The significance of PTEN's protein phosphatase activity

Many hundreds of research papers over the last ten years have established the significance of PTEN's lipid phosphatase activity in mediating many of its effects on specific cellular processes in many different cell types, including cell growth, proliferation, survival, and migration ([Backman et al., 2002], [Iijima et al., 2002], [Leslie and Downes, 2002] and [Salmena et al., 2008]). In some cases, detailed signalling mechanisms have been identified by which these PtdInsP₃-dependent effects are manifest ([Kolsch et al., 2008], [Manning and Cantley, 2007] and [Tee and Blenis, 2005]). Further, in some settings, in vivo data from, for example genetic deletion of PTEN, relates closely with independent manipulation of the PI3K/Akt signalling pathway ([Bayascas et al., 2005], [Chen et al., 2006], [Crackower et al., 2002] and [Ma et al., 2005]). Together these studies indicate that the dominant effects of PTEN function are mediated through its regulation of PtdInsP₃-dependent signalling, but that its protein phosphatase activity also contributes in some settings. These conclusions are of great importance given the intense efforts underway to develop PI3K (EC 2.7.1.153) inhibitors as cancer therapeutics. The experiments reviewed here have firmly established that the protein phosphatase activity of PTEN plays a role in the regulation of cellular processes including migration. On the other hand, it has not been established beyond doubt that PTEN acts on substrates other than itself; no such substrates have been confidently identified and effector mechanisms for PTEN's protein phosphatase activity are currently unclear. The goal for future research must be firstly to understand the signalling mechanisms by which PTEN protein phosphatase activity acts: whether this is through identifying substrates, or working out how autodephosphorylation mediates its effects. Secondly, and critically, the significance of PTEN's protein phosphatase activity must be established in vivo. This can be achieved through relating the phenotypes intervening with both PTEN and with protein phosphatase effector pathways when they are identified, and through the generation of mouse models expressing substrate selective PTEN mutants. We should then be able to answer the important question of whether PTEN's protein phosphatase activity contributes to tumour suppression.     

Phosphatidic acid phosphohydrolase in the regulation of inflammatory signaling

Since the cloning and characterization of PAP-1 by Carman's group, a preponderance of new information has emerged pertaining to the molecular function of the enzyme and its physiological function in lipid metabolism ([Carman and Han, 2006] and [Donkor et al., 2007]). As a consequence of this development, PAP-1 and lipin research have informed us further regarding lipid metabolism and adipose tissue development ([Carman and Han, 2006] and [Reue and Zhang, 2008]). In recent years, the field of inflammatory lipid signaling has undergone a great expansion and has come to identify a wide variety of protein and metabolic inflammatory mediators, including PAP-1 as well as PLD, PKC, PLC and DAG. The focus of this review was to summarize experimental evidence supporting a role for PAP-1 in inflammatory signaling, which is summarized in Scheme 2 (Grkovich et al., in press). Further clarification is needed to identify the precise signaling functions and downstream targets of DAG forming enzymes, such as PLD/PAP-1 and PLC, as evidence supports both as participants in inflammatory signaling in different systems. Clarification of these regulatory questions should lead to a more complete understanding of inflammatory signaling while helping to identify future pharmacological targets.     

Chemotherapeutic agents and gene expression in cardiac myocytes

It is becoming apparent that anti-cancer chemotherapies are increasingly associated with cardiac dysfunction or even congestive heart failure (Minotti et al., 2004; Eliott, 2006; Suter et al., 2004; Ren, 2005). Our data suggest that one of the contributing factors to the cardiotoxicitiy of these drugs may be the activation of the AhR-response (including the increased expression of Cyp1a1) and/or other detoxification program in cardiac myocytes themselves. The induction of such responses may have secondary effects (e.g. to increase the level of intracellular oxidative stress), which may influence the contractility or even survival of cardiac myocytes. Furthermore, the specific response of cardiac myocytes, both with respect to the metabolizing enzymes and the export channels, potentially differs from other cells (e.g. we failed to detect any increase in expression of other “classical” AhR-responsive genes, Ugt1a1 and Ugt1a6). This could account for, for example, the observation that doxoribicinol (the 13-hydroxy form of doxorubicin) accumulates in cardiac myocytes but not in hepatocytes (Del Tacca et al., 1985; Olson et al., 1988). Given the vulnerability of the heart and the almost irreparable damage that can be done by severe oxidative stress, further studies would seem to be merited specifically on the effects of chemotherapeutic agents on cardiac myocytes.     

