Abstract: Selective autophagy can be mediated via receptor molecules that link specific cargoes to the autophagosomal membranes decorated by ubiquitin-like microtubule-associated protein light chain 3 (LC3) modifiers. Although several autophagy receptors have been identified, little is known about mechanisms controlling their functions in vivo. In this work, we found that phosphorylation of an autophagy receptor, optineurin, promoted selective autophagy of ubiquitin-coated cytosolic Salmonella enterica. The protein kinase TANK binding kinase 1 (TBK1) phosphorylated optineurin on serine-177, enhancing LC3 binding affinity and autophagic clearance of cytosolic Salmonella. Conversely, ubiquitin- or LC3-binding optineurin mutants and silencing of optineurin or TBK1 impaired Salmonella autophagy, resulting in increased intracellular bacterial proliferation. We propose that phosphorylation of autophagy receptors might be a general mechanism for regulation of cargo-selective autophagy.

Abstract: O-GlcNAcylation is the addition of β-D-N-acetylglucosamine to serine or threonine residues of nuclear and cytoplasmic proteins. O-linked N-acetylglucosamine (O-GlcNAc) was not discovered until the early 1980s and still remains difficult to detect and quantify. Nonetheless, O-GlcNAc is highly abundant and cycles on proteins with a timescale similar to protein phosphorylation. O-GlcNAc occurs in organisms ranging from some bacteria to protozoans and metazoans, including plants and nematodes up the evolutionary tree to man. O-GlcNAcylation is mostly on nuclear proteins, but it occurs in all intracellular compartments, including mitochondria. Recent glycomic analyses have shown that O-GlcNAcylation has surprisingly extensive cross talk with phosphorylation, where it serves as a nutrient/stress sensor to modulate signaling, transcription, and cytoskeletal functions. Abnormal amounts of O-GlcNAcylation underlie the etiology of insulin resistance and glucose toxicity in diabetes, and this type of modification plays a direct role in neurodegenerative disease. Many oncogenic proteins and tumor suppressor proteins are also regulated by O-GlcNAcylation. Current data justify extensive efforts toward a better understanding of this invisible, yet abundant, modification. As tools for the study of O-GlcNAc become more facile and available, exponential growth in this area of research will eventually take place.

Abstract: Mitogen-activated protein kinases (MAPKs) are serine-threonine protein kinases that play the major role in signal transduction from the cell surface to the nucleus. MAPKs, which consist of growth factor-regulated extracellular signal-related kinases (ERKs), and the stress-activated MAPKs, c-jun NH2-terminal kinases (JNKs) and p38 MAPKs, are part of a three-kinase signaling module composed of the MAPK, an MAPK kinase (MAP2K) and an MAPK kinase (MAP3K). MAP3Ks phosphorylate MAP2Ks, which in turn activate MAPKs. MAPK phosphatases (MKPs), which recognize the TXY amino acid motif present in MAPKs, dephosphorylate and deactivate MAPKs. MAPK pathways are known to be influenced not only by receptor ligand interactions, but also by different stressors placed on the cell. One type of stress that induces potential activation of MAPK pathways is the oxidative stress caused by reactive oxygen species (ROS). Generally, increased ROS production in a cell leads to the activation of ERKs, JNKs, or p38 MAPKs, but the mechanisms by which ROS can activate these kinases are unclear. Oxidative modifications of MAPK signaling proteins and inactivation and/or degradation of MKPs may provide the plausible mechanisms for activation of MAPK pathways by ROS, which will be reviewed in this paper.

Abstract: The evolutionarily conserved serine-threonine kinase mammalian target of rapamycin (mTOR) plays a critical role in regulating many pathophysiological processes. Functional characterization of the mTOR signaling pathways, however, has been hampered by the paucity of known substrates. We used large-scale quantitative phosphoproteomics experiments to define the signaling networks downstream of mTORC1 and mTORC2. Characterization of one mTORC1 substrate, the growth factor receptor-bound protein 10 (Grb10), showed that mTORC1-mediated phosphorylation stabilized Grb10, leading to feedback inhibition of the phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated, mitogen-activated protein kinase (ERK-MAPK) pathways. Grb10 expression is frequently down-regulated in various cancers, and loss of Grb10 and loss of the well-established tumor suppressor phosphatase PTEN appear to be mutually exclusive events, suggesting that Grb10 might be a tumor suppressor regulated by mTORC1.

