14-3-3:Shc Scaffolds Integrate Phosphoserine and Phosphotyrosine Signaling to Regulate Phosphatidylinositol 3-Kinase Activation and Cell Survival

Integrated cascades of protein tyrosine and serine/threonine phosphorylation play essential roles in transducing signals in response to growth factors and cytokines. How adaptor or scaffold proteins assemble signaling complexes through both phosphotyrosine and phosphoserine/threonine residues to regulate specific signaling pathways and biological responses is unclear. We show in multiple cell types that endogenous 14-3-3ζ is phosphorylated on Tyr¹⁷⁹ in response to granulocyte macrophage colony-stimulating factor. Importantly, 14-3-3ζ can function as an intermolecular bridge that couples to phosphoserine residues and also directly binds the SH2 domain of Shc via Tyr¹⁷⁹. The assembly of these 14-3-3:Shc scaffolds is specifically required for the recruitment of a phosphatidylinositol 3-kinase signaling complex and the regulation of CTL-EN cell survival in response to cytokine. The biological significance of these findings was further demonstrated using primary bone marrow-derived mast cells from 14-3-3ζ^-/- mice. We show that cytokine was able to promote Akt phosphorylation and viability of primary mast cells derived from 14-3-3ζ^-/- mice when reconstituted with wild type 14-3-3ζ, but the Akt phosphorylation and survival response was reduced in cells reconstituted with the Y179F mutant. Together, these results show that 14-3-3:Shc scaffolds can act as multivalent signaling nodes for the integration of both phosphoserine/threonine and phosphotyrosine pathways to regulate specific cellular responses.The ability of a cell to respond to extrinsic stimuli critically hinges on its ability to regulate specific intracellular protein-protein interactions in a reversible manner. Such signals are relayed within the cell through the assembly of signaling complexes that are built using protein scaffolds. One important mechanism by which this occurs is via the binding of Src homology 2 (SH2)⁵ or phosphotyrosine-binding (PTB) domains to phosphotyrosine residues (1, 2). Importantly, the ability of individual SH2 or PTB domains to recognize specific phosphotyrosine motifs in different proteins enables the assembly of purpose-built signaling complexes that promote signaling via specific pathways (3). In some cases, signaling proteins not only contain more than one SH2 and/or PTB domain but are also themselves tyrosine-phosphorylated, leading to a network of phosphotyrosine-mediated protein-protein interactions.Although less well studied, phosphoserine/threonine-binding proteins are also important for the assembly of signaling complexes. For example, the 14-3-3 family of proteins is able to bind phosphoserine/threonine residues in a sequence-specific context (RSX(S/T)XP and RXXX(S/T)XP, where (S/T) is phosphoserine/threonine) (4, 5). The 14-3-3 proteins have been proposed to function as “modifiers” or “sequestrators”; however, because of their dimeric structure, they have also been proposed to function as “adaptor” or “scaffold” proteins through their ability to bring together two serine/threonine phosphorylated proteins (4–7). Additionally, a number of phosphoserine/threonine-binding modules such as tryptophan-tryptophan (WW), Forkhead-associated (FHA), Polo box (PBD), and BRCA1 C-terminal (BRCT) domains have been shown to interact with phosphoserine/threonine residues within a sequence-specific context and have also been proposed to be important for the assembly of multi-protein signaling complexes (8).The genes/cassettes encoding each phosphotyrosine- and phosphoserine/threonine-binding protein/module arose as a separate evolutionary event, and the DNA encoding these modules has been subject to frequent duplication and shuffling. For example, the 14-3-3 family of proteins is ubiquitously expressed in mammalian tissues and is composed of seven different isoforms, each encoded by a separate gene (6). In addition, duplication and shuffling of SH2, PTB, WW, FHA, PBD, and BCRT cassettes has led to their wide distribution among signaling proteins. Yet, despite the frequent duplication and shuffling of the DNA encoding these domains throughout evolution, proteins that contain both a phosphotyrosine-binding cassette (e.g. SH2 or PTB) and a phosphoserine/threonine-binding cassette (e.g. 14-3-3, WW, FHA, PBD, and BCRT) have not been identified. This is perhaps surprising given the highly integrated nature of phosphotyrosine and phosphoserine/threonine signaling and would suggest that alternative strategies to regulate integration are at play.We show here that 14-3-3ζ is tyrosine-phosphorylated, enabling it to interact with Shc and provide a scaffold for the assembly of signaling complexes via both phosphoserine/threonine and phosphotyrosine residues. Our results show that Tyr¹⁷⁹ of 14-3-3ζ directly binds to the SH2 domain of Shc and that this interaction is critical for the assembly of a phosphatidylinositol (PI) 3-kinase signaling complex in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation. Moreover, we show that Tyr¹⁷⁹ of 14-3-3ζ is necessary and sufficient for the ability of GM-CSF to regulate PI 3-kinase and cell survival in the CTL-EN line. Furthermore, reconstitution of primary mast cells derived from 14-3-3ζ^-/- mice with wild type (wt) or mutant 14-3-3ζ demonstrated an important role for Tyr¹⁷⁹ in cytokine-mediated Akt phosphorylation and cell survival. These multivalent 14-3-3:Shc scaffolds provide a novel mechanism by which phosphoserine/threonine and phosphotyrosine pathways can be integrated for the regulation of specific cellular responses.     

