Structural snapshots of the catalytic cycle of the phosphodiesterase Autotaxin.

Autotaxin (ATX) is a secreted phosphodiesterase that produces the signalling lipid lysophosphatidic acid (LPA). The bimetallic active site of ATX is structurally related to the alkaline phosphatase superfamily. Here, we present a new crystal structure of ATX in complex with orthovanadate (ATX-VO5), which binds the Oγ nucleophile of Thr209 and adopts a trigonal bipyramidal conformation, following the nucleophile attack onto the substrate. We have now a portfolio of ATX structures we discuss as intermediates of the catalytic mechanism: the new ATX-VO5 structure; a unique structure where the nucleophile Thr209 is phosphorylated (ATX-pThr). Comparing these to a complex with the LPA product (ATX-LPA) and with a complex with a phosphate ion (ATX-PO4), that represent the Michaelis complex of the reaction, we observe movements of Thr209, changes in the relative displacement of the zinc ions, and a water molecule that likely fulfils the second nucleophilic attack. We propose that ATX follows the associative two-step in-line displacement mechanism.

The polybasic insertion in autotaxin α confers specific binding to heparin and cell surface heparan sulfate proteoglycans.

Autotaxin (ATX) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), playing a key role in diverse physiological and pathological processes. ATX exists in distinct splice variants, but isoform-specific functions remain elusive. Here we characterize the ATXα isoform, which differs from the canonical form (ATXß) in having a 52-residue polybasic insertion of unknown function in the catalytic domain. We find that the ATXα insertion is susceptible to cleavage by extracellular furin-like endoproteases, but cleaved ATXα remains structurally and functionally intact due to strong interactions within the catalytic domain. Through ELISA and surface plasmon resonance assays, we show that ATXα binds specifically to heparin with high affinity (K(d) ~10(-8) M), whereas ATXß does not; furthermore, heparin moderately enhanced the lysophospholipase D activity of ATXα. We further show that ATXα, but not ATXß, binds abundantly to SKOV3 carcinoma cells. ATXα binding was abolished after treating the cells with heparinase III, but not after chondroitinase treatment. Thus, the ATXα insertion constitutes a cleavable heparin-binding domain that mediates interaction with heparan sulfate proteoglycans, thereby targeting LPA production to the plasma membrane.

Regulation of connexin43 gap junctional communication by phosphatidylinositol 4,5-bisphosphate.

Cell-cell communication through connexin43 (Cx43)-based gap junction channels is rapidly inhibited upon activation of various G protein-coupled receptors; however, the mechanism is unknown. We show that Cx43-based cell-cell communication is inhibited by depletion of phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P(2)) from the plasma membrane. Knockdown of phospholipase Cbeta3 (PLCbeta3) inhibits PtdIns(4,5)P(2) hydrolysis and keeps Cx43 channels open after receptor activation. Using a translocatable 5-phosphatase, we show that PtdIns(4,5)P(2) depletion is sufficient to close Cx43 channels. When PtdIns(4,5)P(2) is overproduced by PtdIns(4)P 5-kinase, Cx43 channel closure is impaired. We find that the Cx43 binding partner zona occludens 1 (ZO-1) interacts with PLCbeta3 via its third PDZ domain. ZO-1 is essential for PtdIns(4,5)P(2)-hydrolyzing receptors to inhibit cell-cell communication, but not for receptor-PLC coupling. Our results show that PtdIns(4,5)P(2) is a key regulator of Cx43 channel function, with no role for other second messengers, and suggest that ZO-1 assembles PLCbeta3 and Cx43 into a signaling complex to allow regulation of cell-cell communication by localized changes in PtdIns(4,5)P(2).

Structural basis of substrate discrimination and integrin binding by autotaxin.

Autotaxin (ATX, also known as ectonucleotide pyrophosphatase/phosphodiesterase-2, ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemoattractant for many cell types. ATX-LPA signaling is involved in various pathologies including tumor progression and inflammation. However, the molecular basis of substrate recognition and catalysis by ATX and the mechanism by which it interacts with target cells are unclear. Here, we present the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. We have identified a hydrophobic lipid-binding pocket and mapped key residues for catalysis and selection between nucleotide and phospholipid substrates. We have shown that ATX interacts with cell-surface integrins through its N-terminal somatomedin B-like domains, using an atypical mechanism. Our results define determinants of substrate discrimination by the ENPP family, suggest how ATX promotes localized LPA signaling and suggest new approaches for targeting ATX with small-molecule therapeutic agents.

Spatiotemporal regulation of chloride intracellular channel protein CLIC4 by RhoA.

Chloride intracellular channel (CLIC) 4 is a soluble protein structurally related to omega-type glutathione-S-transferases (GSTs) and implicated in various biological processes, ranging from chloride channel formation to vascular tubulogenesis. However, its function(s) and regulation remain unclear. Here, we show that cytosolic CLIC4 undergoes rapid but transient translocation to discrete domains at the plasma membrane upon stimulation of G(13)-coupled, RhoA-activating receptors, such as those for lysophosphatidic acid, thrombin, and sphingosine-1-phosphate. CLIC4 recruitment is strictly dependent on Galpha(13)-mediated RhoA activation and F-actin integrity, but not on Rho kinase activity; it is constitutively induced upon enforced RhoA-GTP accumulation. Membrane-targeted CLIC4 does not seem to enter the plasma membrane or modulate transmembrane chloride currents. Mutational analysis reveals that CLIC4 translocation depends on at least six conserved residues, including reactive Cys35, whose equivalents are critical for the enzymatic function of GSTs. We conclude that CLIC4 is regulated by RhoA to be targeted to the plasma membrane, where it may function not as an inducible chloride channel but rather by displaying Cys-dependent transferase activity toward a yet unknown substrate.

Direct measurement of cyclic AMP diffusion and signaling through connexin43 gap junctional channels.

Gap junctions (GJ) are clusters of transmembrane channels that allow direct cell-to-cell transfer of ions and small molecules. The GJ-permeant signaling molecule cAMP is of particular interest because of its numerous cellular effects. However, to assess the biological relevance of GJ-mediated cAMP transfer, quantitative aspects must be determined. Here we employed cAMP indicators based on fluorescence resonance energy transfer (FRET) to study propagation of cAMP signals to neighbor cells through connexin43 (Cx43)-based gap junctions in Rat-1 cells quantitatively. Intracellular cAMP levels were selectively raised in single cells by either photorelease of caged cAMP or stimulation of G(s)-coupled receptors. cAMP elevations spread to adjacent cells within seconds in a Cx43-dependent manner. We determined that Rat-1 cells follow cAMP rises in surrounding monolayer cells to approx. 40% in amplitude. This degree of cAMP transfer sufficed to evoke a well-characterized response to cAMP in neighbor cells, i.e. the PKA-mediated phosphorylation of the ER transcription factor in A431 carcinoma cells. We conclude that contacting cells can cooperatively regulate cAMP-sensitive processes via gap junctional diffusion of cAMP.