Cell proliferation and migration are modulated by Cdk-1-phosphorylated endothelial-monocyte activating polypeptide II

Background

Endothelial-Monocyte Activating Polypeptide (EMAP II) is a secreted protein with well-established anti-angiogenic activities. Intracellular EMAP II expression is increased during fetal development at epithelial/mesenchymal boundaries and in pathophysiologic fibroproliferative cells of bronchopulmonary dysplasia, emphysema, and scar fibroblast tissue following myocardial ischemia. Precise function and regulation of intracellular EMAP II, however, has not been explored to date.

Methodology/Principal Findings

Here we show that high intracellular EMAP II suppresses cellular proliferation by slowing progression through the G2M cell cycle transition in epithelium and fibroblast. Furthermore, EMAP II binds to and is phosphorylated by Cdk1, and exhibits nuclear/cytoplasmic partitioning, with only nuclear EMAP II being phosphorylated. We observed that extracellular secreted EMAP II induces endothelial cell apoptosis, where as excess intracellular EMAP II facilitates epithelial and fibroblast cells migration.

Conclusions/Significance

Our findings suggest that EMAP II has specific intracellular effects, and that this intracellular function appears to antagonize its extracellular anti-angiogenic effects during fetal development and pulmonary disease progression.     

Functional Proteomics Identifies Acinus L as a Direct Insulin- and Amino Acid-Dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Substrate

