main etiological agent in Alzheimer''s disease. SORLA controls the sorting of APP between the TGN and endosomal compartments, thereby regulating proteolytic breakdown into amyloidogenic and nonamyloidogenic products (1, 17, 24).In addition to the brain, expression of SORLA has also been reported in other tissues, including testis, pancreas, smooth muscle, and kidney (11, 15, 30). In the kidney, expression was mapped to the collecting duct epithelium (23). However, the relevance of the receptor for renal physiology remained enigmatic.In this study, we addressed the physiological role of SORLA in the kidney. Contrary to previous reports, we detected significant expression of SORLA in epithelial cells of the thick ascending limb (TAL) of Henle''s loop, the distal convoluted tubule, and the connecting tubule. Lack of receptor expression in SORLA-deficient mice results in an inability to properly reabsorb sodium and chloride under osmotic stress induced by thirsting. The underlying cellular defect was linked to an inability of the TAL to activate Na⁺-K⁺-Cl⁻ cotransporter 2 (NKCC2), the major sodium transporter in the distal nephron. Interaction of SORLA with Ste-20-related proline-alanine-rich kinase (SPAK), the kinase that activates NKCC2, and missorting of SPAK in SORLA-deficient cells suggest a novel regulatory pathway in which intracellular trafficking of SPAK by SORLA is essential for NKCC2 activation and for renal ion handling.     

Urotensin-II Receptor Stimulation of Cardiac L-type Ca2+ Channels Requires the βγ Subunits of Gi/o-protein and Phosphatidylinositol 3-Kinase-dependent Protein Kinase C β1 Isoform

Recent studies have demonstrated that urotensin-II (U-II) plays important roles in cardiovascular actions including cardiac positive inotropic effects and increasing cardiac output. However, the mechanisms underlying these effects of U-II in cardiomyocytes still remain unknown. We show by electrophysiological studies that U-II dose-dependently potentiates L-type Ca²⁺ currents (I_Ca,L) in adult rat ventricular myocytes. This effect was U-II receptor (U-IIR)-dependent and was associated with a depolarizing shift in the voltage dependence of inactivation. Intracellular application of guanosine-5′-O-(2-thiodiphosphate) and pertussis toxin pretreatment both abolished the stimulatory effects of U-II. Dialysis of cells with the QEHA peptide, but not scrambled peptide SKEE, blocked the U-II-induced response. The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin as well as the class I PI3K antagonist {"type":"entrez-nucleotide","attrs":{"text":"CH132799","term_id":"45009640","term_text":"CH132799"}}CH132799 blocked the U-II-induced I_Ca,L response. Protein kinase C antagonists calphostin C and chelerythrine chloride as well as dialysis of cells with 1,2bis(2aminophenoxy)ethaneN,N,N′,N′-tetraacetic acid abolished the U-II-induced responses, whereas PKCα inhibition or PKA blockade had no effect. Exposure of ventricular myocytes to U-II markedly increased membrane PKCβ₁ expression, whereas inhibition of PKCβ₁ pharmacologically or by shRNA targeting abolished the U-II-induced I_Ca,L response. Functionally, we observed a significant increase in the amplitude of sarcomere shortening induced by U-II; blockade of U-IIR as well as PKCβ inhibition abolished this effect, whereas Bay K8644 mimicked the U-II response. Taken together, our results indicate that U-II potentiates I_Ca,L through the βγ subunits of G_i/o-protein and downstream activation of the class I PI3K-dependent PKCβ₁ isoform. This occurred via the activation of U-IIR and contributes to the positive inotropic effect on cardiomyocytes.     

Inhibition of Noncanonical Ca2+ Oscillation/Calcineurin/GSK-3β Pathway Contributes to Anti-Inflammatory Effect of Sigma-1 Receptor Activation

Neurochemical Research - Further understanding the mechanism for microglia activation is necessary for developing novel anti-inflammatory strategies. Our previous study found that the activation of...     

