Intestinal Anion Exchanger Down-regulated in Adenoma (DRA) Is Inhibited by Intracellular Calcium

The Na/H exchanger 3 (NHE3) and the Cl/HCO₃ exchanger down-regulated in adenoma (DRA) together facilitate intestinal electroneutral NaCl absorption. Elevated Ca²⁺_i inhibits NHE3 through mechanisms involving the PDZ domain proteins NHE3 kinase A regulatory protein (E3KARP) or PDZ kidney 1 (PDZK1). DRA also possesses a PDZ-binding motif, but the roles of interactions with E3KARP or PDZK1 and Ca²⁺_i in DRA regulation are unknown. Wild type DRA and a mutant lacking the PDZ interaction motif (DRA-ETKFminus) were expressed constitutively in human embryonic kidney (HEK) and inducibly in Caco-2/BBE cells. DRA-mediated Cl/HCO₃ exchange was measured as intracellular pH changes. Ca²⁺_i was assessed fluorometrically. DRA was induced 8–16-fold and was delivered to the apical surface of polarized Caco-2 cells. Putative anion transporter 1 and cystic fibrosis transmembrane regulator did not contribute to Cl/HCO₃ exchange in transfected Caco-2 cells. The calcium ionophore 4Br-{"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 inhibited DRA and DRA-ETKFminus in HEK cells, but only full-length DRA was inhibited in Caco-2 cells. In contrast, 100 μm UTP, which increased Ca²⁺_i, inhibited full-length DRA but not DRA-ETKFminus in Caco-2 and HEK cells. In HEK cells, which express little PDZK1, additional transfection of PDZK1 was required for UTP to inhibit DRA. As HEK cells do not express cystic fibrosis transmembrane regulator or NHE3, the data indicate that Ca²⁺_i-dependent DRA inhibition is not because of modulation of other transport activities. In polarized epithelium, this inhibition requires interaction of DRA with PDZK1. Together with data from PDZK1^−/− mice, these data underscore the prominent role of PDZK1 in Ca²⁺_i-mediated inhibition of colonic NaCl absorption.In the gastrointestinal tract electroneutral NaCl absorption occurs in the distal ileum and proximal colon by parallel Na/H exchange and Cl/HCO₃ exchange. Studies in knock-out mice have confirmed that NHE3² (Na/H exchanger, isoform 3; SLC9A3) and DRA (down-regulated in adenoma; SLC26A3) are the primary transporters responsible for these events (1, 2). The importance of the latter is emphasized by the human genetic disorder congenital chloride diarrhea (3), in which a nonfunctional DRA leads to life-threatening diarrhea. DRA is also expressed in the duodenum (in the lower villus) and in the pancreas (4–6), where it is involved in chloride and bicarbonate secretion together with CFTR (4–7). All three transport proteins, NHE3, DRA, and CFTR, have PDZ interaction motifs that facilitate binding to several members of the NHERF class of PDZ adapter proteins (8, 9).Electroneutral NaCl absorption is regulated largely in parallel but reciprocally with electrogenic chloride secretion (10). In different systems NHE3 is acutely regulated by cAMP, cGMP, intracellular calcium, lysophosphatidic acid, and epidermal growth factor (11) as well as by tumor necrosis factor-α (12). Notably, some of these regulatory processes are mediated through the interaction of NHE3 with several members of the NHERF class of PDZ adapter proteins (8, 11).Relatively little is known about the acute regulation of DRA. In the context of chloride and bicarbonate secretion, DRA is activated by cAMP, if it is expressed in a complex with CFTR and PDZ adapter proteins (PDZK1, also known as CAP70, and/or NHERF) (6, 7, 13). It is expected that DRA is inhibited in vivo in parallel with NHE3 during NaCl absorption, and in Caco-2/BBE cells transfected with NHE3 and DRA, this coupled inhibition has recently been shown (14). In Xenopus oocytes DRA is refractory to regulation by the calmodulin antagonist calmidazolium (10 μm), the PP2A inhibitor calyculin A (100 nm), or actin-modifying agents (13). Other data suggest that direct phosphorylation may regulate DRA, as mutation of tyrosine 756 (Y756F) increases DRA activity, although no signaling pathway has been suggested (13). Thus the regulation of DRA remains poorly understood. Moreover, no data address whether DRA regulation can occur independently or is always dependent on regulation of partner transporters, i.e. CFTR or NHE3, to which it is functionally and structurally coupled.Here we show that DRA activity is inhibited by elevations of Ca²⁺_i, that this regulation is independent of CFTR and NHE3, and that regulation requires interactions between DRA and the PDZ adapter protein PDZK1.     

Conserved Glutamate Residues Glu-343 and Glu-519 Provide Mechanistic Insights into Cation/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter hCNT3