Polarity proteins regulate mammalian cell–cell junctions and cancer pathogenesis

Polarity pathways regulate important functions during the formation and maintenance of cell–cell junctions and during morphogenesis. In addition, cell polarity pathways are emerging as critical regulators of initiation and progression of carcinoma by functioning as tumor suppressors, downstream of oncogenes, or promoters of the metastatic process (Figure 2). It is highly likely that further analysis of cell polarity proteins and the pathways they control will identify novel biomarkers and potential drug targets for managing and treating patients with carcinoma.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

• of special interest

•• of outstanding interest

LECs go crazy in embryo development

We have reviewed studies in which LEC TFs have been used to explore totipotency via SE and regulation of the maturation phase during zygotic embryogenesis. LEC TFs are master regulators of the maturation phase, activating genes encoding seed proteins that define this phase of embryo development. Regulation of the maturation phase seems to involve a feedback loop between the LEC TFs and hormones. LEC TFs stimulate ABA levels and activate genes that repress GA levels, contributing to the high ABA to GA ratio characteristic of the maturation phase. High ABA levels in turn stimulate LEC TFs to activate seed protein genes, and the reduction in GA levels might facilitate LEC TF activity. Although the LEC TFs are master regulators of the maturation phase, LEC genes are initially expressed before the onset of the maturation phase. The cellular process that initiates the maturation phase is not known. Nor is it known how LEC TFs interact with ABA and GA at the molecular level.SE is an outstanding example of totipotency in plants. Ectopic expression of LEC genes causes vegetative or reproductive cells to change their fate and undergo somatic embryo development. LEC TFs, via LEC2, activate auxin biosynthetic enzymes, and we propose that an increase in endogenous auxin levels serves to induce SE (Figure 3). How exogenous or endogenous auxin acts as the induction signal remains to be determined. We suggest that LEC TFs enable cells to become competent to respond to the induction signal by inactivating GA and, perhaps, by increasing ABA levels (Figure 3). Thus, a potential thread between the roles of LEC TFs in the maturation phase and SE might be their involvement in controlling the ABA to GA balance. It remains to be determined whether and how ABA and GA influence embryogenic competence. Although many questions remain, substantial progress has been made in determining how the LEC TFs ‘go crazy’ during embryo development.     

Cancer cell sensitivity to arginine deprivation in vitro is not determined by endogenous levels of arginine metabolic enzymes

Single amino acid Arg (arginine) deprivation is currently considered as a therapeutic approach to treat certain types of tumours; the molecular mechanisms that underlie tumour cell sensitivity or resistance to Arg restriction are still little understood. Here, we address the question of whether endogenous levels of key Arg metabolic enzymes [catabolic: arginases, ARG1 (arginase type 1) and ARG2 (arginase type 2), and anabolic: OTC (ornithine transcarbamylase) and ASS (argininosuccinate synthetase)] affect cellular responses to arginine deprivation in vitro. Human epithelial cancer cells of different organs of origin exhibiting variable sensitivity to Arg deprivation provided the experimental models. Neither the basal expression status of the analysed enzymes, nor their changes upon arginine withdrawal correlated with cancer cell sensitivity to arginine deprivation. However, the ability to utilize exogenous Arg precursors (ornithine and citrulline) for growth in Arg‐deficient medium strongly correlated with expression of the corresponding enzymes, OTC and ASS. We also observed that OTC expression was below the level of detection in all the types of tumour cells analysed, suggesting that in vitro, at least for them, Arg is an essential amino acid.     