The serine/threonine kinase mammalian target of rapamycin (mTOR) governs growth, metabolism, and aging in response to insulin and amino acids (aa), and is often activated in metabolic disorders and cancer. Much is known about the regulatory signaling network that encompasses mTOR, but surprisingly few direct mTOR substrates have been established to date. To tackle this gap in our knowledge, we took advantage of a combined quantitative phosphoproteomic and interactomic strategy. We analyzed the insulin- and aa-responsive phosphoproteome upon inhibition of the mTOR complex 1 (mTORC1) component raptor, and investigated in parallel the interactome of endogenous mTOR. By overlaying these two datasets, we identified acinus L as a potential novel mTORC1 target. We confirmed acinus L as a direct mTORC1 substrate by co-immunoprecipitation and MS-enhanced kinase assays. Our study delineates a triple proteomics strategy of combined phosphoproteomics, interactomics, and MS-enhanced kinase assays for the de novo-identification of mTOR network components, and provides a rich source of potential novel mTOR interactors and targets for future investigation.The serine/threonine kinase mammalian target of rapamycin (mTOR)¹ is conserved in all eukaryotes from yeast to mammals (1). mTOR is a central controller of cellular growth, whole body metabolism, and aging, and is frequently deregulated in metabolic diseases and cancer (2). Consequently, mTOR as well as its upstream and downstream cues are prime candidates for targeted drug development to alleviate the causes and symptoms of age-related diseases (3, 4). The identification of novel mTOR regulators and effectors thus remains a major goal in biomedical research. A vast body of literature describes a complex signaling network around mTOR. However, our current comparatively detailed knowledge of mTOR''s upstream cues contrasts with a rather limited set of known direct mTOR substrates.mTOR exists in two structurally and functionally distinct multiprotein complexes, termed mTORC1 and mTORC2. Both complexes contain mTOR kinase as well as the proteins mLST8 (mammalian lethal with SEC thirteen 8) (5–7), and deptor (DEP domain-containing mTOR-interacting protein) (8). mTORC1 contains the specific scaffold protein raptor (regulatory-associated protein of mTOR) (9, 10), whereas mTORC2 contains the specific binding partners rictor (rapamycin-insensitive companion of mTOR) (5–7), mSIN1 (TORC2 subunit MAPKAP1) (11–13), and PRR5/L (proline rich protein 5/-like) (14–16). The small macrolide rapamycin acutely inhibits mTORC1, but can also have long-term effects on mTORC2 (17, 18). More recently, ATP-analogs (19) that block both mTOR complexes, such as Torin 1 (20), have been developed. As rapamycin has already been available for several decades, our knowledge of signaling events associated with mTORC1 as well as its metabolic inputs and outputs is much broader as compared with mTORC2. mTORC1 responds to growth factors (insulin), nutrients (amino acids, aa) and energy (ATP). In response, mTORC1 activates anabolic processes (protein, lipid, nucleotide synthesis) and blocks catabolic processes (autophagy) to ultimately allow cellular growth (21). The insulin signal is transduced to mTORC1 via the insulin receptor (IR), and the insulin receptor substrate (IRS), which associates with class I phosphoinositide 3-kinases (PI3Ks). Subsequent phosphatidylinositol 3,4,5 trisphosphate (PIP3) binding leads to relocalization of the AGC kinases phosphoinositide-dependent protein kinase 1 (PDK1) and Akt (also termed protein kinase B, PKB) to the plasma membrane, where PDK1 phosphorylates Akt at T308 (22, 23). In response, Akt phosphorylates and inhibits the heterocomplex formed by the tuberous sclerosis complex proteins 1 and 2 (TSC1-TSC2) (24, 25). TSC1-TSC2 is the inhibitory, GTPase-activating protein for the mTORC1-inducing GTPase Ras homolog enriched in brain (rheb) (26–30), which activates mTORC1 at the lysosome. mTORC1 localization depends on the presence of aa, which in a rag GTPase-dependent manner induce mTORC1 relocalization to lysosomes (31, 32). Low energy levels are sensed by the AMP-dependent kinase (AMPK), which in turn phosphorylates the TSC1-TSC2 complex (33) and raptor (34), thereby inhibiting mTORC1.mTORC1 phosphorylates its well-described downstream substrate S6-kinase (S6K) at T389, the proline-rich Akt substrate of 40 kDa (PRAS40) at S183, and the translational repressor 4E-binding protein (4E-BP) at T37/46 (35–41). Unphosphorylated 4E-BP binds and inhibits the translation initiation factor 4G (eIF4G), which within the eIF4F complex mediates the scanning process of the ribosome to reach the start codon. Phosphorylation by mTORC1 inhibits 4E-BP''s interaction with eIF4E, thus allowing for assembly of eIF4F, and translation initiation (42, 43). More recently, also the IR-activating growth factor receptor-bound protein 10 (Grb10) (44, 45), the autophagy-initiating Unc-51-like kinase ULK1 (46), and the trifunctional enzymatic complex CAD composed of carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase (47, 48), which is required for nucleotide synthesis, have been described as direct mTORC1 substrates.mTORC2 activation is mostly described to be mediated by insulin, and this is mediated by a PI3K variant that is distinct from the PI3K upstream of mTORC1 (49, 50). Furthermore, mTORC2 responds to aa (5, 51). In response, mTORC2 phosphorylates the AGC kinases Akt at S473 (52–55), and serum and glucocorticoid kinase SGK (56) and protein kinase C alpha (PKCalpha) (7) within their hydrophobic motifs (57, 58), to control cellular motility (5–7), hepatic glycolysis, and lipogenesis (59). In addition, mTOR autophosphorylation at S2481 has been established as an mTORC2 readout in several cell lines including HeLa cells (49).Given the multiplicity of effects via which mTOR controls cellular and organismal growth and metabolism, it is surprising that only relatively few direct mTOR substrates have been established to date. Proteomic studies are widely used to identify novel interactors and substrates of protein kinases. Two studies have recently shed light on the interaction of rapamycin and ATP-analog mTOR inhibitors with TSC2 inhibition in mammalian cells (44, 45), and one study has analyzed the effects of raptor and rictor knockouts in non-stimulated cells (48).In this work, we report a functional proteomics approach to study mTORC1 substrates. We used an inducible raptor knockdown to inhibit mTORC1 in HeLa cells, and analyzed the effect in combination with insulin and aa induction by quantitative phosphoproteomics using stable isotope labeling by amino acids in cell culture (SILAC) (60). In parallel, we purified endogenous mTOR complexes and studied the interactome of mTOR by SILAC-MS. Through comparative data evaluation, we identified acinus L as a potential novel aa/insulin-sensitive mTOR substrate. We further validated acinus L by co-immunoprecipitation and MS-enhanced kinase assays as a new direct mTORC1 substrate.     

Complexin synchronizes primed vesicle exocytosis and regulates fusion pore dynamics

ComplexinII (CpxII) and SynaptotagminI (SytI) have been implicated in regulating the function of SNARE proteins in exocytosis, but their precise mode of action and potential interplay have remained unknown. In this paper, we show that CpxII increases Ca²⁺-triggered vesicle exocytosis and accelerates its secretory rates, providing two independent, but synergistic, functions to enhance synchronous secretion. Specifically, we demonstrate that the C-terminal domain of CpxII increases the pool of primed vesicles by hindering premature exocytosis at submicromolar Ca²⁺ concentrations, whereas the N-terminal domain shortens the secretory delay and accelerates the kinetics of Ca²⁺-triggered exocytosis by increasing the Ca²⁺ affinity of synchronous secretion. With its C terminus, CpxII attenuates fluctuations of the early fusion pore and slows its expansion but is functionally antagonized by SytI, enabling rapid transmitter discharge from single vesicles. Thus, our results illustrate how key features of CpxII, SytI, and their interplay transform the constitutively active SNARE-mediated fusion mechanism into a highly synchronized, Ca²⁺-triggered release apparatus.     