Abstract: Contact inhibition of cell growth is essential for embryonic development and maintenance of tissue architecture in adult organisms, and the growth of tumors is characterized by a loss of contact inhibition of proliferation. The recently identified Hippo signaling pathway has been implicated in contact inhibition of proliferation as well as organ size control. The modulation of the phosphorylation and nuclear localization of Yes-associated protein (YAP) by the highly conserved kinase cascade of the Hippo signaling pathway has been intensively studied. However, cell-surface receptors regulating the Hippo signaling pathway in mammals are not well understood. In this study, we show that Hippo signaling pathway components are required for E-cadherin–dependent contact inhibition of proliferation. Knockdown of the Hippo signaling components or overexpression of YAP inhibits the decrease in cell proliferation caused by E-cadherin homophilic binding at the cell surface, independent of other cell–cell interactions. We also demonstrate that the E-cadherin/catenin complex functions as an upstream regulator of the Hippo signaling pathway in mammalian cells. Expression of E-cadherin in MDA-MB-231 cells restores the density-dependent regulation of YAP nuclear exclusion. Knockdown of β-catenin in densely cultured MCF10A cells, which mainly depletes E-cadherin–bound β-catenin, induces a decrease in the phosphorylation of S127 residue of YAP and its nuclear accumulation. Moreover, E-cadherin homophilic binding independent of other cell interactions is sufficient to control the subcellular localization of YAP. Therefore, Our results indicate that, in addition to its role in cell–cell adhesion, E-cadherin-mediated cell–cell contact directly regulates the Hippo signaling pathway to control cell proliferation.

Abstract: Phosphorylation of G protein–coupled receptors (GPCRs, which are also known as seven-transmembrane spanning receptors) by GPCR kinases (GRKs) plays essential roles in the regulation of receptor function by promoting interactions of the receptors with β-arrestins. These multifunctional adaptor proteins desensitize GPCRs, by reducing receptor coupling to G proteins and facilitating receptor internalization, and mediate GPCR signaling through β-arrestin–specific pathways. Detailed mapping of the phosphorylation sites on GPCRs targeted by individual GRKs and an understanding of how these sites regulate the specific functional consequences of β-arrestin engagement may aid in the discovery of therapeutic agents targeting individual β-arrestin functions. The β 2 -adrenergic receptor (β 2 AR) has many serine and threonine residues in the carboxyl-terminal tail and the intracellular loops, which are potential sites of phosphorylation. We monitored the phosphorylation of the β 2 AR at specific sites upon stimulation with an agonist that promotes signaling by both G protein–mediated and β-arrestin–mediated pathways or with a biased ligand that promotes signaling only through β-arrestin–mediated events in the presence of the full complement of GRKs or when either GRK2 or GRK6 was depleted. We correlated the specific and distinct patterns of receptor phosphorylation by individual GRKs with the functions of β-arrestins and propose that the distinct phosphorylation patterns established by different GRKs establish a “barcode” that imparts distinct conformations to the recruited β-arrestin, thus regulating its functional activities.

Abstract: There is a strong rationale to therapeutically target the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway in breast cancer since it is highly deregulated in this disease and it also mediates resistance to anti-HER2 therapies. However, initial studies with rapalogs, allosteric inhibitors of mTORC1, have resulted in limited clinical efficacy probably due to the release of a negative regulatory feedback loop that triggers AKT and ERK signaling. Since activation of AKT occurs via PI3K, we decided to explore whether PI3K inhibitors prevent the activation of these compensatory pathways. Using HER2-overexpressing breast cancer cells as a model, we observed that PI3K inhibitors abolished AKT activation. However, PI3K inhibition resulted in a compensatory activation of the ERK signaling pathway. This enhanced ERK signaling occurred as a result of activation of HER family receptors as evidenced by induction of HER receptors dimerization and phosphorylation, increased expression of HER3 and binding of adaptor molecules to HER2 and HER3. The activation of ERK was prevented with either MEK inhibitors or anti-HER2 monoclonal antibodies and tyrosine kinase inhibitors. Combined administration of PI3K inhibitors with either HER2 or MEK inhibitors resulted in decreased proliferation, enhanced cell death and superior anti-tumor activity compared with single agent PI3K inhibitors. Our findings indicate that PI3K inhibition in HER2-overexpressing breast cancer activates a new compensatory pathway that results in ERK dependency. Combined anti-MEK or anti-HER2 therapy with PI3K inhibitors may be required in order to achieve optimal efficacy in HER2-overexpressing breast cancer. This approach warrants clinical evaluation.