Expression and function of phospholipase C in breast carcinoma

Phosphoinositide-specific phospholipase C (PLC) control the levels of their substrate phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂), and its hydrolysis products diacylglycerol (DAG) and Ins(1,4,5)P₃, second messengers key to growth control and cell movement. The former is modulated by breakdown of plasma membrane and nuclear phosphoinositides, while the latter is mediated by phosphoinositide-driven remodeling of the actin cytoskeleton. The roles of PLC in the etiology and progression of breast carcinoma, however, are poorly understood. Previous studies reported a correlation between PLCβ₂ expression and breast tumor grade, making PLCβ₂ a potential marker for clinical outcome (Bertagnolo et al., 2006). While over-expression of PLCβ₂ is not sufficient to induce transformation of normal breast epithelial cells, it appears to play a role in promoting cell migration (Bertagnolo et al., 2007).Here we examined the expression of this and other PLC mRNA (β₁–β₄, δ₁, δ₃ and δ₄, γ₁ and γ₂) in normal breast epithelial lines, MCF-10A, and compared that pattern to breast tumor lines MDA-MB-231 and to T47D, using real-time relative-quantification PCR. Our results show that PLCγ₁, γ₂ and δ₁ and δ3 are more highly expressed in the transformed cell lines compared to MCF-10A when normalized to mRNA encoding various house keeping proteins; whereas PLCβ₂ mRNA levels were considerably lower than other PLC subtypes, including PLCβ₁ in the metastatic lines. Examination of PLC mRNA levels from normal and cancerous human breast tissue showed a similar pattern of expression, however, when staging or tumor size was considered, PLCδ₁ and δ₃ expression were positively correlated.To test whether PLCδ₁ or δ₃ played any role in tumor cell proliferation or cell migration, we transfected cells with siRNA specifically targeting these isoforms. RNAi mediated knockdown of either PLC isoform, reduced proliferation of the MDA-MB-231 cells. Morphological changes including cell rounding, and surface blebbing and nuclear fragmentation were observed. These changes were accompanied by reductions in cell migration activities. On the other hand, PLCδ₁ knockdown failed to cause comparable morphological changes in the normal MCF-10A line, but did reduce cell proliferation and migration. Taken together, these data are consistent with the idea that PLCδ₁ and δ₃ isoforms support the growth and migration of normal and neoplastic mammary epithelial cells in vitro.     

Very high light resistant mutants of Chlamydomonas reinhardtii: Responses of Photosystem II,nonphotochemical quenching and xanthophyll pigments to light and CO2

We have isolated very high light resistant nuclear mutants (VHL ^R) in Chlamydomonas reinhardtii, that grow in 1500–2000 mol photons m^–2 s^–1 (VHL) lethal to wildtype. Four nonallelic mutants have been characterized in terms of Photosystem II (PS II) function, nonphotochemical quenching (NPQ) and xanthophyll pigments in relation to acclimation and survival under light stress. In one class of VHL ^R mutants isolated from wild type (S4 and S9), VHL resistance was accompanied by slower PS II electron transfer, reduced connectivity between PS II centers and decreased PS II efficiency. These lesions in PS II function were already present in the herbicide resistant D1 mutant A251L (L ^*) from which another class of VHL ^R mutants (L4 and L30) were isolated, confirming that optimal PS II function was not critical for survival in very high light. Survival of all four VHL ^R mutants was independent of CO₂ availability, whereas photoprotective processes were not. The de-epoxidation state (DPS) of the xanthophyll cycle pigments in high light (HL, 600 mol photons m^–2 s^–1) was strongly depressed when all genotypes were grown in 5% CO₂. In S4 and S9 grown in air under HL and VHL, high DPS was well correlated with high NPQ. However when the same genotypes were grown in 5% CO₂, high DPS did not result in high NPQ, probably because high photosynthetic rates decreased thylakoid pH. Although high NPQ lowered the reduction state of PS II in air compared to 5% CO₂ at HL in wildtype, S4 and S9, this did not occur during growth of S4 and S9 in VHL. L ^* and VHL ^R mutants L4 and L30, also showed high DPS with low NPQ when grown air or 5% CO₂, possibly because they were unable to maintain sufficiently high pH due to constitutively impaired PS II electron transport. Although dissipation of excess photon energy through NPQ may contribute to VHL resistance, there is little evidence that the different genes conferring the VHL ^R phenotype affect this form of photoprotection. Rather, the decline of chlorophyll per biomass in all VHL ^R mutants grown under VHL suggests these genes may be involved in regulating antenna components and photosystem stoichiometries.This revised version was published online in October 2005 with corrections to the Cover Date.     