Human concentrative nucleoside transporter 3 (hCNT3) utilizes electrochemical gradients of both Na⁺ and H⁺ to accumulate pyrimidine and purine nucleosides within cells. We have employed radioisotope flux and electrophysiological techniques in combination with site-directed mutagenesis and heterologous expression in Xenopus oocytes to identify two conserved pore-lining glutamate residues (Glu-343 and Glu-519) with essential roles in hCNT3 Na⁺/nucleoside and H⁺/nucleoside cotransport. Mutation of Glu-343 and Glu-519 to aspartate, glutamine, and cysteine severely compromised hCNT3 transport function, and changes included altered nucleoside and cation activation kinetics (all mutants), loss or impairment of H⁺ dependence (all mutants), shift in Na⁺:nucleoside stoichiometry from 2:1 to 1:1 (E519C), complete loss of catalytic activity (E519Q) and, similar to the corresponding mutant in Na⁺-specific hCNT1, uncoupled Na⁺ currents (E343Q). Consistent with close-proximity integration of cation/solute-binding sites within a common cation/permeant translocation pore, mutation of Glu-343 and Glu-519 also altered hCNT3 nucleoside transport selectivity. Both residues were accessible to the external medium and inhibited by p-chloromercuribenzene sulfonate when converted to cysteine.Physiologic nucleosides and the majority of synthetic nucleoside analogs with antineoplastic and/or antiviral activity are hydrophilic molecules that require specialized plasma membrane nucleoside transporter (NT)³ proteins for transport into or out of cells (1–4). NT-mediated transport is required for nucleoside metabolism by salvage pathways and is a critical determinant of the pharmacologic actions of nucleoside drugs (3–6). By regulating adenosine availability to purinoreceptors, NTs also modulate a diverse array of physiological processes, including neurotransmission, immune responses, platelet aggregation, renal function, and coronary vasodilation (4, 6, 7). Two structurally unrelated NT families of integral membrane proteins exist in human and other mammalian cells and tissues as follows: the SLC28 concentrative nucleoside transporter (CNT) family and the SLC29 equilibrative nucleoside transporter (ENT) family (3, 4, 6, 8, 9). ENTs are normally present in most, possibly all, cell types (4, 6, 8). CNTs, in contrast, are found predominantly in intestinal and renal epithelia and other specialized cell types, where they have important roles in absorption, secretion, distribution, and elimination of nucleosides and nucleoside drugs (1–3, 5, 6, 9).The CNT protein family in humans is represented by three members, hCNT1, hCNT2, and hCNT3. Belonging to a CNT subfamily phylogenetically distinct from hCNT1/2, hCNT3 utilizes electrochemical gradients of both Na⁺ and H⁺ to accumulate a broad range of pyrimidine and purine nucleosides and nucleoside drugs within cells (10, 11). hCNT1 and hCNT2, in contrast, are Na⁺-specific and transport pyrimidine and purine nucleosides, respectively (11–13). Together, hCNT1–3 account for the three major concentrative nucleoside transport processes of human and other mammalian cells. Nonmammalian members of the CNT protein family that have been characterized functionally include hfCNT, a second member of the CNT3 subfamily from the ancient marine prevertebrate the Pacific hagfish Eptatretus stouti (14), CeCNT3 from Caenorhabditis elegans (15), CaCNT from Candida albicans (16), and the bacterial nucleoside transporter NupC from Escherichia coli (17). hfCNT is Na⁺- but not H⁺-coupled, whereas CeCNT3, CaCNT, and NupC are exclusively H⁺-coupled. Na⁺:nucleoside coupling stoichiometries are 1:1 for hCNT1 and hCNT2 and 2:1 for hCNT3 and hfCNT3 (11, 14). H⁺:nucleoside coupling ratios for hCNT3 and CaCNT are 1:1 (11, 16).Although much progress has been made in molecular studies of ENT proteins (4, 6, 8), studies of structurally and functionally important regions and residues within the CNT protein family are still at an early stage. Topological investigations suggest that hCNT1–3 and other eukaryote CNT family members have a 13 (or possibly 15)-transmembrane helix (TM) architecture, and multiple alignments reveal strong sequence similarities within the C-terminal half of the proteins (18). Prokaryotic CNTs lack the first three TMs of their eukaryotic counterparts, and functional expression of N-terminally truncated human and rat CNT1 in Xenopus oocytes has established that these three TMs are not required for Na⁺-dependent uridine transport activity (18). Consistent with this finding, chimeric studies involving hCNT1 and hfCNT (14) and hCNT1 and hCNT3 (19) have demonstrated that residues involved in Na⁺- and H⁺-coupling reside in the C-terminal half of the protein. Present in this region of the transporter, but of unknown function, is a highly conserved (G/A)XKX₃NEFVA(Y/M/F) motif common to all eukaryote and prokaryote CNTs.By virtue of their negative charge and consequent ability to interact directly with coupling cations and/or participate in cation-induced and other protein conformational transitions, glutamate and aspartate residues play key functional and structural roles in a broad spectrum of mammalian and bacterial cation-coupled transporters (20–30). Little, however, is known about their role in CNTs. This study builds upon a recent mutagenesis study of conserved glutamate and aspartate residues in hCNT1 (31) to undertake a parallel in depth investigation of corresponding residues in hCNT3. By employing the multifunctional capability of hCNT3 as a template for these studies, this study provides novel mechanistic insights into the molecular mechanism(s) of CNT-mediated cation/nucleoside cotransport, including the role of the (G/A)XKX₃NEFVA(Y/M/F) motif.     

Semaphorin3A Signaling Mediated by Fyn-dependent Tyrosine Phosphorylation of Collapsin Response Mediator Protein 2 at Tyrosine 32

Collapsin response mediator protein 2 (CRMP2) is an intracellular protein that mediates signaling of Semaphorin3A (Sema3A), a repulsive axon guidance molecule. Fyn, a Src-type tyrosine kinase, is involved in the Sema3A signaling. However, the relationship between CRMP2 and Fyn in this signaling pathway is still unknown. In our research, we demonstrated that Fyn phosphorylated CRMP2 at Tyr³² residues in HEK293T cells. Immunohistochemical analysis using a phospho-specific antibody at Tyr³² of CRMP showed that Tyr³²-phosphorylated CRMP was abundant in the nervous system, including dorsal root ganglion neurons, the molecular and Purkinje cell layer of adult cerebellum, and hippocampal fimbria. Overexpression of a nonphosphorylated mutant (Tyr³² to Phe³²) of CRMP2 in dorsal root ganglion neurons interfered with Sema3A-induced growth cone collapse response. These results suggest that Fyn-dependent phosphorylation of CRMP2 at Tyr³² is involved in Sema3A signaling.Collapsin response mediator proteins (CRMPs)⁴ have been identified as intracellular proteins that mediate Semaphorin3A (Sema3A) signaling in the nervous system (1). CRMP2 is one of the five members of the CRMP family. CRMPs also mediate signal transduction of NT3, Ephrin, and Reelin (2–4). CRMPs interact with several intracellular molecules, including tubulin, Numb, kinesin1, and Sra1 (5–8). CRMPs are involved in axon guidance, axonal elongation, cell migration, synapse maturation, and the generation of neuronal polarity (1, 2, 4, 5).CRMP family proteins are known to be the major phosphoproteins in the developing brain (1, 9). CRMP2 is phosphorylated by several Ser/Thr kinases, such as Rho kinase, cyclin-dependent kinase 5 (Cdk5), and glycogen synthase kinase 3β (GSK3β) (2, 10–13). The phosphorylation sites of CRMP2 by these kinases are clustered in the C terminus and have already been identified. Rho kinase phosphorylates CRMP2 at Thr⁵⁵⁵ (10). Cdk5 phosphorylates CRMP2 at Ser⁵²², and this phosphorylation is essential for sequential phosphorylations by GSK3β at Ser⁵¹⁸, Thr⁵¹⁴, and Thr⁵⁰⁹ (2, 11–13). These phosphorylations disrupt the interaction of CRMP2 with tubulin or Numb (2, 3, 13). The sequential phosphorylation of CRMP2 by Cdk5 and GSK3β is an essential step in Sema3A signaling (11, 13). Furthermore, the neurofibrillary tangles in the brains of people with Alzheimer disease contain hyperphosphorylated CRMP2 at Thr⁵⁰⁹, Ser⁵¹⁸, and Ser⁵²² (14, 15).CRMPs are also substrates of several tyrosine kinases. The phosphorylation of CRMP2 by Fes/Fps and Fer has been shown to be involved in Sema3A signaling (16, 17). Phosphorylation of CRMP2 at Tyr⁴⁷⁹ by a Src family tyrosine kinase Yes regulates CXCL12-induced T lymphocyte migration (18). We reported previously that Fyn is involved in Sema3A signaling (19). Fyn associates with PlexinA2, one of the components of the Sema3A receptor complex. Fyn also activates Cdk5 through the phosphorylation at Tyr¹⁵ of Cdk5 (19). In dorsal root ganglion (DRG) neurons from fyn-deficient mice, Sema3A-induced growth cone collapse response is attenuated compared with control mice (19). Furthermore, we recently found that Fyn phosphorylates CRMP1 and that this phosphorylation is involved in Reelin signaling (4). Although it has been shown that CRMP2 is involved in Sema3A signaling (1, 11, 13), the relationship between Fyn and CRMP2 in Sema3A signaling and the tyrosine phosphorylation site(s) of CRMPs remain unknown.Here, we show that Fyn phosphorylates CRMP2 at Tyr³². Using a phospho-specific antibody against Tyr³², we determined that the residue is phosphorylated in vivo. A nonphosphorylated mutant CRMP2Y32F inhibits Sema3A-induced growth cone collapse. These results indicate that tyrosine phosphorylation by Fyn at Tyr³² is involved in Sema3A signaling.     