Abstract: Many biological processes are regulated through the selective dephosphorylation of proteins. Protein serine-threonine phosphatases are assembled from catalytic subunits bound to diverse regulatory subunits that provide substrate specificity and subcellular localization. We describe a small molecule, guanabenz, that bound to a regulatory subunit of protein phosphatase 1, PPP1R15A/GADD34, selectively disrupting the stress-induced dephosphorylation of the α subunit of translation initiation factor 2 (eIF2α). Without affecting the related PPP1R15B-phosphatase complex and constitutive protein synthesis, guanabenz prolonged eIF2α phosphorylation in human stressed cells, adjusting the protein production rates to levels manageable by available chaperones. This favored protein folding and thereby rescued cells from protein misfolding stress. Thus, regulatory subunits of phosphatases are drug targets, a property used here to restore proteostasis in stressed cells.

Abstract: To elucidate cellular events underlying the pluripotency of human embryonic stem cells (hESCs), we performed parallel quantitative proteomic and phosphoproteomic analyses of hESCs during differentiation initiated by a diacylglycerol analog or transfer to media that had not been conditioned by feeder cells. We profiled 6521 proteins and 23,522 phosphorylation sites, of which almost 50% displayed dynamic changes in phosphorylation status during 24 hours of differentiation. These data are a resource for studies of the events associated with the maintenance of hESC pluripotency and those accompanying their differentiation. From these data, we identified a core hESC phosphoproteome of sites with similar robust changes in response to the two distinct treatments. These sites exhibited distinct dynamic phosphorylation patterns, which were linked to known or predicted kinases on the basis of the matching sequence motif. In addition to identifying previously unknown phosphorylation sites on factors associated with differentiation, such as kinases and transcription factors, we observed dynamic phosphorylation of DNA methyltransferases (DNMTs). We found a specific interaction of DNMTs during early differentiation with the PAF1 (polymerase-associated factor 1) transcriptional elongation complex, which binds to promoters of the pluripotency and known DNMT target genes encoding OCT4 and NANOG, thereby providing a possible molecular link for the silencing of these genes during differentiation.

Abstract: When brassinosteroid levels are low, the GSK3-like kinase BIN2 phosphorylates and inactivates the BZR1 transcription factor to inhibit growth in plants. Brassinosteroid promotes growth by inducing dephosphorylation of BZR1, but the phosphatase that dephosphorylates BZR1 has remained unknown. Here, using tandem affinity purification, we identified protein phosphatase 2A (PP2A) as a BZR1-interacting protein. Genetic analyses demonstrated a positive role for PP2A in brassinosteroid signalling and BZR1 dephosphorylation. Members of the B' regulatory subunits of PP2A directly interact with BZR1's putative PEST domain containing the site of the bzr1-1D mutation. Interaction with and dephosphorylation by PP2A are enhanced by the bzr1-1D mutation, reduced by two intragenic bzr1-1D suppressor mutations, and abolished by deletion of the PEST domain. This study reveals a crucial function for PP2A in dephosphorylating and activating BZR1 and completes the set of core components of the brassinosteroid-signalling cascade from cell surface receptor kinase to gene regulation in the nucleus.

Abstract: Glycogen synthase kinase-3 (GSK-3) is a widely expressed and highly conserved serine/threonine protein kinase encoded in mammals by two genes that generate two related proteins: GSK-3α and GSK-3β. GSK-3 is active in cells under resting conditions and is primarily regulated through inhibition or diversion of its activity. While GSK-3 is one of the few protein kinases that can be inactivated by phosphorylation, the mechanisms of GSK-3 regulation are more varied and not fully understood. Precise control appears to be achieved by a combination of phosphorylation, localization, and sequestration by a number of GSK-3-binding proteins. GSK-3 lies downstream of several major signaling pathways including the phosphatidylinositol 3’ kinase pathway, the Wnt pathway, Hedgehog signaling and Notch. Specific pools of GSK-3, which differ in intracellular localization, binding partner affinity and relative amount are differentially sensitized to several distinct signaling pathways and these sequestration mechanisms contribute to pathway insulation and signal specificity. Dysregulation of signaling pathways involving GSK-3 is associated with the pathogenesis of numerous neurological and psychiatric disorders and there are data suggesting GSK-3 isoform-selective roles in several of these. Here, we review the current knowledge of GSK-3 regulation and targets and discuss the various animal models that have been employed to dissect the functions of GSK-3 in brain development and function through the use of conventional or conditional knock-out mice as well as transgenic mice. These studies have revealed fundamental roles for these protein kinases in memory, behavior and neuronal fate determination and provide insights into possible therapeutic interventions.