Action of Levan Fructotransferase of Arthrobacter ureafaciens on a Mixture of Branched Levanpentasaccharides

Two coryneform bacteria, Arthrobacter globiformis IFO 12137 (ATCC 8010) and Brevibacterium helvolum IFO 12073, which have the arginine oxygenase pathway, could utilize L-ornithine, L-citrulline, and D-arginine. The cells of the bacteria grown on these amino acids contained high levels of guanidinobutyrase and induced levels of the enzymes of the preceding steps of the pathway. 4-Guanidinobutyrate induced guanidinobutyrase but failed to induce the other enzymes, indicating that it was the direct inducer of guanidinobutyrase. These amino acids and L-arginine also induced L-arginine: 2-ketoglutarate aminotransferase. 4-Aminobutyrate was formed on incubation of L-citrulline with L-citrulline-grown cells of A. globiformis in the presence of gabaculine; its amount was about 50% of the L-citrulline degraded. The L-arginine-grown cells produced 4-aminobutyrate and urea from L-arginine in the presence of aminooxyacetate or gabaculine; the amount of 4-aminobutyratewas 80% or more of that of the L-arginine degraded. When the oxygenase pathway was blocked with thioglycolate, the degradation of L-arginine and the formation of urea and 4-aminobutyrate were greatly suppressed. These results indicate that these amino acids are degraded via the arginine oxygenase and the arginine aminotransferase pathways and the major route is the former. Agmatine was degraded in these bacteria and induced agmatine deiminase, carbamoylputrescine hydrolase, putrescine oxidase, and aminobutyraldehyde dehydrogenase. None of the enzymes was induced by L-arginine.     

Death by over-eating: The Gaucher disease associated gene GBA1, identified in a screen for mediators of autophagic cell death,is necessary for developmental cell death in Drosophila midgut

Autophagy is critical for homeostasis and cell survival during stress, but can also lead to cell death, a little understood process that has been shown to contribute to developmental cell death in lower model organisms, and to human cancer cell death. We recently reported¹ Dasari SK, Bialik S, Levin-Zaidman S, Levin-Salomon V, Merrill AH, Jr., Futerman AH, Kimchi A. Signalome-wide rnai screen identifies gba1 as a positive mediator of autophagic cell death. Cell Death Differ. 2017;24(7):1288-1302. https://doi.org/10.1038/cdd.2017.80. PMID:28574511[Crossref], [PubMed], [Web of Science ®] [Google Scholar] on our thorough molecular and morphologic characterization of an autophagic cell death system involving resveratrol treatment of lung carcinoma cells. To gain mechanistic insight into this death program, we performed a signalome-wide RNAi screen for genes whose functions are necessary for resveratrol-induced death. The screen identified GBA1, the gene encoding the lysosomal enzyme glucocerebrosidase, as an important mediator of autophagic cell death. Here we further show the physiological relevance of GBA1 to developmental cell death in midgut regression during Drosophila metamorphosis. We observed a delay in midgut cell death in two independent Gba1a RNAi lines, indicating the critical importance of Gba1a for midgut development. Interestingly, loss-of-function GBA1 mutations lead to Gaucher Disease and are a significant risk factor for Parkinson Disease, which have been associated with defective autophagy. Thus GBA1 is a conserved element critical for maintaining proper levels of autophagy, with high levels leading to autophagic cell death.     

