@article{oh_huber_shen_athwal_wu_huber_2009, title={Overexpression of a directed mutant of 14-3-3 omega in Arabidopsis leaves affects phosphorylation and protein content of nitrate reductase}, volume={87}, ISSN={["1916-2804"]}, DOI={10.1139/b09-003}, abstractNote={The 14-3-3 family of proteins are highly conserved signaling proteins in eukaryotes that bind to their client proteins, usually through specific phosphorylated target sequences. While the 14-3-3 proteins are thought to interact with a wide array of cellular proteins, there have been few studies addressing the in-vivo role of 14-3-3. As one approach to study this in-vivo role, we generated transgenic Arabidopsis plants constitutively overexpressing a directed mutant of 14-3-3 isoform ω that inhibits phosphorylated nitrate reductase (pNR) in a largely divalent-cation-independent manner in vitro. The transgenic plants had increased relative phosphorylation of NR at the regulatory Ser-534 site and decreased NR activity measured in the presence of 5 mmol·L –1 MgCl 2 relative to nontransgenic plants. In addition, total NR protein was increased and the protein half-life was increased about two-fold. Two-dimensional difference gel electrophoresis analysis of proteins extracted from leaves of plants expressing the mutant 14-3-3 identified numerous cellular proteins that were altered in abundance. In particular, several β-glucosidase and glutathione S-transferase isoforms were decreased in abundance relative to wild type plants suggesting a possible alteration in stress or defense responses.}, number={7}, journal={BOTANY}, author={Oh, Man-Ho and Huber, Joan L. and Shen, Wei and Athwal, Gurdeep S. and Wu, Xia and Huber, Steven C.}, year={2009}, month={Jul}, pages={691–701} }
 @article{athwal_huber_2002, title={Divalent cations and polyamines bind to loop 8 of 14-3-3 proteins, modulating their interaction with phosphorylated nitrate reductase}, volume={29}, ISSN={["1365-313X"]}, DOI={10.1046/j.0960-7412.2001.01200.x}, abstractNote={Summary Binding of 14‐3‐3 proteins to nitrate reductase phosphorylated on Ser 543 (phospho‐NR) inhibits activity and is responsible for the inactivation of nitrate reduction that occurs in darkened leaves. The 14‐3‐3‐dependent inactivation of phospho‐NR is known to require millimolar concentrations of a divalent cation such as Mg 2+ at pH 7.5. We now report that micromolar concentrations of the polyamines, spermidine 4+ and spermine 3+ , can substitute for divalent cations in modulating 14‐3‐3 action. Effectiveness of the polyamines decreased with a decrease of polycation charge: spermine 4+ > spermidine 3+ >>> cadavarine 2+ ≈ putrescine 2+ ≈ agmatine 2+ ≈ N 1 ‐acetylspermidine 2+ , indicating that two primary and at least one secondary amine group were required. C‐terminal truncations of GF14ω, which encodes the Arabidopsis 14‐3‐3 isoform ω, indicated that loop 8 (residues 208–219) is the likely cation‐binding site. Directed mutagenesis of loop 8, which contains the EF hand‐like region identified in earlier studies, was performed to test the role of specific amino acid residues in cation binding. The E208A mutant resulted in a largely divalent cation‐independent inhibition of phospho‐NR activity, whereas the D219A mutant was fully Mg 2+ ‐dependent but had decreased affinity for the cation. Mutations and C‐terminal truncations that affected the Mg 2+ dependence of phospho‐NR inactivation had similar effects on polyamine dependence. The results implicate loop 8 as the site of divalent cation and polyamine binding, and suggest that activation of 14‐3‐3s occurs, at least in part, by neutralization of negative charges associated with acidic residues in the loop. We propose that binding of polyamines to 14‐3‐3s could be involved in their regulation of plant growth and development.}, number={2}, journal={PLANT JOURNAL}, author={Athwal, GS and Huber, SC}, year={2002}, month={Jan}, pages={119–129} }