Abstract: Contact inhibition of cell growth is essential for embryonic development and maintenance of tissue architecture in adult organisms, and the growth of tumors is characterized by a loss of contact inhibition of proliferation. The recently identified Hippo signaling pathway has been implicated in contact inhibition of proliferation as well as organ size control. The modulation of the phosphorylation and nuclear localization of Yes-associated protein (YAP) by the highly conserved kinase cascade of the Hippo signaling pathway has been intensively studied. However, cell-surface receptors regulating the Hippo signaling pathway in mammals are not well understood. In this study, we show that Hippo signaling pathway components are required for E-cadherin–dependent contact inhibition of proliferation. Knockdown of the Hippo signaling components or overexpression of YAP inhibits the decrease in cell proliferation caused by E-cadherin homophilic binding at the cell surface, independent of other cell–cell interactions. We also demonstrate that the E-cadherin/catenin complex functions as an upstream regulator of the Hippo signaling pathway in mammalian cells. Expression of E-cadherin in MDA-MB-231 cells restores the density-dependent regulation of YAP nuclear exclusion. Knockdown of β-catenin in densely cultured MCF10A cells, which mainly depletes E-cadherin–bound β-catenin, induces a decrease in the phosphorylation of S127 residue of YAP and its nuclear accumulation. Moreover, E-cadherin homophilic binding independent of other cell interactions is sufficient to control the subcellular localization of YAP. Therefore, Our results indicate that, in addition to its role in cell–cell adhesion, E-cadherin-mediated cell–cell contact directly regulates the Hippo signaling pathway to control cell proliferation.

Abstract: mTOR kinase inhibitors block mTORC1 and mTORC2 and thus do not cause the mTORC2 activation of AKT observed with rapamycin We now show, however, that these drugs have a biphasic effect on AKT Inhibition of mTORC2 leads to AKT S473 dephosphorylation and a rapid but transient inhibition of AKT T308 phosphorylation and AKT signaling However, inhibition of mTOR kinase also relieves feedback inhibition of RTKs leading to subsequent PI3K activation and rephosphorylation of AKT T308 sufficient to reactivate AKT activity and signaling Thus, catalytic inhibition of mTOR kinase leads to a new steady state characterized by profound inhibition of mTORC1 and accumulation of activated AKT phosphorylated on T308 but not S473 Combined inhibition of mTOR kinase and the induced RTKs fully abolishes AKT signaling and results in profound cell death and tumor regression in vivo These findings reveal the adaptive capabilities of oncogenic signaling networks and the limitations of monotherapy for inhibiting feedback-regulated pathways

Abstract: Although originally considered toxic, hydrogen sulfide (H(2)S) has been implicated in mediating various biological processes. Nevertheless, its cellular targets and mode of action are not well understood. Protein tyrosine phosphatases (PTPs), which regulate numerous signal transduction pathways, use an essential cysteine residue at the active site, which is characterized by a low pK(a) and is susceptible to reversible oxidation. Here, we report that PTP1B was reversibly inactivated by H(2)S, in vitro and in cells, through sulfhydration of the active-site cysteine residue. Unlike oxidized PTP1B, the sulfhydrated enzyme was preferentially reduced in vitro by thioredoxin, compared to glutathione or dithiothreitol. Sulfhydration of PTP1B in cells required the presence of cystathionine γ-lyase (CSE), a critical enzyme in H(2)S production, and resulted in inhibition of phosphatase activity. Suppression of CSE decreased H(2)S production and decreased the phosphorylation of tyrosine-619 in PERK [protein kinase-like endoplasmic reticulum (ER) kinase], thus reducing its activation in response to ER stress. PERK, which phosphorylates the eukaryotic translational initiation factor 2, leading to attenuation of protein translation, was a direct substrate of PTP1B. In addition, CSE knockdown led to activation of the nonreceptor tyrosine kinase SRC, previously shown to be mediated by PTP1B. These effects of suppressing H(2)S production on the response to ER stress were abrogated by a small-molecule inhibitor of PTP1B. Together, these data define a signaling function for H(2)S in inhibiting PTP1B activity and thereby promoting PERK activity during the response to ER stress.

