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Andrew M Scharenberg

Publications and source records attributed to Andrew M Scharenberg.

At least 19 recordsLinked to original sources
PubMed2007

SLC41A2 encodes a plasma-membrane Mg2+ transporter.

The TRPM7 (transient receptor potential melastatin 7) ion channel has been implicated in the uptake of Mg2+ into vertebrate cells, as elimination of TRPM7 expression through gene targeting in DT40 B-lymphocytes renders them unable to grow in the absence of supplemental Mg2+. However, a residual capacity of TRPM7-deficient cells to accumulate Mg2+ and proliferate when provided with supplemental Mg2+ suggests the existence of Mg2+ uptake mechanism(s) other than TRPM7. Evaluation of the expression of several members of the SLC41 (solute carrier family 41) family, which exhibit homology with the MgtE class of prokaryotic putative bivalent-cation transporters, demonstrated that one, SLC41A2 (solute carrier family 41 member 2), is expressed in both wild-type and TRPM7-deficient DT40 cells. Characterization of heterologously expressed SLC41A2 protein indicated that it is a plasma-membrane protein with an N-terminus-outside/C-terminus-inside 11-TM (transmembrane)-span topology, consistent with its functioning as a trans-plasma-membrane transporter. In contrast with a previous report of ion-channel activity associated with SLC41A2 expression in oocytes, investigation of whole cell currents in SLC41A2-expressing DT40 cells revealed no novel currents of any type associated with SLC41A2 expression. However, expression of SLC41A2 in TRPM7-deficient cells under the control of a doxycycline-inducible promoter was able to conditionally enhance their net uptake of 26Mg2+ and conditionally and dose-dependently provide them with the capacity to grow in the absence of supplemental Mg2+, observations strongly supporting a model whereby SLC41A2 directly mediates trans-plasma-membrane Mg2+ transport. Overall, our results suggest that SLC41A2 functions as a plasma-membrane Mg2+ transporter in vertebrate cells.

Animals↗
PubMed2006

TRPML cation channels regulate the specialized lysosomal compartment of vertebrate B-lymphocytes.

B-lymphocytes possess a specialized lysosomal compartment, the regulated transformation of which has been implicated in B-cell antigen presentation. Members of the mucolipin (TRPML) family of cation channels have been implicated in regulated vesicular transport in several tissues, but a role for TRPML function in lymphocyte vesicular transport physiology has not been previously described. To address the role of TRPML proteins in lymphocyte vesicular transport, we analyzed the lysosomal compartment in cultured B-lymphocytes engineered to lack TRPML1 or after expression of N- or C-terminal GFP fusion proteins of TRPML1 or TRPML2. Consistent with previous analyses of lymphocytes derived from human patients with mutations in TRPML1, we were not able to detect abnormalities in the lysosomes of TRPML1-deficient DT40 B-lymphocytes. However, while N-terminal GFP fusions of TRPML2 localized to normal appearing lysosomes, C-terminal GFP fusions of either TRPML1 or TRPML2 acted to antagonize endogenous TRPML function, localizing to large vesicular structures, the histological properties of which were indistinguishable from the enlarged lysosomes observed in affected tissues of TRPML1-deficient humans. Endocytosed B-cell receptors were delivered to these enlarged lysosomes, demonstrating that a TRPML-dependent process is required for normal regulation of the specialized lysosome compartment of vertebrate B-lymphocytes.

Animals↗
PubMed2006

Metabolite of SIR2 reaction modulates TRPM2 ion channel.