Rapid and repeatable host plant shifts drive reproductive isolation following a recent human-mediated introduction of the apple maggot fly,Rhagoletis pomonella

Ecological speciation via host-shifting is often invoked as a mechanism for insect diversification, but the relative importance of this process is poorly understood. The shift of Rhagoletis pomonella in the 1850s from the native downy hawthorn, Crataegus mollis, to introduced apple, Malus pumila, is a classic example of sympatric host race formation, a hypothesized early stage of ecological speciation. The accidental human-mediated introduction of R. pomonella into the Pacific Northwest (PNW) in the late 1970s allows us to investigate how novel ecological opportunities may trigger divergent adaptation and host race formation on a rapid timescale. Since the introduction, the fly has spread in the PNW, where in addition to apple, it now infests native black hawthorn, Crataegus douglasii, and introduced ornamental hawthorn, Crataegus monogyna. We use this “natural experiment” to test for genetic differentiation among apple, black, and ornamental hawthorn flies co-occurring at three sympatric sites. We report evidence that populations of all three host-associations are genetically differentiated at the local level, indicating that partial reproductive isolation has evolved in this novel habitat. Our results suggest that conditions suitable for initiating host-associated divergence may be common in nature, allowing for the rapid evolution of new host races when ecological opportunity arises.     

Molecular mechanism of 14-3-3 protein-mediated inhibition of plant nitrate reductase

14-3-3 proteins regulate key processes in eukaryotic cells including nitrogen assimilation in plants by tuning the activity of nitrate reductase (NR), the first and rate-limiting enzyme in this pathway. The homodimeric NR harbors three cofactors, each of which is bound to separate domains, thus forming an electron transfer chain. 14-3-3 proteins inhibit NR by binding to a conserved phosphorylation site localized in the linker between the heme and molybdenum cofactor-containing domains. Here, we have investigated the molecular mechanism of 14-3-3-mediated NR inhibition using a fragment of the enzyme lacking the third domain, allowing us to analyze electron transfer from the heme cofactor via the molybdenum center to nitrate. The kinetic behavior of the inhibited Mo-heme fragment indicates that the principal point at which 14-3-3 acts is the electron transfer from the heme to the molybdenum cofactor. We demonstrate that this is not due to a perturbation of the reduction potentials of either the heme or the molybdenum center and conclude that 14-3-3 most likely inhibits nitrate reductase by inducing a conformational change that significantly increases the distance between the two redox-active sites.     

ABSORPTION AND UTILIZATION OF IRRADIANCE BY CYANOBACTERIAL MATS IN TWO ICE-COVERED ANTARCTIC LAKES WITH CONTRASTING LIGHT CLIMATES

We investigated the under-ice light climate and the efficiency with which light was absorbed and utilized by benthic algal mats in Lakes Hoare and Vanda, two perennially ice-covered lakes in the McMurdo Dry Valleys area of Southern Victoria Land, Antarctica. The ice cover and water column of Lake Vanda were much more transparent than those of Lake Hoare (18% vs. 2% transmission though ice and attenuation coefficients for downwelling irradiance of 0.05 vs. 0.12 m ^{− 1}, respectively). In both lakes the under-ice spectra were dominated by blue-green wavelengths. The benthic flora under perennial ice covers of both lakes comprised thick mucilaginous mats, dominated by cyanobacteria. The mats were well suited to absorb the dominant blue-green wavelengths of the under-ice light, with phycoerythrin being present at high concentrations. The pigment systems of the benthic mats absorbed 30%–50% of the light that reached them, varying with depth and lake. There was a tendency for the percentage of absorption to increase as ambient irradiance decreased. The efficiency of utilization of absorbed irradiance was examined by constructing absorbed irradiance/oxygen evolution curves to estimate community quantum yield. Mats from 13 m in Lake Hoare showed the highest quantum yields, approaching 1 mol of carbon fixed for every 8 mol quanta absorbed under light-limiting conditions. Lake Vanda mats had lower quantum yields, but these increased with depth. Calculated in situ irradiance occasionally exceeded the measured saturating irradiance for oxygen evolution in both lakes, thus efficiency in situ was below the maximum at times. As in other environments, optimization strategies allowed efficient capture and utilization of the lower and middle ranges of experienced irradiance but led to a compromised capacity to use the highest irradiances encountered at each depth.     

Host choice in the phoretic mite Parasitellus fucorum (Mesostigmata: Parasitidae): which bumblebee caste is the best?