Abstract: Maintaining sufficient levels of ATP (the immediate source of cellular energy) is essential for the proper functioning of all living cells As a consequence, cells require mechanisms to balance energy demand with supply In eukaryotic cells the AMP-activated protein kinase (AMPK) cascade has an important role in this homeostasis AMPK is activated by a fall in ATP (concomitant with a rise in ADP and AMP), which leads to the activation of catabolic pathways and the inhibition of anabolic pathways Here we review the role of AMPK as an energy sensor and consider the recent finding that ADP, as well as AMP, causes activation of mammalian AMPK We also review recent progress in structural studies on phosphorylated AMPK that provides a mechanism for the regulation of AMPK in which AMP and ADP protect it against dephosphorylation Finally, we briefly survey some of the outstanding questions concerning the regulation of AMPK

Abstract: The plasma membrane sodium/proton exchanger Salt-Overly-Sensitive 1 (SOS1) is a critical salt tolerance determinant in plants. The SOS2–SOS3 calcium-dependent protein kinase complex up-regulates SOS1 activity, but the mechanistic details of this crucial event remain unresolved. Here we show that SOS1 is maintained in a resting state by a C-terminal auto-inhibitory domain that is the target of SOS2–SOS3. The auto-inhibitory domain interacts intramolecularly with an adjacent domain of SOS1 that is essential for activity. SOS1 is relieved from auto-inhibition upon phosphorylation of the auto-inhibitory domain by SOS2–SOS3. Mutation of the SOS2 phosphorylation and recognition site impeded the activation of SOS1 in vivo and in vitro. Additional amino acid residues critically important for SOS1 activity and regulation were identified in a genetic screen for hypermorphic alleles.

Abstract: Protein glycosylation is an important posttranslational process, which regulates protein folding and functional expression. Studies have shown that abnormal glycosylation in tumor cells affects cancer progression and malignancy. In the current study, we have identified sialylated proteins using an alkynyl sugar probe in two different lung cancer cell lines, CL1-0 and CL1-5 with distinct invasiveness derived from the same parental cell line. Among the identified sialylated proteins, epidermal growth factor receptor (EGFR) was chosen to understand the effect of sialylation on its function. We have determined the differences in glycan sequences of EGFR in both cells and observed higher sialylation and fucosylation of EGFR in CL1-5 than in CL1-0. Further study suggested that overexpression of sialyltransferases in CL1-5 and α1,3-fucosyltransferases (FUT4 or FUT6) in CL1-5 and A549 cells would suppress EGFR dimerization and phosphorylation upon EGF treatment, as compared to the control and CL1-0 cells. Such modulating effects on EGFR dimerization were further confirmed by sialidase or fucosidase treatment. Thus, increasing sialylation and fucosylation could attenuate EGFR-mediated invasion of lung cancer cells. However, incorporation of the core fucose by α1,6-fucosylatransferase (FUT8) would promote EGFR dimerization and phosphorylation.

Abstract: S-type anion channels are direct targets of abscisic acid (ABA) signaling and contribute to chloride and nitrate release from guard cells, which in turn initiates stomatal closure. SLAC1 was the first component of the guard cell S-type anion channel identified. However, we found that guard cells of Arabidopsis SLAC1 mutants exhibited nitrate conductance. SLAH3 (SLAC1 homolog 3) was also present in guard cells, and coexpression of SLAH3 with the calcium ion (Ca2+)-dependent kinase CPK21 in Xenopus oocytes mediated nitrate-induced anion currents. Nitrate, calcium, and phosphorylation regulated SLAH3 activity. CPK21-dependent SLAH3 phosphorylation and activation were blocked by ABI1, a PP2C-type protein phosphatase that is inhibited by ABA and inhibits the ABA signaling pathway in guard cells. We reconstituted the ABA-stimulated phosphorylation of the SLAH3 amino-terminal domain by CPK21 in vitro by including the ABA receptor-phosphatase complex RCAR1-ABI1 in the reactions. We propose that ABA perception by the complex consisting of ABA receptors of the RCAR/PYR/PYL family and ABI1 releases CPK21 from inhibition by ABI1, and then CPK21 is further activated by an increase in the cytosolic Ca2+ concentration, leading to its phosphorylation of SLAH3. Thus, the identification of SLAH3 as the nitrate-, calcium-, and ABA-sensitive guard cell anion channel provides insights into the relationship among stomatal response to drought, signaling by nitrate, and nitrate metabolism.