Differential 14-3-3 Affinity Capture Reveals New Downstream Targets of Phosphatidylinositol 3-Kinase Signaling

We devised a strategy of 14-3-3 affinity capture and release, isotope differential (d₀/d₄) dimethyl labeling of tryptic digests, and phosphopeptide characterization to identify novel targets of insulin/IGF1/phosphatidylinositol 3-kinase signaling. Notably four known insulin-regulated proteins (PFK-2, PRAS40, AS160, and MYO1C) had high d₀/d₄ values meaning that they were more highly represented among 14-3-3-binding proteins from insulin-stimulated than unstimulated cells. Among novel candidates, insulin receptor substrate 2, the proapoptotic CCDC6, E3 ubiquitin ligase ZNRF2, and signaling adapter SASH1 were confirmed to bind to 14-3-3s in response to IGF1/phosphatidylinositol 3-kinase signaling. Insulin receptor substrate 2, ZNRF2, and SASH1 were also regulated by phorbol ester via p90RSK, whereas CCDC6 and PRAS40 were not. In contrast, the actin-associated protein vasodilator-stimulated phosphoprotein and lipolysis-stimulated lipoprotein receptor, which had low d₀/d₄ scores, bound 14-3-3s irrespective of IGF1 and phorbol ester. Phosphorylated Ser¹⁹ of ZNRF2 (RTRAYpS¹⁹GS), phospho-Ser⁹⁰ of SASH1 (RKRRVpS⁹⁰QD), and phospho- Ser⁴⁹³ of lipolysis-stimulated lipoprotein receptor (RPRARpS⁴⁹³LD) provide one of the 14-3-3-binding sites on each of these proteins. Differential 14-3-3 capture provides a powerful approach to defining downstream regulatory mechanisms for specific signaling pathways.Activated tyrosine kinase receptors generally drive cells to assimilate nutrients; regulate partitioning of the assimilate to make storage polymers and biosynthetic precursors and for energy production; and promote cellular survival, growth, division, movement, and differentiation. From this spectrum, each cell displays a specific subset of responses depending on the hormone, specific receptors, cross-talk from other signaling pathways, metabolic conditions, and cellular complement of effector proteins. For example, insulin stimulates glucose uptake and glycogen synthesis in skeletal muscle, whereas IGF1¹ promotes survival, growth, and proliferation of many cell types (1, 2).Many of these cellular responses are mediated via PI 3-kinase, which generates phosphatidylinositol 3,4,5-trisphosphate, promoting the activation of AGC protein kinases such as PKB/Akt and other signaling components (1, 3). PI 3-kinase is activated by binding to tyrosine-phosphorylated receptors such as the platelet-derived growth factor receptor or via adaptor molecules such as insulin receptor substrates, which are phosphorylated by the activated insulin receptor. Deregulated PI 3-kinase and downstream signaling has been linked to problems with wound healing, immune responses, neurodegeneration, and cardiovascular disease; decreased PI 3-kinase signaling may underlie insulin resistance and type II diabetes; and this pathway is often activated in human tumors (4, 5). To help pinpoint drug targets for these diseases we must define the mechanisms linking PI 3-kinase and other signaling pathways to downstream effectors and understand specificity with respect to different hormone/cell type combinations.Many missing substrates of PI 3-kinase/AGC kinases must be found to explain all the cellular responses to insulin and growth factors (3). Several targets of PI 3-kinase/PKB signaling, including TSC2 (6), PRAS40 (7), AS160 (8), and FYVE domain-containing phosphatidylinositol 3-phosphate 5-kinase (9) were identified using the anti-PAS antibody, which loosely recognizes the minimal phosphorylated consensus for PKB, which is RXRXX(pS/pT) where pS is phosphoserine and pT is phosphothreonine. Another helpful feature for identifying new downstream targets is that phosphorylation by PKB sometimes creates binding sites for 14-3-3s, which are dimeric proteins that bind to specific phosphorylated sites on target proteins. Thus PKB promotes the binding of 14-3-3s to proteins including PFKFB2 cardiac PFK-2 (10, 11), BimEL (12), β-catenin (13), p27(Kip1) (14), PRAS40 (7), FOXO1 (15), Miz1 (16), TBC1D4 (AS160 (17, 18), and TBC1D1 (19). Functionally 14-3-3s can trigger changes in the conformations of their targets and alter how targets interact with other proteins. Consistent with 14-3-3/target interactions being important in cellular responses to growth factors and insulin, reagents that compete with targets for binding to 14-3-3s inhibit the IGF1-stimulated increase in the glycolytic stimulator fructose-2,6-bisphosphate (10) and PKB-dependent cell survival (20).Some 14-3-3-binding sites on the above named proteins can also be phosphorylated by other basophilic protein kinases (21). For example, AS160 and TBC1D1 are two related RabGAPs (GTPase-activating protein for Rabs) regulated by multisite phosphorylation that regulate trafficking of GluT4 transporter to the plasma membrane for uptake of glucose. The two 14-3-3-binding sites on AS160 can be phosphorylated by PKB, p90RSK, serum- and glucocorticoid-inducible kinase, and other kinases, whereas one of the 14-3-3-binding sites on TBC1D1 is also a substrate of the energy-sensing kinase AMP-activated protein kinase (17–19). Thus, the relative sensitivity of glucose trafficking to insulin and AMP-activated protein kinase activators in different tissues may depend in part on the distribution of AS160 and TBC1D1. Other insulin-regulated 14-3-3 targets, such as myosin 1C (22), are also convergence points for phosphorylation by more than one AGC and/or Ca²⁺/calmodulin-dependent protein kinase.Here many more proteins than those already identified were found to display 14-3-3 and/or PAS binding signals when the PI 3-kinase pathway was activated in cells against a “background” of other proteins whose 14-3-3 and PAS binding status was unaffected by PI 3-kinase signaling. We aimed to pick out the PI 3-kinase-regulated proteins, which was challenging given the hundreds of 14-3-3 binding partners in mammalian cells (10, 23–27). We used 14-3-3 affinity capture and release, identified phosphopeptides, and devised a quantitative proteomics approach in which 14-3-3-binding proteins from insulin-stimulated versus unstimulated cells were labeled with formaldehyde containing light or heavy isotopes, respectively. Biochemical checking of candidates from these screens, which included proteins with links to diabetes, cancers, and neurodegenerative disorders, confirmed the identification of novel downstream targets of PI 3-kinase, some of which are also convergence points for regulation by MAPK/p90RSK signaling.     

Deletion of the Chloride Transporter Slc26a7 Causes Distal Renal Tubular Acidosis and Impairs Gastric Acid Secretion