The transient receptor potential melastatin-related channel 2 (TRPM2) is a nonselective cation channel, whose prolonged activation by oxidative and nitrative agents leads to cell death. Here, we show that the drug puromycin selectively targets TRPM2-expressing cells, leading to cell death. Our data suggest that the silent information regulator 2 (Sir2 or sirtuin) family of enzymes mediates this susceptibility to cell death. Sirtuins are protein deacetylases that regulate gene expression, apoptosis, metabolism, and aging. These NAD+-dependent enzymes catalyze a reaction in which the acetyl group from substrate is transferred to the ADP-ribose portion of NAD+ to form deacetylated product, nicotinamide, and the metabolite OAADPr, whose functions remain elusive. Using cell-based assays and RNA interference, we show that puromycin-induced cell death is greatly diminished by nicotinamide (a potent sirtuin inhibitor), and by decreased expression of sirtuins SIRT2 and SIRT3. Furthermore, we demonstrate using channel current recordings and binding assays that OAADPr directly binds to the cytoplasmic domain of TRPM2 and activates the TRPM2 channel. ADP-ribose binds TRPM2 with similarly affinity, whereas NAD+ displays almost negligible binding. These studies provide the first evidence for the potential role of sirtuin-generated OAADPr in TRPM2 channel gating.

Adenosine Diphosphate Ribose↗
PubMed2006

Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes.

We have taken a knockout approach to interrogate the function of protein kinase D (PKD) serine/threonine kinases in lymphocytes. DT40 B cells express two PKD family members, PKD1 and PKD3, which are both rapidly activated by the B-cell antigen receptor (BCR). DT40 cells with single or dual deletions of PKD1 and/or PKD3 were viable, allowing the role of individual PKD isoforms in BCR signal transduction to be assessed. One proposed downstream target for PKD1 in lymphocytes is the class II histone deacetylases (HDACs). Regulation of chromatin accessibility via class II histone deacetylases is an important mechanism controlling gene expression patterns, but the molecules that control this key process in B cells are not known. Herein, we show that phosphorylation and nuclear export of the class II histone deacetylases HDAC5 and HDAC7 are rapidly induced following ligation of the BCR or after treatment with phorbol esters (a diacylglycerol mimetic). Loss of either PKD1 or PKD3 had no impact on HDAC phosphorylation, but loss of both PKD1 and PKD3 abrogated antigen receptor-induced class II HDAC5/7 phosphorylation and nuclear export. These studies reveal an essential and redundant role for PKD enzymes in controlling class II HDACs in B lymphocytes and suggest that PKD serine kinases are a critical link between the BCR and epigenetic control of chromatin.

Animals↗
PubMed2005

TRPM2 and TRPM7: channel/enzyme fusions to generate novel intracellular sensors.

Within the transient receptor potential (Trp) superfamily of ion channels, three members of the Trp (melastatin) (TRPM) subfamily stand out as their amino acid sequences indicate that they possess both ion channel and enzymatic functions. Recently, progress has been made in understanding the relationships between these disparate functionalities for two of these proteins, TRPM2 and TRPM7. TRPM2 appears to have adapted an ADP-ribose hydrolase (ADPRibase) enzyme's structure as a means of binding ADP-ribose and conveying information about the accumulation of ADP-ribose to the cell via the activation of Na(+)/Ca(2+) entry through the channel domain. While the ADPRibase activity of TRPM2's enzymatic domain is not required for channel gating, whether a converse relationship exists, wherein channel gating or ion flow modulates the enzymatic domain's ADPRibase activity, is not known. In contrast, TRPM7 appears to have evolved to place a Mg(2+)-regulated protein kinase domain in close proximity to a Mg(2+)-permeant ion channel, such that the kinase domain's phosphotransferase activity is able respond to local changes in free Mg(2+) occurring as the result of the flux of Mg(2+) through the channel. As with TRPM2, the activity of TRPM7's enzymatic domain is not required for gating of its channel domain, although evidence exists that it may have an alternative means of influencing channel gating. These insights into the functional relationships between the channel and enzymatic domains of TRPM2 and TRPM7 suggest informative models for their roles in vertebrate cell physiology.

Adenosine Diphosphate Ribose↗
PubMed2004

Accumulation of free ADP-ribose from mitochondria mediates oxidative stress-induced gating of TRPM2 cation channels.