Abstract: The role of protein phosphorylation in the life cycle of malaria parasites is slowly emerging. Here we combine global phospho-proteomic analysis with kinome-wide reverse genetics to assess the importance of protein phosphorylation in Plasmodium falciparum asexual proliferation. We identify 1177 phosphorylation sites on 650 parasite proteins that are involved in a wide range of general cellular activities such as DNA synthesis, transcription and metabolism as well as key parasite processes such as invasion and cyto-adherence. Several parasite protein kinases are themselves phosphorylated on putative regulatory residues, including tyrosines in the activation loop of PfGSK3 and PfCLK3; we show that phosphorylation of PfCLK3 Y526 is essential for full kinase activity. A kinome-wide reverse genetics strategy identified 36 parasite kinases as likely essential for erythrocytic schizogony. These studies not only reveal processes that are regulated by protein phosphorylation, but also define potential anti-malarial drug targets within the parasite kinome.

Abstract: Phosphatidic acid (PA) is a critical phospholipid constituent in eukaryotic cell membranes, that accounts for 1–4 % of the total lipid.1 This lipophilic glycerophospholipid has a phosphate head group, and as such serves not only a structural capacity in lipid bilayers, but also participates as an intermediate in lipid metabolism and as a signaling molecule. Because of the small head group, PA facilitates changes in lipid bilayer curvature that are important for membrane fusion events, such as vesicular trafficking and endocytosis.2 PA is also a precursor to other lipid signaling molecules including diacylglycerol (DAG) and lysophosphatidic acid (LPA). As a lipid second messenger, PA activates signaling proteins and acts as a node within the membrane to which signaling proteins translocate. Several signaling proteins, including Raf-13,4 and mTOR,5 directly bind PA to mediate translocation or activation, respectively. PA has been implicated in signaling cascades involving cell growth, proliferation, and survival. Aberrant PA signaling has been identified in multiple cancers,6 neurodegeneration,7 and platelet aggregation,8 which makes proteins that mediate cellular levels of PA attractive as potential therapeutic targets. PA can be generated de novo9,10,11 by sequential enzyme-catalyzed acylations of glycerol-3-phosphate, or in response to cell signaling pathways (Figure 1). Every glycerophospholipid generated in eukaryotic membranes transitions through PA, a pathway characterized by Eugene Kennedy and his colleagues more than half a century ago.11,12 Signal generated PA is formed by enzymes that modify existing lipids. These enzymes include lysophosphatidic acid acyltransferase (LPAAT) which acylates LPA, DAG kinase which phosphorylates DAG at the sn-3 position, and phospholipase D (PLD) which hydrolyzes the headgroup of a phospholipid, generally phosphatidylcholine (PC), triggering the release of choline. Figure 1 PLD activity, an enzyme catalyzed hydrolysis of a phosphodiester bond, was first described in plants,13,14,15,16 and subsequently many enzymes from a range of viral, prokaryotic and eukaryotic organisms have been described as possessing PLD activity. To date, more than 4000 PLD enzymes have been entered in NCBI GenBank. The majority of these enzymes hydrolyze phosphodiester bonds within phospholipids such as PC (classified as EC 3.1.4.417), but there are other enzymes ascribed to having PLD activity that hydrolyze neutral lipids and even polynucleotide backbone. A large subset of enzymes with PLD activity share a conserved HxKxxxxDx6GSxN motif (HKD),18 or a variation thereof, that is responsible for catalytic activity. These enzymes are members of the PLD superfamily, and are proposed to follow a similar reaction mechanism. Non-HKD enzymes exhibiting PLD activity have divergent structures and catalytic mechanisms. These non-HKD enzymes are discussed here as a means of comparison. In this comprehensive review of the PLD superfamily, specific emphasis is given to the conventional mammalian isoforms, PLD1 and PLD2, and the tools with which these enzymes are studied. The merits of PLD as a potential therapeutic target are also reviewed, as are implications for modulation of PLD activity in cell signaling pathways, whole organisms, and aberrant or disease-related models.

Abstract: The enzymes involved in metabolism and signaling are regulated by posttranslational modifications that influence their catalytic activity, rates of turnover, and targeting to subcellular locations. Most prominent among these has been phosphorylation/dephosphorylation, but now a distinct class of modification coming to the fore is a set of versatile redox modifications of key cysteine residues. Here we review the chemical, structural, and regulatory aspects of such redox regulation of enzymes and discuss examples of how these regulatory modifications often work in concert with phosphorylation/dephosphorylation events, making redox dependence an integral part of many cell signaling processes. Included are the emerging roles played by peroxiredoxins, a family of cysteine-based peroxidases that now appear to be major players in both antioxidant defense and cell signaling. Antioxid. Redox Signal. 14, 1065–1077.