Host caste recognition may be important for the dispersal of phoretic mites associated with social insects. All developmental stages of the mite Parasitellus fucorum (Acari: Mesostigmata: Parasitidae) live in the nests of bumblebees (Hymenoptera: Apidae: Bombus). Dispersal occurs by specialised phoretic instars, deutonymphs, which attach to adult bumblebees. Since bumblebee colonies are annual and only young queens overwinter, deutonymphs that are able to discriminate between bumblebee castes and preferentially attach to queens should be favoured by selection. In the field, deutonymphs of P. fucorum were found to be phoretic on bumblebee workers and queens, and in behavioural experiments all castes proved to be attractive as carriers for the mites. However, they preferred queens that had hibernated as carriers when they could choose between workers and queens. In a further experiment, when given a choice, deutonymphs switched from males to young queens but never transferred from a queen to a male. These results suggest that deutonymphs preferentially attach to queens but may also use other castes for transport. Those dispersing on workers and males may try to switch to queens later. Host-switching is possible during copulation and on flowers, where bees of all castes forage. Received: 14 November 1997 / Accepted: 16 February 1998     

Vascular mediators in chronic lung disease of infancy: Role of endothelial monocyte activating polypeptide II (EMAP II)

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of prematurity. Over the years, the BPD phenotype has evolved, but despite various advances in neonatal management approaches, the reduction in the BPD burden is minimal. With the advent of surfactant, glucocorticoids, and new ventilation strategies, BPD has evolved from a disease of structural injury into a new BPD, marked by an arrest in alveolar growth in the lungs of extremely premature infants. This deficient alveolar growth has been associated with a diminution of pulmonary vasculature. Several investigators have described the epithelial / vascular co‐dependency and the significant role of crosstalk between vessel formation, alveologenesis, and lung dysplasia's; hence identification and study of factors that regulate pulmonary vascular emergence and inflammation has become crucial in devising effective therapeutic approaches for this debilitating condition. The potent antiangiogenic and proinflammatory protein Endothelial Monocyte Activating Polypeptide II (EMAP II) has been described as a mediator of pulmonary vascular and alveolar formation and its expression is inversely related to the periods of vascularization and alveolarization in the developing lung. Hence the study of EMAP II could play a vital role in studying and devising appropriate therapeutics for diseases of aberrant lung development, such as BPD. Herein, we review the vascular contribution to lung development and the implications that vascular mediators such as EMAP II have in distal lung formation during the vulnerable stage of alveolar genesis. Birth Defects Research (Part A) 100:180–188, 2014. © 2014 Wiley Periodicals, Inc.     

Substrate specificity of the Plasmodium falciparum glycosylphosphatidylinositol biosynthetic pathway and inhibition by species-specific suicide substrates

The substrate specificities of the early glycosylphosphatidylinositol biosynthetic enzymes of Plasmodium were determined using substrate analogues of D-GlcN(alpha)1-6-D-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol (GlcN-PI). Similarities between the Plasmodium and mammalian (HeLa) enzymes were observed. These are as follows: (i) The presence and orientation of the 2'-acetamido/amino and 3'-OH groups are essential for substrate recognition for the de-N-acetylase, inositol acyltransferase, and first mannosyltransferase enzymes. (ii) The 6'-OH group of the GlcN is dispensable for the de-N-acetylase, inositol acyltransferase, all four of the mannosyltransferases, and the ethanolamine phosphate transferase. (iii) The 4'-OH group of GlcNAc is not required for recognition, but substitution interferes with binding to the de-N-acetylase. The 4'-OH group of GlcN is essential for the inositol acyltransferase and first mannosyltransferase. (iv) The carbonyl group of the natural 2-O-hexadecanyl ester of GlcN-(acyl)PI is essential for substrate recognition by the first mannosyltransferase. However, several differences were also discovered: (i) Plasmodium-specific inhibition of the inositol acyltransferase was detected with GlcN-[L]-PI, while GlcN-(2-O-alkyl)PI weakly inhibited the first mannosyltransferase in a competitive manner. (ii) The Plasmodium de-N-acetylase can act on analogues containing N-benzoyl, GalNAc, or betaGlcNAc whereas the human enzyme cannot. Using the parasite specificity of the later two analogues with the known nonspecific de-N-acetylase suicide inhibitor [Smith, T. K., et al. (2001) EMBO J. 20, 3322-3332], GalNCONH(2)-PI and GlcNCONH(2)-beta-PI were designed and found to be potent (IC(50) approximately 0.2 microM), Plasmodium-specific suicide substrate inhibitors. These inhibitors could be potential lead compounds for the development of antimalaria drugs.