Effects of iron and copper ions in promotion of selective abscission and ethylene production by citrus fruit and the inactivation of indoleacetic Acid

Application of Cu²⁺ (<10^-2 M) and Fe³⁺ ions as aqueous solutions of chloride salts promoted fruit abscission, erratic rind damage, and ethylene production of various citrus species with little to no defoliation. Mixing of 10^-5 M Cu²⁺ or Fe³⁺ ions with equimolar indole-3-acetic acid resulted in a reduction of the ultraviolet absorption at 220 nanometers, and an increase at 245 nanometers. Ultraviolet irradiation accelerated the change by Fe³⁺ and Cu²⁺ ions in the absorption of indole-3-acetic acid. Pretreatment of indole-3-acetic acid with Fe³⁺ and Cu²⁺ ions for 6 hours resulted in more than 90% reduction in its growth-promoting activity in the Avena bioassay, even when cations were removed by chromatography. Acceleration of abscission by Fe³⁺ and Cu²⁺ ions could be related to both promotion of ethylene production and direct inactivation of auxin.     

The covalent binding of 3-methylindole metabolites to bovine tissue

Tritiated 3-methylindole (3MI) was administered intravenously to calves. Total and covalent bound radioactivity were measured in different tissues. Pulmonary tissue showed the highest concentration of covalent bound radioactivity. (G-³H) or (methyl-¹⁴C) 3MI became covalently bound to microsomal protein when incubated with bovine lung microsomes. This covalent binding was dependent on time, temperature, oxygen and NADPH and was inhibited by SKF-525A, cytochrome c, a carbon monoxide enriched atmosphere and cysteine. The microsomal enzyme system catalysing covalent binding of 3MI has the classical characteristics of a cytochrome P-450 dependent mixed function oxidase. Metabolic activation of 3MI to a highly electrophilic intermediate, may be fundamental in the pathogenesis of 3MI induced pulmonary damage.     

Microfilaments and tropomyosin of cultured mammalian cells: isolation and characterization

Microfilaments were isolated from cultured mammalian cells, utilizing procedures similar to those for isolation of "native" thin filaments from muscle. Isolated microfilaments from rat embryo, baby hamster kidney (BHK- 21), and Swiss mouse 3T3 cells appeared structurally similar to muscle thin filaments, exhibiting long, 6 nm Diam profiles with a beaded, helical substructure. An arrowhead pattern was observed after reaction of isolated microfilaments with rabbit skeletal muscle myosin subfragment 1. Under appropriate conditions, isolated microfilaments will aggregate into a form that resembles microfilament bundles seen in situ cultured cells. Isolated microfilaments represent a complex of proteins including actin. Some of these components have been tentatively identified, based on coelectrophoresis with purified proteins, as myosin, tropomyosin, and a high molecular weight actin-binding protein. The tropomyosin components of isolated microfilaments were unexpected; polypeptides comigrated on SDS-polyacrylamide gels with both muscle and nonmuscle types of tropomyosin. In order to identify more specifically these subunits, we isolated and partially characterized tropomyosin from three cell types. BHK-21 cell tropomyosin was similar to other nonmuscle tropomyosins, as judged by several criteria. However, tropomyosin isolated from rate embryo and 3T3 cells contained subunits that comigrated with both skeletal muscle and nonmuscle types of myosin, whereas the BHK cell protein consistently contained a minor muscle-like subunit. The array of tropomyosin subunits present in a cell culture was reflected in the polypeptide chain pattern seen on SDS-polyacrylamide gels of microfilaments isolated from that culture. These studies provide a starting point for correlating changes in the ultrastructural organization of microfilaments with alterations in their protein composition.     

Zooplankton excretion and NH 4 + cycling in near-surface waters of the Southern Ocean. I. Ross sea,austral summer 1977–1978

Summary Ammonium (NH ₄ ⁺ ) is a preferred nitrogen source for Antarctic phytoplankton growth and the principal nitrogenous waste excreted by zooplankton and micronekton. In austral summer 1977–1978, NH ₄ ⁺ was present at average concentrations of 0.1–0.3 g-at liter^-1 in the upper 50 m of the Ross Sea and was excreted by resident mixed zooplankton at rates of 1.6 g-at NH ₄ ⁺ g^-1 wet weight h^-1. Zooplankton biomass sampled by vertically-towed 0.5 m nets averaged 0.06 ml m^-3 in the upper 200 m over the Ross Sea shelf and 0.09 ml m^-3 in the upper 200 m north of the shelf-slope front. Olson (1980) has reported that phytoplankton nitrogenous requirements were 110 and 170 g-at NH ₄ ⁺ m^-3 day^-1 in these respective regions. A synthesis of these data predicts that average summertime standing crops of zooplankton might regenerate only about 2% of the daily NH ₄ ⁺ requirements of Ross Sea phytoplankton. For comparison, the potential NH ₄ ⁺ excretion impact of microheterotrophs, seabirds, and local aggregations of zooplankton are discussed and contrasted with non-biogenic inputs of NH ₄ ⁺ associated with basal meltwater from the Ross Ice Shelf and the seasonal melting of the sea ice pack.     