Enhancer modularity and the evolution of new traits

ABSTRACT

Animals have modular cis-regulatory regions in their genomes, and expression of a single gene is often regulated by multiple enhancers residing in such a region. In the laboratory, and also in natural populations, loss of an enhancer can result in a loss of gene expression. Although only a few examples have been well characterized to date, some studies have suggested that an evolutionary gain of a new enhancer function can establish a new gene expression domain. Our recent study showed that Drosophila guttifera has more enhancers and additional expression domains of the wingless gene during the pupal stage, compared to D. melanogaster, and that these new features appear to have evolved in the ancestral lineage leading to D. guttifera.¹ Koshikawa S, Giorgianni MW, Vaccaro K, Kassner VA, Yoder JH, Werner T, Carroll SB. Gain of cis-regulatory activities underlies novel domains of wingless gene expression in Drosophila. Proc Natl Acad Sci USA 2015; 112:7524-9; PMID:26034272; http://dx.doi.org/10.1073/pnas.1509022112[Crossref], [PubMed], [Web of Science ®] , [Google Scholar] Gain of a new expression domain of a developmental regulatory gene (toolkit gene), such as wingless, can cause co-option of the expression of its downstream genes to the new domain, resulting in duplication of a preexisting structure at this new body position. Recently, with the advancement of evo-devo studies, we have learned that the developmental regulatory systems are strikingly similar across various animal taxa, in spite of the great diversity of the animals' morphology. Even behind “new” traits, co-options of essential developmental genes from known systems are very common. We previously provided concrete evidence of gains of enhancer activities of a developmental regulatory gene underlying gains of new traits.¹ Koshikawa S, Giorgianni MW, Vaccaro K, Kassner VA, Yoder JH, Werner T, Carroll SB. Gain of cis-regulatory activities underlies novel domains of wingless gene expression in Drosophila. Proc Natl Acad Sci USA 2015; 112:7524-9; PMID:26034272; http://dx.doi.org/10.1073/pnas.1509022112[Crossref], [PubMed], [Web of Science ®] , [Google Scholar] Broad occurrence of this scenario is testable and should be validated in the future.     

A novel gene expression pathway regulated by nuclear phosphoinositides

Star-PAP is a recently identified nuclear speckle localized non-canonical poly(A) polymerase that has a functional interaction with PIPKIα, and whose activity is modulated by the PIPKIα product, PI4,5P₂. Similar to other poly(A) polymerases, such as the canonical PAPα and the non-canonical GLD2 PAP, Star-PAP resides in a large complex of proteins involved in the 3′ end formation of mRNAs (Fig. 4). The Star-PAP complex shares components with the canonical PAPα complex though it contains unique associated proteins such as PIPKIα and CKIα. The Star-PAP complex assembles into a highly stable 3′ end processing machine upon oxidative stress induction. This assembled complex shows enhanced enzyme activity and hypersensitivity to exogenous PI4,5P₂, implying that an activated Star-PAP is distinctly modified and/or contains unique factors as compared to Star-PAP purified from resting cells.     

Examination of Testicular Gene Expression Patterns in Yorkshire Pigs with High and Low Levels of Boar Taint

Boar taint refers to the objectionable odor and flavor in meat of some uncastrated male pigs, which is primarily due to high levels of androstenone, a steroid produced in the testis, and 3-methylindole (skatole) which is produced by bacterial degradation of tryptophan in the intestinal tract. We determined testicular gene expression patterns of Yorkshire pigs with high and low levels of boar taint using swine DNA microarrays with two-color hybridization. The microarrays contained 19486 annotated probes; the expressions of 8719 genes were detected. Fifty-three genes were significantly up-regulated in the high boar taint group and four were significantly down-regulated (p < 0.05; fold change > ±1.55). Gene ontology (GO) analysis short-listed 11 significant GO terms (p < 0.05), most of which are associated with steroid metabolism and mitochondrial components. Comparing the results of this study with published work on Duroc and Norwegian Landrace boars, ¹ Moe M , Meuwissen T , Lien S , Bendixen C , Wang X , Conley LN , Berget I , Tajet H , Grindflek E . Gene expression profiles in testis of pigs with extreme high and low levels of androstenone . BMC Genomics . 2007 ; 8 : 405 . [Google Scholar] eleven genes (HSB17B4, FDX1, CYP11A1, DHRS4, PRDX1, CYB5, CYP17A1, FTL, IDI1, SULT2A1, and RDH12) were over-expressed in all three breeds with a high androstenone level. The current findings confirmed a number of candidate genes identified in previous functional studies and suggest several new genes differentially expressed with different levels of boar taint.     