TRPM2 is a member of the transient receptor potential melastatin-related (TRPM) family of cation channels, which possesses both ion channel and ADP-ribose hydrolase functions. TRPM2 has been shown to gate in response to oxidative and nitrosative stresses, but the mechanism through which TRPM2 gating is induced by these types of stimuli is not clear. Here we show through structure-guided mutagenesis that TRPM2 gating by ADP-ribose and both oxidative and nitrosative stresses requires an intact ADP-ribose binding cleft in the C-terminal nudix domain. We also show that oxidative/nitrosative stress-induced gating can be inhibited by pharmacological reagents predicted to inhibit NAD hydrolysis to ADP-ribose and by suppression of ADP-ribose accumulation by cytosolic or mitochondrial overexpression of an enzyme that specifically hydrolyzes ADP-ribose. Overall, our data are most consistent with a model of oxidative and nitrosative stress-induced TRPM2 activation in which mitochondria are induced to produce free ADP-ribose and release it to the cytosol, where its subsequent accumulation induces TRPM2 gating via interaction within a binding cleft in the C-terminal NUDT9-H domain of TRPM2.

Adenosine Diphosphate Ribose↗
PubMed2004

Receptor-mediated regulation of the TRPM7 channel through its endogenous protein kinase domain.

TRPM7 is a ubiquitously expressed and constitutively active divalent cation-selective ion channel, whose basal activity is regulated by intracellular levels of Mg(2+) and Mg.ATP. We have investigated receptor-mediated mechanisms that may actively regulate TRPM7 activity. We here report that TRPM7 currents are suppressed by intracellular GTPgammaS, suggesting the involvement of heterotrimeric G proteins. TRPM7 currents are also inhibited by stimulating endogenous muscarinic receptors, which is mediated by G(i) because the inhibitory effect is blunted by pertussis toxin. Conversely, stimulation of endogenous G(s)-coupled beta-adrenergic receptors potentiates TRPM7 currents, whereas G(q)-coupled thrombin receptors have little effect. Consistent with the involvement of G(s)/G(i) in controlling adenylyl cyclase activity, elevations of intracellular cAMP levels enhance TRPM7 activity and prevent receptor-mediated modulation of TRPM7 activity by muscarinic and adrenergic agonists. This cAMP-dependent effect requires the functional integrity of both protein kinase A (PKA) and the endogenous kinase domain of TRPM7 because cAMP-mediated effects are abolished when treating cells with the PKA inhibitors H89 or KT5720 as well as in cells expressing phosphotransferase-deficient TRPM7 constructs. These mutant channels are also much less susceptible to GTPgammaS-mediated inhibition, suggesting that the main regulatory effect occurs through G(i)- and G(s)-mediated changes in cAMP. Taken together, our results demonstrate that TRPM7 activity is up- and down-regulated through its endogenous kinase in a cAMP- and PKA-dependent manner.

Calcium↗
PubMed2004

Dual-function ion channel/protein kinases: novel components of vertebrate magnesium regulatory mechanisms.

Although magnesium is the dominant divalent intracellular cation and is required for the function of diverse types of enzymes that participate in virtually every cellular process, the molecular mechanisms that regulate its homeostasis are poorly understood. Electrophysiologic and biochemical investigations of a novel dual-function ion channel/kinase protein have recently converged with the identification of the gene locus for an autosomal recessive form of inherited hypomagnesemia to provide new insight into vertebrate magnesium regulatory mechanisms.

Animals↗
PubMed2003

Regulation of vertebrate cellular Mg2+ homeostasis by TRPM7.

TRPM7 is a polypeptide with intrinsic ion channel and protein kinase domains whose targeted deletion causes cells to experience growth arrest within 24 hr and eventually die. Here, we show that while TRPM7's kinase domain is not essential for activation of its channel, a functional coupling exists such that structural alterations of the kinase domain alter the sensitivity of channel activation to Mg(2+). Investigation of the relationship between Mg(2+) and the cell biological role of TRPM7 revealed that TRPM7-deficient cells become Mg(2+) deficient, that both the viability and proliferation of TRPM7-deficient cells are rescued by supplementation of extracellular Mg(2+), and that the capacity of heterologously expressed TRPM7 mutants to complement TRPM7 deficiency correlates with their sensitivity to Mg(2+). Overall, our results indicate that TRPM7 has a central role in Mg(2+) homeostasis as a Mg(2+) uptake pathway regulated through a functional coupling between its channel and kinase domains.