SLC26A7 (human)/Slc26a7 (mouse) is a recently identified chloride-base exchanger and/or chloride transporter that is expressed on the basolateral membrane of acid-secreting cells in the renal outer medullary collecting duct (OMCD) and in gastric parietal cells. Here, we show that mice with genetic deletion of Slc26a7 expression develop distal renal tubular acidosis, as manifested by metabolic acidosis and alkaline urine pH. In the kidney, basolateral Cl⁻/HCO₃⁻ exchange activity in acid-secreting intercalated cells in the OMCD was significantly decreased in hypertonic medium (a normal milieu for the medulla) but was reduced only mildly in isotonic medium. Changing from a hypertonic to isotonic medium (relative hypotonicity) decreased the membrane abundance of Slc26a7 in kidney cells in vivo and in vitro. In the stomach, stimulated acid secretion was significantly impaired in isolated gastric mucosa and in the intact organ. We propose that SLC26A7 dysfunction should be investigated as a potential cause of unexplained distal renal tubular acidosis or decreased gastric acid secretion in humans.The collecting duct segment of the distal kidney nephron plays a major role in systemic acid base homeostasis by acid secretion and bicarbonate absorption. The acid secretion occurs via H⁺-ATPase and H-K-ATPase into the lumen and bicarbonate is absorbed via basolateral Cl⁻/HCO₃⁻ exchangers (1–4). The tubules, which are located within the outer medullary region of the kidney collecting duct (OMCD),² have the highest rate of acid secretion among the distal tubule segments and are therefore essential to the maintenance of acid base balance (2).The gastric parietal cell is the site of generation of acid and bicarbonate through the action of cytosolic carbonic anhydrase II (5, 6). The intracellular acid is secreted into the lumen via gastric H-K-ATPase, which works in conjunction with a chloride channel and a K⁺ recycling pathway (7–10). The intracellular bicarbonate is transported to the blood via basolateral Cl⁻/HCO₃⁻ exchangers (11–14).SLC26 (human)/Slc26 (mouse) isoforms are members of a conserved family of anion transporters that display tissue-specific patterns of expression in epithelial cells (15–24). Several SLC26 members can function as chloride/bicarbonate exchangers. These include SLC26A3 (DRA), SLC26A4 (pendrin), SLC26A6 (PAT1 or CFEX), SLC26A7, and SLC26A9 (25–31). SLC26A7 and SLC26A9 can also function as chloride channels (32–34).SLC26A7/Slc26a7 is predominantly expressed in the kidney and stomach (28, 29). In the kidney, Slc26a7 co-localizes with AE1, a well-known Cl⁻/HCO₃⁻ exchanger, on the basolateral membrane of (acid-secreting) A-intercalated cells in OMCD cells (29, 35, 36) (supplemental Fig. 1). In the stomach, Slc26a7 co-localizes with AE2, a major Cl⁻/HCO₃⁻ exchanger, on the basolateral membrane of acid secreting parietal cells (28). To address the physiological function of Slc26a7 in the intact mouse, we have generated Slc26a7 ko mice. We report here that Slc26a7 ko mice exhibit distal renal tubular acidosis and impaired gastric acidification in the absence of morphological abnormalities in kidney or stomach.     

Protein-tyrosine Phosphatase-�� and Src Functionally Link Focal Adhesions to the Endoplasmic Reticulum to Mediate Interleukin-1-induced Ca2+ Signaling

Calcium (Ca²⁺) signaling by the pro-inflammatory cytokine interleukin-1 (IL-1) is dependent on focal adhesions, which contain diverse structural and signaling proteins including protein phosphatases. We examined here the role of protein-tyrosine phosphatase (PTP) α in regulating IL-1-induced Ca²⁺ signaling in fibroblasts. IL-1 promoted recruitment of PTPα to focal adhesions and endoplasmic reticulum (ER) fractions, as well as tyrosine phosphorylation of the ER Ca²⁺ release channel IP₃R. In response to IL-1, catalytically active PTPα was required for Ca²⁺ release from the ER, Src-dependent phosphorylation of IP₃R1 and accumulation of IP₃R1 in focal adhesions. In pulldown assays and immunoprecipitations PTPα was required for the association of PTPα with IP₃R1 and c-Src, and this association was increased by IL-1. Collectively, these data indicate that PTPα acts as an adaptor to mediate functional links between focal adhesions and the ER that enable IL-1-induced Ca²⁺ signaling.The interleukin-1 (IL-1)³ family of pro-inflammatory cytokines mediates host responses to infection and injury. Impaired control of IL-1 signaling leads to chronic inflammation and destruction of extracellular matrices (1, 2), as seen in pathological conditions such as pulmonary fibrosis (3), rheumatoid arthritis (4, 5), and periodontitis (6). IL-1 elicits multiple signaling programs, some of which trigger Ca²⁺ release from the endoplasmic reticulum (ER) as well as expression of multiple cytokines and inflammatory factors including c-Fos and c-Jun (7, 8), and matrix metalloproteinases (9, 10), which mediate extracellular matrix degradation via mitogen-activated protein kinase-regulated pathways (11).In anchorage-dependent cells including fibroblasts and chondrocytes, focal adhesions (FAs) are required for IL-1-induced Ca²⁺ release from the ER and activation of ERK (12–14). FAs are actin-enriched adhesive domains composed of numerous (>50) scaffolding and signaling proteins (15–17). Many FA proteins are tyrosine-phosphorylated, including paxillin, focal adhesion kinase, and src family kinases, all of which are crucial for the assembly and disassembly of FAs (18–21). Protein-tyrosine phosphorylation plays a central role in regulating many cellular processes including adhesion (22, 23), motility (24), survival (25), and signal transduction (26–29). Phosphorylation of proteins by kinases is balanced by protein-tyrosine phosphatases (PTP), which can enhance or attenuate downstream signaling by dephosphorylation of tyrosine residues (30–32).PTPs can be divided into two main categories: receptor-like and intracellular PTPs (33). Two receptor-like PTPs have been localized to FA (leukocyte common antigen-related molecule and PTPα). Leukocyte common antigen-related molecule can dephosphorylate and mediate degradation of p130^cas, which ultimately leads to cell death (34, 35). PTPα contains a heavily glycosylated extracellular domain, a transmembrane domain, and two intracellular phosphatase domains (33, 36). The amino-terminal domain predominantly mediates catalytic activity, whereas the carboxyl-terminal domain serves a regulatory function (37, 38). PTPα is enriched in FA (23) and is instrumental in regulating FA dynamics (39) via activation of c-Src/Fyn kinases by dephosphorylating the inhibitory carboxyl tyrosine residue, namely Tyr⁵²⁹ (22, 40–42) and facilitation of integrin-dependent assembly of Src-FAK and Fyn-FAK complexes that regulate cell motility (43). Although PTPα has been implicated in formation and remodeling of FAs (44, 45), the role of PTPα in FA-dependent signaling is not defined.Ca²⁺ release from the ER is a critical step in integrin-dependent IL-1 signal transduction and is required for downstream activation of ERK (13, 46). The release of Ca²⁺ from the ER depends on the inositol 1,4,5-triphosphate receptor (IP₃R), which is an IP₃-gated Ca²⁺ channel (47). All of the IP₃R subtypes (subtypes 1–3) have been localized to the ER, as well as other the plasma membrane and other endomembranes (48–50). Further, IP₃R may associate with FAs, enabling the anchorage of the ER to FAs (51, 52). However, the molecule(s) that provide the structural link for this association has not been defined.FA-restricted, IL-1-triggered signal transduction in anchorage-dependent cells may rely on interacting proteins that are enriched in FAs and the ER (53). Here, we examined the possibility that PTPα associates with c-Src and IP₃R to functionally link FAs to the ER, thereby enabling IL-1 signal transduction.     