Translational control of gurken mRNA in Drosophila development

Localized mRNA translation is a widespread mechanism for targeting protein synthesis, important for cell fate, motility and pathogenesis. In Drosophila, the spatiotemporal control of gurken/TGF-α mRNA translation is required for establishing the embryonic body axes. A number of recent studies have highlighted key aspects of the mechanism of gurken mRNA translational control at the dorsoanterior corner of the mid-stage oocyte. Orb/CPEB and Wispy/GLD-2 are required for polyadenylation of gurken mRNA, but unlocalized gurken mRNA in the oocyte is not fully polyadenylated.¹ Norvell A, Wong J, Randolph K, Thompson L. Wispy and Orb cooperate in the cytoplasmic polyadenylation of localized gurken mRNA. Dev Dyn Off Publ Am Assoc Anat 2015; 244:1276-1285. [Google Scholar] At the dorsoanterior corner, Orb and gurken mRNA have been shown to be enriched at the edge of Processing bodies, where translation occurs.² Weil TT, Parton RM, Herpers B, Soetaert J, Veenendaal T, Xanthakis D, Dobbie IM, Halstead JM, Hayashi R, Rabouille C, et al. Drosophila patterning is established by differential association of mRNAs with P bodies. Nat Cell Biol 2012; 14:1305-1313; PMID:23178881; http://dx.doi.org/10.1038/ncb2627[Crossref], [PubMed], [Web of Science ®] [Google Scholar] Over-expression of Orb in the adjacent nurse cells, where gurken mRNA is transcribed, is sufficient to cause mis-expression of Gurken protein.³ Davidson A, Parton RM, Rabouille C, Weil TT, Davis I. Localized translation of gurken/TGF-α mRNA during axis specification is controlled by access to Orb/CPEB on processing bodies. Cell Rep 2016; 14:2451-2462; PMID:26947065; http://dx.doi.org/10.1016/j.celrep.2016.02.038[Crossref], [PubMed], [Web of Science ®] [Google Scholar] In orb mutant egg chambers, reducing the activity of CK2, a Serine/Threonine protein kinase, enhances the ventralized phenotype, consistent with perturbation of gurken translation.⁴ Wong LC, Costa A, McLeod I, Sarkeshik A, Yates J 3rd, Kyin S, Perlman D, Schedl P, et al. The functioning of the drosophila CPEB protein Orb is regulated by phosphorylation and requires casein kinase 2 activity. PLoS One 2011; 6:e24355; PMID:21949709; http://dx.doi.org/10.1371/journal.pone.0024355[Crossref], [PubMed], [Web of Science ®] [Google Scholar] Here we show that sites phosphorylated by CK2 overlap with active Orb and with Gurken protein expression. Together with our new findings we consolidate the literature into a working model for gurken mRNA translational control and review the role of kinases, cell cycle factors and polyadenylation machinery highlighting a multitude of conserved factors and mechanisms in the Drosophila egg chamber.     

L-Amino acid oxidase from Naja naja oxiana venom

A new l-amino acid oxidase (LAAO) was isolated from the Central Asian cobra Naja naja oxiana venom by size exclusion, ion exchange and hydrophobic chromatography. The N-terminal sequence and the internal peptide sequences share high similarity with other snake venom l-amino acid oxidases, especially with those isolated from elapid venoms. The enzyme is stable at low temperatures (− 20 °C, − 70 °C) and loses its activity by heating at 70 °C. Specific substrates for the isolated protein are l-phenylalanine, l-tryptophan, l-methionine and l-leucine. The enzyme has antibacterial activity inhibiting the growth of Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria. N. naja oxiana LAAO dose-dependently inhibited ADP- or collagen-induced platelet aggregation with IC₅₀ of 0.094 μM and 0.036 μM, respectively. The antibacterial and anti-aggregating activity was abolished by catalase.