Adenosine Triphosphate↗
PubMed2003

TRPM7 provides an ion channel mechanism for cellular entry of trace metal ions.

Trace metal ions such as Zn(2+), Fe(2+), Cu(2+), Mn(2+), and Co(2+) are required cofactors for many essential cellular enzymes, yet little is known about the mechanisms through which they enter into cells. We have shown previously that the widely expressed ion channel TRPM7 (LTRPC7, ChaK1, TRP-PLIK) functions as a Ca(2+)- and Mg(2+)-permeable cation channel, whose activity is regulated by intracellular Mg(2+) and Mg(2+).ATP and have designated native TRPM7-mediated currents as magnesium-nucleotide-regulated metal ion currents (MagNuM). Here we report that heterologously overexpressed TRPM7 in HEK-293 cells conducts a range of essential and toxic divalent metal ions with strong preference for Zn(2+) and Ni(2+), which both permeate TRPM7 up to four times better than Ca(2+). Similarly, native MagNuM currents are also able to support Zn(2+) entry. Furthermore, TRPM7 allows other essential metals such as Mn(2+) and Co(2+) to permeate, and permits significant entry of nonphysiologic or toxic metals such as Cd(2+), Ba(2+), and Sr(2+). Equimolar replacement studies substituting 10 mM Ca(2+) with the respective divalent ions reveal a unique permeation profile for TRPM7 with a permeability sequence of Zn(2+) approximately Ni(2+) >> Ba(2+) > Co(2+) > Mg(2+) >/= Mn(2+) >/= Sr(2+) >/= Cd(2+) >/= Ca(2+), while trivalent ions such as La(3+) and Gd(3+) are not measurably permeable. With the exception of Mg(2+), which exerts strong negative feedback from the intracellular side of the pore, this sequence is faithfully maintained when isotonic solutions of these divalent cations are used. Fura-2 quenching experiments with Mn(2+), Co(2+), or Ni(2+) suggest that these can be transported by TRPM7 in the presence of physiological levels of Ca(2+) and Mg(2+), suggesting that TRPM7 represents a novel ion-channel mechanism for cellular metal ion entry into vertebrate cells.

Animals↗
PubMed2002

Regulation of protein kinase Cnu by the B-cell antigen receptor.

Diacylglycerol-dependent signaling plays an important role in signal transduction through T- and B-lymphocyte antigen receptors. Recently, a novel serine-threonine kinase of the protein kinase C (PKC) family has been described and designated as PKCnu. PKCnu has two putative diacylglycerol binding C1 domains, suggesting that it may participate in a novel diacylglycerol-mediated signaling pathway. Here we show that both endogenous and recombinant PKCnu are trans-located to the plasma membrane and activated by the diacylglycerol mimic phorbol 12-myristate 13-acetate. Mutational analysis demonstrates that PKCnu activation is dependent on trans-phosphorylation of two conserved activation loop serine residues. We also find that PKCnu is an important physiologic target of the B-cell receptor (BCR), because PKCnu is found to be abundantly expressed in chicken and human B-cell lines and, in addition, exhibits robust activation after BCR engagement. Genetic and pharmacologic analyses of BCR-mediated PKCnu activation indicate that it requires intact phospholipase Cgamma and PKC signaling pathways. Furthermore, in co-transfection assays, PKCnu can be trans-phosphorylated by the novel PKC isozymes PKCepsilon, PKCeta, or PKCtheta but not the classical PKC enzyme, PKCalpha. Taken together, these results suggest that PKCnu is an important component of signaling pathways downstream from novel PKC enzymes after B-cell receptor engagement.

Animals↗
PubMed2002

NUDT9, a member of the Nudix hydrolase family, is an evolutionarily conserved mitochondrial ADP-ribose pyrophosphatase.