Copper-Binding Compounds from Methylosinus trichosporium OB3b

Two copper-binding compounds/cofactors (CBCs) were isolated from the spent media of both the wild type and a constitutive soluble methane monooxygenase (sMMO^C) mutant, PP319 (P. A. Phelps et al., Appl. Environ. Microbiol. 58:3701–3708, 1992), of Methylosinus trichosporium OB3b. Both CBCs are small polypeptides with molecular masses of 1,218 and 779 Da for CBC-L₁ and CBC-L₂, respectively. The amino acid sequence of CBC-L₁ is S?MYPGS?M, and that of CBC-L₂ is SPMP?S. Copper-free CBCs showed absorption maxima at 204, 275, 333, and 356 with shoulders at 222 and 400 nm. Copper-containing CBCs showed a broad absorption maximum at 245 nm. The low-temperature electron paramagnetic resonance (EPR) spectra of copper-containing CBC-L₁ showed the presence of a copper center with an EPR splitting constant between those of type 1 and type 2 copper centers (g_⊥ = 2.087, g_∥ = 2.42 G, |A_∥| = 128 G). The EPR spectrum of CBC-L₂ was more complex and showed two spectrally distinct copper centers. One signal can be attributed to a type 2 Cu²⁺ center (g_⊥ = 2.073, g_∥ = 2.324 G, |A_∥| = 144 G) which could be saturated at higher powers, while the second shows a broad, nearly isotropic signal near g_⊥ = 2.063. In wild-type strains, the concentrations of CBCs in the spent media were highest in cells expressing the pMMO and stressed for copper. In contrast to wild-type strains, high concentrations of CBCs were observed in the extracellular fraction of the sMMO^C mutants PP319 and PP359 regardless of the copper concentration in the culture medium.In methanotrophs, the relationship between the concentration of copper and expression of the two different methane monooxygenases (MMOs) is well characterized (8, 11, 45, 49, 50). Under low copper-to-biomass ratios, methane oxidation activity is observed in the soluble fraction, and the enzyme is referred to as the soluble methane monooxygenase (sMMO). At higher copper-to-biomass ratios, methane oxidation activity is observed in the membrane fraction, and the enzyme is referred to as the membrane-associated or particulate methane monooxygenase (pMMO). The polypeptides and structural genes for both enzymes have been characterized (4, 18–22, 24, 25, 32, 34–40, 43–45, 47–49, 51, 62, 63). In addition to expression of the two MMOs, four other physiological traits have been identified in cells expressing the pMMO that are affected by the copper concentration in the culture medium. First, the concentration of copper in the culture media is directly related to pMMO activity in cell-free fractions, although the levels of expression of pMMO polypeptides vary in different methanotrophs (1, 8, 30, 36, 50, 63). For example, the expression levels of the three pMMO polypeptides in Methylococcus capsulatus Bath remained constant with varying copper concentrations (8, 36), whereas in Methylomicrobium albus BG8, the expression level of the putative pMMO polypeptides increased with increased copper in the culture medium (8). Second, the concentrations of membrane-associated copper and iron show a proportional increase as the copper concentration in the culture medium is increased (36, 63). Third, the formation and level of intracytoplasmic membranes in cells cultured in copper-supplemented media are dependent on the copper concentration in the culture media (8, 11, 40, 48). Lastly, the K_s for methane oxidation by pMMO is altered by the copper concentration in the culture media (33a).Berson and Lidstrom (1) have recently noted that in spite of the central role of copper in the physiology of methanotrophs, the mechanism(s) of copper acquisition remains vague. Although true, a few studies have suggested the existence of a specific copper acquisition system in M. capsulatus Bath and M. trichosporium OB3b. The first indication of a specific copper uptake system was provided from phenotypic characterization of the constitutive sMMO mutants (sMMO^C) isolated by Phelps et al. (42). Fitch et al. (17) found that in M. trichosporium OB3b, these sMMO^C mutants were defective in copper uptake and showed preliminary evidence for an extracellular copper-complexing agent. Working with the same mutants, Téllez et al. partially purified this copper-complexing agent and determined that it was a small molecule with a molecular mass of approximately 500 Da with an association constant with copper of 1.4 × 10¹⁶ M⁻¹ (55). Other evidence for a specific copper uptake system was provided by the copper-binding cofactor (CBC) from M. capsulatus Bath (63). During the isolation of the pMMO from M. capsulatus Bath, CBC was identified in association with the purified enzyme, in the washed membrane fraction, and in the extracellular fraction. The CBC was determined to be a small polypeptide with a molecular mass of 1,232 Da. In M. capsulatus Bath, the cellular location of the CBC varied depending on the copper concentration in the culture medium and on the expression of the pMMO.This paper ties together and extends these observations on specific copper acquisition systems in M. trichosporium OB3b and M. capsulatus Bath. Here we describe the initial isolation and characterization of two copper-complexing agents, called CBC-L₁ and CBC-L₂, from the M. trichosporium OB3b wild type and sMMO^C mutant PP319. CBC-L₁ from M. trichosporium OB3b was identical to the CBC previously identified during the isolation of the pMMO from M. capsulatus Bath. This paper is also the first report of a second CBC, CBC-L₂, which may have been present as a contaminant in previous CBC preparations from M. capsulatus Bath. One or both of the CBCs appear to be the same copper-complexing agent partially purified by Téllez et al. (55). Lastly, this report describes the effect of the copper concentration in the culture medium on copper uptake, the expression of both MMOs, and extracellular concentration of the CBC in wild-type and sMMO^C mutant strains of M. trichosporium OB3b.     

Structure of Insoluble Rat Sperm Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) via Heterotetramer Formation with Escherichia coli GAPDH Reveals Target for Contraceptive Design