Role of the nitric oxide/cyclic GMP/Ca2+ signaling pathway in the pyrogenic effect of interleukin-1β

Interleukin-1β (IL-1β) has a wide spectrum of inflammatory, metabolic, haemopoietic, and immunological properties. Because it produces fever when injected into animals and humans, it is considered an endogenous pyrogen. There is evidence to suggest that Ca²⁺ plays a critical role in the central mechanisms of thermoregulation, and in the intracellular signaling pathways controlling fever induced by IL-1β and other pyrogens. Data from different labs indicate that Ca²⁺ and Na⁺ determine the temperature set point in the posterior hypothalamus (PH) of various mammals and that changes in Ca²⁺ and PGE₂ concentrations in the cerebrospinal fluid (CSF) of these animals are associated with IL-1β-induced fever. Antipyretic drugs such as acetylsalicylic acid, dexamethasone, and lipocortin 5-(204–212) peptide counteract IL-1β-induced fever and abolish changes in Ca²⁺ and PGE₂ concentrations in CSF. In vitro studies have established that activation of the nitric oxide (NO)/cyclic GMP (cGMP) pathway is part of the signaling cascade transducing Ca²⁺ mobilization in response to IL-1β and that the ryanodine (RY)- and inositol-(1,4,5)-trisphosphate (IP₃)-sensitive pools are the main source of the mobilized Ca²⁺. It is concluded that the NO/cGMP/Ca²⁺ pathway is part of the signaling cascade subserving some of the multiple functions of IL-1β.     

Activation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) is needed for the TGFβ-induced chondrogenic and osteogenic differentiation of mesenchymal stem cells

The role of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway on the osteogenesis of progenitor and stem cells has received a lot of attention due to conflicting results in the literature. ERK1/2 has been reported to be both activating and inhibitory to the osteogenesis of different cell types under varying culture conditions. This study focused specifically on the role of ERK1/2 on the chondrogenesis and osteogenesis of mesenchymal stem cells (MSC) induced by cytokine exposure. Bone marrow-derived MSC were cultured in three-dimensional fibrin gel scaffolds and stimulated down the chondrogenic and osteogenic programs by addition of TGF-β3 to and osteogenic buffer media. Cells were cultured under control conditions (no cytokine supplementation), treated with TGF-β3 or treated with PD98059 + TGF-β3 for 7 days. RT-PCR results show that addition of TGF-β3 significantly upregulates the phosphorylation of ERK1/2 and induces the cells down the chondrogenic and osteogenic pathways (as demonstrated by the significant upregulation of aggrecan, sox9, collagen types 1 & 2 gene expressions). Inhibition of ERK1/2 phosphorylation with PD98059 led to the abolishment of the upregulation of chondrogenic and osteogenic-specific gene expressions. These results demonstrate that ERK1/2 is needed for the chondrogenic and osteogenic differentiation of MSC as induced by TGF-β3 supplementation.     

Structural Dynamics of the Activation of Elongation Factor 2 Kinase by Ca2 +-Calmodulin

Eukaryotic elongation factor 2 kinase (eEF-2K), the only known calmodulin (CaM)-activated α-kinase, phosphorylates eukaryotic elongation factor 2 (eEF-2) on a specific threonine (Thr-56) diminishing its affinity for the ribosome and reducing the rate of nascent chain elongation during translation. Despite its critical cellular role, the precise mechanisms underlying the CaM-mediated activation of eEF-2K remain poorly defined. Here, employing a minimal eEF-2K construct (TR) that exhibits activity comparable to the wild-type enzyme and is fully activated by CaM in vitro and in cells, and using a variety of complimentary biophysical techniques in combination with computational modeling, we provide a structural mechanism by which CaM activates eEF-2K. Native mass analysis reveals that CaM, with two bound Ca^2 + ions, forms a stoichiometric 1:1 complex with TR. Chemical crosslinking mass spectrometry and small-angle X-ray scattering measurements localize CaM near the N-lobe of the TR kinase domain and the spatially proximal C-terminal helical repeat. Hydrogen/deuterium exchange mass spectrometry and methyl NMR indicate that the conformational changes induced on TR by the engagement of CaM are not localized but are transmitted to remote regions that include the catalytic site and the functionally important phosphate binding pocket. The structural insights obtained from the present analyses, together with our previously published kinetics data, suggest that TR, and by inference, wild-type eEF-2K, upon engaging CaM undergoes a conformational transition resulting in a state that is primed to efficiently auto-phosphorylate on the primary activating T348 en route to full activation.