We have recently characterized the protein product of the human NUDT9 gene as a highly specific ADP-ribose (ADPR) pyrophosphatase. We now report an analysis of the human NUDT9 gene and its potential alternative transcripts along with detailed studies of the enzymatic properties and cell biological behavior of human NUDT9 protein. Our analysis of the human NUDT9 gene and twenty-two distinct cloned NUDT9 transcripts indicates that the full-length NUDT9 alpha transcript is the dominant form, and suggests that an alternative NUDT9 beta transcript occurs as the result of a potentially aberrant splice from a cryptic donor site within the first exon to the splice acceptor site of exon 2. Computer analysis of the predicted protein of the NUDT9 alpha transcript identified an N-terminal signal peptide or subcellular targeting sequence. Using green fluorescence protein tagging, we demonstrate that the predicted human NUDT9 alpha protein is targeted highly specifically to mitochondria, whereas the predicted protein of the NUDT9 beta transcript, which is missing this sequence, exhibits no clear subcellular localization. Investigation of the physical and enzymatic properties of NUDT9 indicates that it is functional as a monomer, optimally active at near neutral pH, and that it requires divalent metal ions and an intact Nudix motif for enzymatic activity. Furthermore, partial proteolysis of NUDT9 suggests that NUDT9 enzymes consist of two distinct domains: a proteolytically resistant C-terminal domain retaining essentially full specific ADPR pyrophosphatase activity and a proteolytically labile N-terminal portion that functions to enhance the affinity of the C-terminal domain for ADPR.

Amino Acid Sequence↗
PubMed2002

Analysis of O-acetyl-ADP-ribose as a target for Nudix ADP-ribose hydrolases.

The Sir2 family of NAD(+)-dependent histone/protein deacetylases has been implicated in a wide range of biological activities, including gene silencing, life span extension, and chromosomal stability. Recent evidence has indicated that these proteins produce a novel metabolite O-acetyl-ADP-ribose (OAADPr) during deacetylation. Cellular studies have demonstrated that this metabolite exhibits biological effects when microinjected in living cells. However, the molecular targets of OAADPr remain to be identified. Here we have analyzed the ADP-ribose-specific Nudix family of hydrolases as potential in vivo metabolizing enzymes of OAADPr. In vitro, we found that the ADP-ribose hydrolases (yeast YSA1, mouse NudT5, and human NUDT9) cleaved OAADPr to the products AMP and acetylated ribose 5'-phosphate. Steady-state kinetic analyses revealed that YSA1 and NudT5 hydrolyzed OAADPr with similar kinetic constants to those obtained with ADP-ribose as substrate. In dramatic contrast, human NUDT9 was 500-fold less efficient (k(cat)/K(m) values) at hydrolyzing OAADPr compared with ADP-ribose. The inability of OAADPr to inhibit the reaction of NUDT9 with ADP-ribose suggests that NUDT9 binds OAADPr with low affinity, likely due to steric considerations of the additional acetylated-ribose moiety. We next explored whether Nudix hydrolytic activities against OAADPr could be observed in cell extracts from yeast and human. Using a detailed analysis of the products generated during the consumption of OAADPr in extracts, we identified two robust enzymatic activities that were not consistent with the known Nudix hydrolases. Instead, we identified cytoplasmic esterase activities that hydrolyze OAADPr to acetate and ADP-ribose, whereas a distinct activity residing in the nucleus is consistent with an OAADPr-specific acetyltransferase. These findings establish for the first time that select members of the ADP-ribose hydrolases are potential targets of OAADPr metabolism. However, the predominate endogenous activities observed from diverse cell extracts represent novel enzymes.

Animals↗
PubMed2002

Activation of Syk tyrosine kinase is required for c-Cbl-mediated ubiquitination of Fcepsilon RI and Syk in RBL cells.

Engagement of the high affinity receptor for IgE (FcepsilonRI) on mast cells and basophils results in FcepsilonRI beta and gamma subunits ubiquitination by an as yet undefined mechanism. Here we show that, upon FcepsilonRI engagement on RBL-2H3 cells Syk undergoes ubiquitination and Syk kinase activity is required for its own ubiquitination and that of FcepsilonRI beta and gamma chains. This requirement was demonstrated by overexpression of Syk wild-type or its kinase-dead mutant in RBL cells or using an Syk-deficient RBL-derived cell line transfected with wild-type or a kinase inactive form of Syk. We also identify c-Cbl as the E3 ligase responsible for both Syk and receptor ubiquitination. Furthermore, we demonstrate that Syk controls tyrosine phosphorylation of Syk-associated Cbl induced after receptor engagement. These data suggest a mutual regulation between Syk and Cbl activities. Finally, we show that a selective inhibitor of proteasome degradation induces persistence of tyrosine-phosphorylated receptor complexes, of activated Syk, and of FcepsilonRI-triggered degranulation. Our results provide a molecular mechanism for down-regulation of engaged receptor complexes by targeting ubiquitinated FcepsilonRI and activated Syk to the proteasome for degradation.