Sperm glyceraldehyde-3-phosphate dehydrogenase has been shown to be a successful target for a non-hormonal contraceptive approach, but the agents tested to date have had unacceptable side effects. Obtaining the structure of the sperm-specific isoform to allow rational inhibitor design has therefore been a goal for a number of years but has proved intractable because of the insoluble nature of both native and recombinant protein. We have obtained soluble recombinant sperm glyceraldehyde-3-phosphate dehydrogenase as a heterotetramer with the Escherichia coli glyceraldehyde-3-phosphate dehydrogenase in a ratio of 1:3 and have solved the structure of the heterotetramer which we believe represents a novel strategy for structure determination of an insoluble protein. A structure was also obtained where glyceraldehyde 3-phosphate binds in the P_s pocket in the active site of the sperm enzyme subunit in the presence of NAD. Modeling and comparison of the structures of human somatic and sperm-specific glyceraldehyde-3-phosphate dehydrogenase revealed few differences at the active site and hence rebut the long presumed structural specificity of 3-chlorolactaldehyde for the sperm isoform. The contraceptive activity of α-chlorohydrin and its apparent specificity for the sperm isoform in vivo are likely to be due to differences in metabolism to 3-chlorolactaldehyde in spermatozoa and somatic cells. However, further detailed analysis of the sperm glyceraldehyde-3-phosphate dehydrogenase structure revealed sites in the enzyme that do show significant difference compared with published somatic glyceraldehyde-3-phosphate dehydrogenase structures that could be exploited by structure-based drug design to identify leads for novel male contraceptives.Glyceraldehyde-3-phosphate dehydrogenase-S (GAPDS³ in rat; GAPDH2 in human) is the sperm-specific isoform of GAPDH (1–3) and the sole GAPDH enzyme in sperm. GAPDS is highly conserved between species showing 94% identity between rat and mouse and 87% identity between rat and human. Within a particular species, GAPDS also shows significant sequence identity to its GAPDH paralogue, 70, 70, and 68% for rat, mouse, and human, respectively. The most striking difference between GAPDS and GAPDH is the presence of an N-terminal polyproline region in GAPDS, which is 97 residues in rat (accession number {"type":"entrez-nucleotide","attrs":{"text":"AJ297631","term_id":"9931190","term_text":"AJ297631"}}AJ297631), 105 in mouse (3), and 72 in human (2). GAPDS is restricted to the principal piece of the sperm flagellum (1, 2, 4) where it is localized to the fibrous sheath (5), an association proposed to be mediated via the N-terminal polyproline extension.GAPDS first came to prominence as a contraceptive target during the 1970s (6–8). Investigations showed that treatment of sperm with α-chlorohydrin or a number of related compounds could inhibit GAPDS activity (9–11), sperm motility (9–13), and the fertilization of oocytes in vitro (14). The metabolite of these compounds, 3-chlorolactaldehyde (15–17), selectively inhibited GAPDS, having no effect on the activity of somatic cell GAPDH (18, 19), providing the specificity required for a potential contraceptive. Questions surrounding these particular compounds were raised when a number of side effects were evident from in vivo trials (7, 20–22); however, the design of small molecule inhibitors of GAPDS may provide a viable alternative. Its potential as a contraceptive target was supported by data from mice where GAPDS^−/− males (23) were infertile because of defects in sperm motility.Glyceraldehyde-3-phosphate dehydrogenases are tetrameric enzymes that catalyze the oxidative phosphorylation of d-glyceraldehyde 3-phosphate (Glc-3-P) into 1,3-diphosphoglycerate in the presence of an NAD cofactor via a two-step chemical mechanism (24). The first models of substrate binding were proposed on the basis of crystal structures of the holoenzyme from lobster (25) and Bacillus stearothermophilus (26), and Moras and co-workers (25) identified two anion-binding sites postulated to correspond to those binding the C-3 phosphate group of d-Glc-3-P (P_s site) and the inorganic phosphate ion (P_i site).Structure-based design of small molecules to inhibit GAPDH is not unprecedented. GAPDH has been targeted from protozoan parasites (27–30), as the bloodstream forms rely solely on glycolysis for energy production (31, 32). A number of mammalian GAPDH structures have also been solved, including rabbit muscle (33, 34), human liver (35), and human placenta (36); however, no structures are available for sperm-specific isoforms of this enzyme.Active heterotetramers of GAPDH between different species have been reported and biochemically characterized previously, both in ratios of 2:2 and 3:1 (37–40). In this study we have successfully obtained crystals of rat recombinant GAPDS as a heterotetramer with Escherichia coli GAPDH in a 1:3 ratio. To understand the basis of inhibition of the sperm isoform by substrate analogue 3-chlorolactaldehyde, a metabolite of α-chlorohydrin, a structure was also determined in the presence of the substrate glyceraldehyde 3-phosphate. The sperm-specific structure was compared with the human placental GAPDH structure (PDB entry 1U8F; Ref. 36) to identify differences that may provide a target for the design of inhibitors specific to the GAPDS protein. The unique structural features identified offer potential candidates for further investigation as inhibitor targets.     

Substituted Cysteine Accessibility Method Analysis of Human Concentrative Nucleoside Transporter hCNT3 Reveals a Novel Discontinuous Region of Functional Importance within the CNT Family Motif (G/A)XKX3NEFVA(Y/M/F)

The human SLC28 family of integral membrane CNT (concentrative nucleoside transporter) proteins has three members, hCNT1, hCNT2, and hCNT3. Na⁺-coupled hCNT1 and hCNT2 transport pyrimidine and purine nucleosides, respectively, whereas hCNT3 mediates transport of both pyrimidine and purine nucleosides utilizing Na⁺ and/or H⁺ electrochemical gradients. These and other eukaryote CNTs are currently defined by a putative 13-transmembrane helix (TM) topology model with an intracellular N terminus and a glycosylated extracellular C terminus. Recent mutagenesis studies, however, have provided evidence supporting an alternative 15-TM membrane architecture. In the absence of CNT crystal structures, valuable information can be gained about residue localization and function using substituted cysteine accessibility method analysis with thiol-reactive reagents, such as p-chloromercuribenzene sulfonate. Using heterologous expression in Xenopus oocytes and the cysteineless hCNT3 protein hCNT3C−, substituted cysteine accessibility method analysis with p-chloromercuribenzene sulfonate was performed on the TM 11–13 region, including bridging extramembranous loops. The results identified residues of functional importance and, consistent with a new revised 15-TM CNT membrane architecture, suggest a novel membrane-associated topology for a region of the protein (TM 11A) that includes the highly conserved CNT family motif (G/A)XKX₃NEFVA(Y/M/F).Specialized nucleoside transporter proteins are required for passage of nucleosides and hydrophilic nucleoside analogs across biological membranes. Physiologically, nucleosides serve as nucleotide precursors in salvage pathways, and pharmacologically nucleoside analogs are used as chemotherapeutic agents in the treatment of cancer and antiviral diseases (1, 2). Additionally, adenosine modulates numerous cellular events via purino-receptor cell signaling pathways, including neurotransmission, vascular tone, immune responses, and other physiological processes (3, 4).Human nucleoside transporter proteins are divided into two families: the SLC29 ENT (equilibrative nucleoside transporter) family and the SLC28 CNT (concentrative nucleoside transporter) family (3, 5–7). hENTs³ mediate bidirectional fluxes of purine and pyrimidine nucleosides down their concentration gradients and are ubiquitously found in most, possibly all, cell types (8). Additionally, the hENT2 isoform is capable of nucleobase transport (9). hCNTs, in contrast, are inwardly directed Na⁺-dependent nucleoside transporters found predominantly in intestinal and renal epithelial and other specialized cell types (10, 11). hCNT1 and hCNT2 are pyrimidine and purine nucleoside-selective, respectively, and couple Na⁺/nucleoside cotransport with 1:1 stoichiometry (12–18). In contrast, hCNT3 is broadly selective for both pyrimidine and purine nucleosides and couples Na⁺/nucleoside cotransport with 2:1 stoichiometry (10, 18, 19). hCNT3 is also capable of H⁺/nucleoside cotransport with a coupling stoichiometry of 1:1, whereby one of the two Na⁺ binding sites also functionally interacts with H⁺ (18, 19).Current models of CNT topology have 13 putative transmembrane helices (TMs) (10, 14, 16, 20). Two additional TMs (designated 5A and 11A) are weakly predicted by computer algorithms (20), and immunocytochemical experiments with site-specific antibodies and studies of native and introduced glycosylation sites have confirmed an intracellular N terminus and an extracellular C terminus (20). Chimeric studies involving hCNTs and hfCNT, a CNT from the ancient marine prevertebrate, the Pacific hagfish Eptatretus stouti, have revealed that the functional domains responsible for CNT nucleoside selectivity and cation coupling reside within the C-terminal TM 7–13 half of the protein (19, 21). NupC, an H⁺-coupled CNT family member from Escherichia coli, lacks TMs 1–3 but otherwise shares a topology similar to that of its eukaryote counterparts (22, 23).A functional cysteineless version of hCNT3 has been generated by mutagenesis of endogenous cysteine residues to serine, resulting in the cysteineless construct hCNT3C− employed originally in a yeast expression system for substituted cysteine accessibility method (SCAM) analysis of TMs 11, 12, and 13 using methanethiosulfonate (MTS) reagents (24). Subsequently, we have also characterized hCNT3C− in the Xenopus oocyte expression system (25) and have initiated SCAM analyses with the alternative thiol-specific reagent p-chloromercuribenzene sulfonate (PCMBS) (26). Measured by transport inhibition, reactivity of introduced cysteine residues with PCMBS, which is both membrane-impermeant and hydrophilic, indicates pore-lining status and access from the extracellular medium; the ability of a permeant to protect against this inhibition denotes location within, or closely adjacent to, the permeant-binding pocket (27, 28). Continuing the investigation of hCNT3 C-terminal membrane topology and function, the present study reports results of PCMBS SCAM analyses of TMs 11–13, including loop regions linking the putative TMs not previously studied using MTS reagents.In earlier structure/function studies of hCNT3, we identified a cluster of conformationally sensitive residue positions in TM 12 (Ile⁵⁵⁴, Tyr⁵⁵⁸, and Cys⁵⁶¹) that exhibit H⁺-activated inhibition by PCMBS, with uridine protection evident for Tyr⁵⁵⁸ and Cys⁵⁶¹ (26). Located deeper within the plane of the membrane, other uridine-protectable residue positions in TM 12 were PCMBS-sensitive in both H⁺- and Na⁺-containing media (26). hCNT3 Glu⁵¹⁹ and the corresponding residue in hCNT1 (Glu⁴⁹⁸) in region TM 11A were also identified as having key roles in permeant and cation binding and translocation (29, 30), and hCNT3 E519C showed inhibition of uridine uptake by PCMBS (30). Centrally positioned within the highly conserved CNT family motif (G/A)XKX₃NEFVA(Y/M/F), residue 519 is proposed to be a direct participant in cation coupling via the common hCNT3 Na⁺/H⁺-binding site that, in other CNTs, is either Na⁺-specific (e.g. hCNT1) or H⁺-specific (e.g. NupC) (30).Building upon the prior work with MTS reagents and other structure/function studies of hCNT3, the present study identified new residues of functional importance in the C-terminal one-third of hCNT3, established the orientations and α-helical structures of TMs 11–13, and determined a novel membrane-associated topology for the TM 11A region of the protein. A revised CNT membrane architecture is proposed.     