DISTURBANCE OF STREAM PERIPHYTON BY PERTURBATIONS IN SHEAR STRESS: TIME TO STRUCTURAL FAILURE AND DIFFERENCES IN COMMUNITY RESISTANCE1

The resistance of stream periphyton to structural disturbance by increases in shear stress (simulating a spate) was investigated in a laboratory flow tank. We monitored loss of biomass from a filamentous community (dominated by Melosira varians) under four different levels of shear stress. In each case, any loss that was going to occur did so within 10 min for this community. In a second experiment, we tested the resistance of four different communities (two dominated by nonfilamentous diatoms and two dominated by filamentous green algae/diatoms) to increases in shear stress. Nine different levels of shear stress were used, ranging from 1- to 70-fold higher than the conditions to which the communities were acclimated. All communities were 14 days old, but some differences in initial biomass occurred that influenced the degree of resistance independently of species composition. Overall, the nonfilamentous diatom communities were the most resistant, and the filamentous communities were the least resistant. The kinetics of the sloughing process varied among community types, with a community dominated by Melosira varians/Gom-phonema parvulum losing 50% of its biomass with only a 3-fold increase in shear stress. In contrast, a community dominated by the nonfilamentous diatoms Fragilaria vaucheriae/Cymbella minuta lost <50% of its biomass after a 70-fold increase in shear stress. Shear stresses required for 50% loss of biomass for the different communities were as follows: 3.6 Newtons.m^?2 for the Melosira varians/Gomphonema parvulum community, 10.0 N.m^?2 for the Spirogyra sp./Gomphoneis her-culeana/Ulothrix zonata community, 50.6 N.m^?2 for the Fragilaria construens/Cymbella minuta/Ach-nanthes minutissima community, and >90.0 N.m^?2for the Fragilaria vaucheriae/Cymbella minuta community. These results show that spates without bedload movement can potentially have widely differing disturbance effects on periphyton loss among streams depending on the initial taxonomic composition of resident communities. These results have important implications for stream ecosystem analysis and modeling.     

Protection, pathogenesis and phenotypic plasticity in Plasmodium falciparum malaria

Why does Plasmodium falciparum cause severe illness in some but not all infections? How is clinical immunity acquired? These questions have intrigued investigators since the clinical epidemiology of malaria was first described. The search for answers to both questions has highlighted the changes that take place at the surface of infected red blood cells during the last half of the erythrocytic cycle. These changes specify the antigenic and adhesive or cytoadherence phenotypes for the infected cell. Now the antigenic and adhesive phenotypes appear to be linked and together undergo clonal variation. In this article David Roberts, Beverley-Ann Biggs, Graham Brown and Christopher Newbold explain how clonal phenotypic variation and the linkage between adhesive and antigenic types contribute to our understanding of naturally acquired immunity and of pathogenesis of severe malaria.     

The consequence of passive administration of an anti-human immunodeficiency virus type 1 neutralizing monoclonal antibody before challenge of chimpanzees with a primary virus isolate.

The anti-gp41 virus neutralizing monoclonal antibody 2F5 was infused into chimpanzees, which were then given an intravenous challenge with a primary human immunodeficiency virus type I (HIV-1) isolate. In two control animals, the infection was established immediately, as evidenced by positive cell-associated DNA PCR and serum RNA PCR tests within 1 week, seroconversion by 4 weeks, and development of lymphadenopathy in this acute phase. Serum RNA PCR tests were negative in one of the two antibody-infused animals until week 8 and in the other antibody-infused animal until week 12; both animals seroconverted at week 14. The peak of measurable virus-specific serum RNA was delayed until week 16 in one antibody-infused animal. Virus-specific RNA in the other animal did not reach levels comparable to those in the other animals through 1 year of follow-up studies. Virus was isolated from the week 16 blood sample from one infused animal. Virus was not isolated from peripheral blood of the second animal but was isolated from lymph node cells taken at week 36. The infection of untreated chimpanzees with this primary isolate appears robust. Use of this isolate should widen the scope of possible experiments in the chimpanzee model. This antibody infusion study indicates that neutralizing antibody, when present at the time of challenge, affects the timing and level of infection and remains influential after it can no longer be detected in the peripheral circulation. It is possible that preexisting, neutralizing antibodies (passively administered or actively elicited) affect the course of acute-phase virus replication in humans. It remains to be established whether these immunologically mediated early effects will influence the course of HIV-1 disease.