Animals↗
PubMed2002

TRPM4 is a Ca2+-activated nonselective cation channel mediating cell membrane depolarization.

Calcium-activated nonselective (CAN) cation channels are expressed in various excitable and nonexcitable cells supporting important cellular responses such as neuronal bursting activity, fluid secretion, and cardiac rhythmicity. We have cloned and characterized a second form of TRPM4, TRPM4b, a member of the TRP channel family, as a molecular candidate of a CAN channel. TRPM4b encodes a cation channel of 25 pS unitary conductance that is directly activated by [Ca2+]i with an apparent K(D) of approximately 400 nM. It conducts monovalent cations such as Na+ and K+ without significant permeation of Ca2+. TRPM4b is activated following receptor-mediated Ca2+ mobilization, representing a regulatory mechanism that controls the magnitude of Ca2+ influx by modulating the membrane potential and, with it, the driving force for Ca2+ entry through other Ca2+-permeable pathways.

Amino Acid Sequence↗
PubMed2002

Dissociation of the store-operated calcium current I(CRAC) and the Mg-nucleotide-regulated metal ion current MagNuM.

Rat basophilic leukaemia cells (RBL-2H3-M1) were used to study the characteristics of the store-operated Ca(2+) release-activated Ca(2+) current (I(CRAC)) and the magnesium-nucleotide-regulated metal cation current (MagNuM) (which is conducted by the LTRPC7 channel). Pipette solutions containing 10 mM BAPTA and no added ATP induced both currents in the same cell, but the time to half-maximal activation for MagNuM was about two to three times slower than that of I(CRAC). Differential suppression of I(CRAC) was achieved by buffering free [Ca(2+)](i) to 90 nM and selective inhibition of MagNuM was accomplished by intracellular solutions containing 6 mM Mg.ATP, 1.2 mM free [Mg(2+)](i) or 100 microM GTP-gamma-S, allowing investigations on these currents in relative isolation. Removal of extracellular Ca(2+) and Mg(2+) caused both currents to be carried significantly by monovalent ions. In the absence or presence of free [Mg(2+)](i), I(CRAC) carried by monovalent ions inactivated more rapidly and more completely than MagNuM carried by monovalent ions. Since several studies have used divalent-free solutions on either side of the membrane to study selectivity and single-channel behaviour of I(CRAC), these experimental conditions would have favoured the contribution of MagNuM to monovalent conductance and call for caution in interpreting results where both I(CRAC) and MagNuM are activated.

Adenosine Triphosphate↗
PubMed2002

Vav3 modulates B cell receptor responses by regulating phosphoinositide 3-kinase activation.

To elucidate the mechanism(s) by which Vav3, a new member of the Vav family proteins, participates in B cell antigen receptor (BCR) signaling, we have generated a B cell line deficient in Vav3. Here we report that Vav3 influences phosphoinositide 3-kinase (PI3K) function through Rac1 in that phosphatidylinositol-3,4,5-trisphosphate (PIP3) generation was attenuated by loss of Vav3 or by expression of a dominant negative form of Rac1. The functional interaction between PI3K and Rac1 was also demonstrated by increased PI3K activity in the presence of GTP-bound Rac1. In addition, we show that defects of calcium mobilization and c-Jun NH2-terminal kinase (JNK) activation in Vav3-deficient cells are relieved by deletion of a PIP3 hydrolyzing enzyme, SH2 domain-containing inositol polyphosphate 5'-phosphatase (SHIP). Hence, our results suggest a role for Vav3 in regulating the B cell responses by promoting the sustained production of PIP3 and thereby calcium flux.

Animals↗