Sac3 Is an Insulin-regulated Phosphatidylinositol 3,5-Bisphosphate Phosphatase: GAIN IN INSULIN RESPONSIVENESS THROUGH Sac3 DOWN-REGULATION IN ADIPOCYTES*

Insulin-regulated stimulation of glucose entry and mobilization of fat/muscle-specific glucose transporter GLUT4 onto the cell surface require the phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P₂) pathway for optimal performance. The reduced insulin responsiveness observed under ablation of the PtdIns(3,5)P₂-synthesizing PIKfyve and its associated activator ArPIKfyve in 3T3L1 adipocytes suggests that dysfunction of the PtdIns(3,5)P₂-specific phosphatase Sac3 may yield the opposite effect. Paradoxically, as uncovered recently, in addition to turnover Sac3 also supports PtdIns(3,5)P₂ biosynthesis by allowing optimal PIKfyve-ArPIKfyve association. These opposing inputs raise the key question as to whether reduced Sac3 protein levels and/or hydrolyzing activity will produce gain in insulin responsiveness. Here we report that small interfering RNA-mediated knockdown of endogenous Sac3 by ∼60%, which resulted in a slight but significant elevation of PtdIns(3,5)P₂ in 3T3L1 adipocytes, increased GLUT4 translocation and glucose entry in response to insulin. In contrast, ectopic expression of Sac3^WT, but not phosphatase-deficient Sac3^D488A, reduced GLUT4 surface abundance in the presence of insulin. Endogenous Sac3 physically assembled with PIKfyve and ArPIKfyve in both membrane and soluble fractions of 3T3L1 adipocytes, but this remained insulin-insensitive. Importantly, acute insulin markedly reduced the in vitro C8-PtdIns(3,5)P₂ hydrolyzing activity of Sac3. The insulin-sensitive Sac3 pool likely controls a discrete PtdIns(3,5)P₂ subfraction as the high pressure liquid chromatography-measurable insulin-dependent elevation in total [³H]inositol-PtdIns(3,5)P₂ was minor. Together, our data identify Sac3 as an insulin-sensitive phosphatase whose down-regulation increases insulin responsiveness, thus implicating Sac3 as a novel drug target in insulin resistance.Insulin simulation of glucose uptake in fat and muscle, which is mediated by the facilitative fat/muscle-specific glucose transporter GLUT4, is essential for maintenance of whole-body glucose homeostasis (1–7). In basal states GLUT4 is localized in the cell interior, cycling slowly between the plasma membrane and one or more intracellular compartments. Insulin action profoundly activates movements of preformed postendosomal GLUT4 storage vesicles toward the cell surface and their subsequent plasma membrane fusion, thereby increasing the rate of glucose transport >10-fold. Defective signaling/execution of GLUT4 translocation is considered to be a common feature in insulin resistance and type 2 diabetes (8, 9). However, the molecular and cellular regulatory mechanisms whereby insulin activates GLUT4 membrane dynamics and glucose transport are still not fully understood. More than 60 protein and phospholipid intermediate players are currently implicated in orchestrating the overall process (1–7). A central role is attributed to the highest phosphorylated member of the phosphoinositide (PI)³ family, i.e. phosphatidylinositol (PtdIns) (3,4,5)P₃ (3). PtdIns(3,4,5)P₃ is generated at the cell surface by the action of wortmannin-sensitive class 1A PI3K that is activated via the insulin-stimulated IR/IR receptor substrate signaling pathway. Inositol polyphosphate 5-phosphatases SHIP or SKIP and 3-phosphatase PTEN rapidly convert PtdIns(3,4,5)P₃ to PtdIns(3,4)P₂ and PtdIns(4,5)P₂, respectively, thereby terminating insulin signal through class 1A PI3K (10–13). The class 1A PI3K-opposing function of these lipid phosphatases has provided an appealing prospect that inhibition of their hydrolyzing activities could produce significant efficacy in the treatment of type 2 diabetes and obesity (14–16).It has recently become apparent that signals by other PIs act in parallel with that of PtdIns(3,4,5)P₃ in integrating the IR-issued signal with GLUT4 surface translocation (3, 4). One such signaling molecule is PtdIns(3,5)P₂, whose functioning as a positive regulator in 3T3L1 adipocyte responsiveness to insulin has been supported by several lines of experimental evidence. Thus, expression of dominant-negative kinase-deficient mutants of PIKfyve, the sole enzyme for PtdIns(3,5)P₂ synthesis (17, 18), inhibits insulin-induced gain of surface GLUT4 without noticeable aberrations of cell morphology (19). Likewise, reduction in the intracellular PtdIns(3,5)P₂ pool through siRNA-mediated PIKfyve depletion reduces GLUT4 cell-surface accumulation and glucose transport activation in response to insulin (20). Concordantly, loss of ArPIKfyve, a PIKfyve activator that physically associates with PIKfyve to facilitate PtdIns(3,5)P₂ intracellular production (21, 22), also decreases insulin-stimulated glucose uptake in 3T3L1 adipocytes (20). Combined ablation of PIKfyve and ArPIKfyve produces a greater decrease in this effect, correlating with a greater reduction in the intracellular PtdIns(3,5)P₂ pool (20). Finally, pharmacological inhibition of PIKfyve activity powerfully reduces the net insulin effect on glucose uptake (23). These observations indicate positive signaling through the PtdIns(3,5)P₂ pathway and suggest that arrested PtdIns(3,5)P₂ turnover might potentiate insulin-regulated activation of glucose uptake.Sac3, a product of a single-copy gene in mammals, is a recently characterized phosphatase implicated in PtdIns(3,5)P₂ turnover (24). Our observations in several mammalian cell types have revealed that Sac3 plays an intricate role in the PtdIns(3,5)P₂ homeostatic mechanism. It is a constituent of the PtdIns(3,5)P₂ biosynthetic PIKfyve-ArPIKfyve complex and facilitates the association of these two (24, 25). Intriguingly, only if the PIKfyve-ArPIKfyve-Sac3 triad (known as the “PAS complex”) is intact will the PIKfyve enzymatic activity be activated (25). Thus, Sac3 not only catalyzes PtdIns(3,5)P₂ turnover but also promotes PtdIns(3,5)P₂ synthesis by functioning as an adaptor for the efficient association of PIKfyve with, and activation by, ArPIKfyve (25). Given these two seemingly opposing inputs, a critical question is whether reduction in Sac3 protein levels or phosphatase activity would facilitate or mitigate insulin action on glucose uptake and GLUT4 translocation. We demonstrate here that reduced levels of Sac3 potentiate, whereas ectopic expression of active Sac3 phosphatase reduces insulin responsiveness of GLUT4 translocation and glucose transport in 3T3L1 adipocytes. Whereas insulin action does not affect the PIKfyve kinase-Sac3 phosphatase association, it markedly inhibits the Sac3 hydrolyzing activity. We suggest that increased PtdIns(3,5)P₂ local availability through Sac3 phosphatase inhibition links insulin signaling to its effect on GLUT4 vesicle dynamics and glucose transport.     

Differential Regulation of Cell Type-specific Apoptosis by Stromelysin-3: A POTENTIAL MECHANISM VIA THE CLEAVAGE OF THE LAMININ RECEPTOR DURING TAIL RESORPTION IN XENOPUS LAEVIS*

Matrix metalloproteinases (MMPs) have been extensively studied because of their functional attributes in development and diseases. However, relatively few in vivo functional studies have been reported on the roles of MMPs in postembryonic organ development. Amphibian metamorphosis is a unique model for studying MMP function during vertebrate development because of its dependence on thyroid hormone (T3) and the ability to easily manipulate this process with exogenous T3. The MMP stromelysin-3 (ST3) is induced by T3, and its expression correlates with cell death during metamorphosis. We have previously shown that ST3 is both necessary and sufficient for larval epithelial cell death in the remodeling intestine. To investigate the roles of ST3 in other organs and especially on different cell types, we have analyzed the effect of transgenic overexpression of ST3 in the tail of premetamorphic tadpoles. We report for the first time that ST3 expression, in the absence of T3, caused significant muscle cell death in the tail of premetamorphic transgenic tadpoles. On the other hand, only relatively low levels of epidermal cell death were induced by precocious ST3 expression in the tail, contrasting what takes place during natural and T3-induced metamorphosis when ST3 expression is high. This cell type-specific apoptotic response to ST3 in the tail suggests distinct mechanisms regulating cell death in different tissues. Furthermore, our analyses of laminin receptor, an in vivo substrate of ST3 in the intestine, suggest that laminin receptor cleavage may be an underlying mechanism for the cell type-specific effects of ST3.The extracellular matrix (ECM),³ the dynamic milieu of the cell microenvironment, plays a critical role in dictating the fate of the cell. The cross-talk between the cell and ECM and the timely catabolism of the ECM are crucial for tissue remodeling during development (1). Matrix metalloproteinases (MMPs), extrinsic proteolytic regulators of the ECM, mediate this process to a large extent. MMPs are a large family of Zn²⁺-dependent endopeptidases potentially capable of cleaving the extracellular as well as nonextracellular proteins (2–9). The MMP superfamily includes collagenases, gelatinases, stromelysins, and membrane-type MMPs based on substrate specificity and domain organization (2–4). MMPs have been implicated to influence a wide range of physiological and pathological processes (10–13). The roles of MMPs appear to be very complex. For example, MMPs have been suggested to play roles in both tumor promotion and suppression (13–19). Unfortunately, relatively few functional studies have been carried out in vivo, especially in relation to the mechanisms involved during vertebrate development.Amphibian metamorphosis presents a fascinating experimental model to study MMP function during postembryonic development. A unique and salient feature of the metamorphic process is the absolute dependence on the signaling of thyroid hormone (20–23). This makes it possible to prevent metamorphosis by simply inhibiting the synthesis of endogenous T3 or to induce precocious metamorphosis by merely adding physiological levels of T3 in the rearing water of premetamorphic tadpoles. Gene expression screens have identified the MMP stromelysin-3 (ST3) as a direct T3 response gene (24–27). Expression studies have revealed a distinct spatial and temporal ST3 expression profile in correlation with metamorphic event, especially cell death (25, 28–31). Organ culture studies on intestinal remodeling have directly substantiated an essential role of ST3 in larval epithelial cell death and ECM remodeling (32). Furthermore, precocious expression of ST3 alone in premetamorphic tadpoles through transgenesis is sufficient to induce ECM remodeling and larval epithelial apoptosis in the tadpole intestine (33). Thus, ST3 appears to be necessary and sufficient for intestinal epithelial cell death during metamorphosis.ST3 was first isolated as a breast cancer-associated gene (34), and unlike most other MMPs, ST3 is secreted as an active protease through a furin-dependent intracellular activation mechanism (35). Like many other MMPs, ST3 is expressed in a number of pathological processes, including most human carcinomas (11, 36–40), as well as in many developmental processes in mammals (10, 34, 41–43), although the physiological and pathological roles of ST3 in vivo are largely unknown in mammals. Interestingly, compared with other MMPs, ST3 has only weak activities toward ECM proteins in vitro but stronger activities against non-ECM proteins like α1 proteinase inhibitor and IGFBP-1 (44–46). Although ST3 may cleave ECM proteins strongly in the in vivo environment, these findings suggest that the cleavage of non-ECM proteins is likely important for its biological roles. Consistently, we have recently identified a cell surface receptor, laminin receptor (LR) as an in vivo substrate of ST3 in the tadpole intestine during metamorphosis (47–49). Analyses of LR expression and cleavage suggest that LR cleavage by ST3 is likely an important mechanism by which ST3 regulates the interaction between the larval epithelial cells and the ECM to induce cell death during intestinal remodeling (47, 48).Here, to investigate the role of ST3 in the apoptosis in other tissues during metamorphosis and whether LR cleavage serves as a mechanism for ST3 to regulate the fate of different cell types, we have analyzed the effects of precocious expression of ST3 in premetamorphic tadpole tail. The tail offers an opportunity to examine the effects of ST3 on different cell types. The epidermis, the fast and slow muscles, and the connective tissue underlying the epidermis in the myotendinous junctions and surrounding the notochord constitute the major tissue types in tail (50). Even though death is the destiny of all these cell types, it is not clear whether they all die through similar or different mechanisms. Microscopic and histochemical analyses have shown that at least the muscle and epidermal cells undergo T3-dependent apoptosis during metamorphosis (23, 29, 51, 52). To study whether ST3 regulates apoptosis of these two cell types, we have made use of the transgenic animals that express a transgenic ST3 under the control of a heat shock-inducible promoter (33). We show that whereas extensive apoptosis is present in both the epidermis and muscles during natural as well as T3-induced metamorphosis, transgenic expression of ST3 induces cell death predominantly in the muscles. Furthermore, we show that LR is expressed in the epidermis and connective tissue but not in muscles of the tadpole tail. More importantly, LR cleavage products are present in the tail during natural metamorphosis but not in transgenic tadpoles overexpressing ST3. These results suggest that ST3 has distinct effects on the epidermis and muscles in the tail, possibly because of the tissue-specific expression and function of LR.