 @article{athwal_lombardo_huber_masters_fu_huber_2000, title={Modulation of 14-3-3 protein interactions with target polypeptides by physical and metabolic effectors}, volume={41}, DOI={10.1093/pcp/41.4.523}, abstractNote={The proteins commonly referred to as 14-3-3s have recently come to prominence in the study of protein:protein interactions, having been shown to act as allosteric or steric regulators and possibly scaffolds. The binding of 14-3-3 proteins to the regulatory phosphorylation site of nitrate reductase (NR) was studied in real-time by surface plasmon resonance, using primarily an immobilized synthetic phosphopeptide based on spinach NR-Ser543. Both plant and yeast 14-3-3 proteins were shown to bind the immobilized peptide ligand in a Mg2+-stimulated manner. Stimulation resulted from a reduction in KD and an increase in steady-state binding level (Req). As shown previously for plant 14-3-3s, fluorescent probes also indicated that yeast BMH2 interacted directly with cations, which bind and affect surface hydrophobicity. Binding of 14-3-3s to the phosphopeptide ligand occurred in the absence of divalent cations when the pH was reduced below neutral, and the basis for enhanced binding was a reduction in KD. At pH 7.5 (+Mg2+), AMP inhibited binding of plant 14-3-3s to the NR based peptide ligand. The binding of AMP to 14-3-3s was directly demonstrated by equilibrium dialysis (plant), and from the observation that recombinant plant 14-3-3s have a low, but detectable, AMP phosphatase activity.}, number={4}, journal={Plant and Cell Physiology}, author={Athwal, G. S. and Lombardo, C. R. and Huber, J. L. and Masters, S. C. and Fu, H. A. and Huber, S. C.}, year={2000}, pages={523–533} }
 @article{athwal_huber_huber_1998, title={Biological significance of divalent metal ion binding to 14-3-3 proteins in relationship to nitrate reductase inactivation}, volume={39}, ISSN={["0032-0781"]}, DOI={10.1093/oxfordjournals.pcp.a029303}, abstractNote={In this report we address two questions regarding the regulation of phosphorylated nitrate reductase (pNR; EC 1.6.6.1) by 14-3-3 proteins. The first concerns the requirement for millimolar concentrations of a divalent cation in order to form the inactive pNR:14-3-3 complex at pH 7.5. The second concerns the reduced requirement for divalent cations at pH 6.5. In answering these questions we highlight a possible general mechanism involved in the regulation of 14-3-3 binding to target proteins. We show that divalent cations (e.g. Ca2+, Mg2+ and Mn2+) bind directly to 14-3-3s, and as a result cause a conformational change, manifested as an increase in surface hydrophobicity. A similar change is also obtained by decreasing the pH from pH 7.5 to pH 6.5, in the absence of divalent cations, and we propose that protonation of amino acid residues brings about a similar effect to metal ion binding. A possible regulatory mechanism, where the 14-3-3 protein has to be "primed" prior to binding a target protein, is discussed.}, number={10}, journal={PLANT AND CELL PHYSIOLOGY}, author={Athwal, GS and Huber, JL and Huber, SC}, year={1998}, month={Oct}, pages={1065–1072} }
 @article{purcino_arellano_athwal_huber_1998, title={Nitrate effect on carbon and nitrogen assimilating enzymes of maize hybrids representing seven eras of breeding}, volume={43}, number={2}, journal={Maydica}, author={Purcino, A. A. C. and Arellano, C. and Athwal, G. S. and Huber, S. C.}, year={1998}, pages={83–94} }
 @article{athwal_huber_huber_1998, title={Phosphorylated nitrate reductase and 14-3-3 proteins - Site of interaction, effects of ions, and evidence for an AMP-binding site on 14-3-3 proteins}, volume={118}, ISSN={["0032-0889"]}, DOI={10.1104/pp.118.3.1041}, abstractNote={The inactivation of phosphorylated nitrate reductase (NR) by the binding of 14-3-3 proteins is one of a very few unambiguous biological functions for 14-3-3 proteins. We report here that serine and threonine residues at the +6 to +8 positions, relative to the known regulatory binding site involving serine-543, are important in the interaction with GF14omega, a recombinant plant 14-3-3. Also shown is that an increase in ionic strength with KCl or inorganic phosphate, known physical effectors of NR activity, directly disrupts the binding of protein and peptide ligands to 14-3-3 proteins. Increased ionic strength attributable to KCl caused a change in conformation of GF14omega, resulting in reduced surface hydrophobicity, as visualized with a fluorescent probe. Similarly, it is shown that the 5' isomer of AMP was specifically able to disrupt the inactive phosphorylated NR:14-3-3 complex. Using the 5'-AMP fluorescent analog trinitrophenyl-AMP, we show that there is a probable AMP-binding site on GF14omega.}, number={3}, journal={PLANT PHYSIOLOGY}, author={Athwal, GS and Huber, JL and Huber, SC}, year={1998}, month={Nov}, pages={1041–1048} }
 @article{toroser_athwal_huber_1998, title={Site-specific regulatory interaction between spinach leaf sucrose-phosphate synthase and 14-3-3 proteins}, volume={435}, ISSN={["0014-5793"]}, DOI={10.1016/S0014-5793(98)01048-5}, abstractNote={We report an Mg2+-dependent interaction between spinach leaf sucrose-phosphate synthase (SPS) and endogenous 14-3-3 proteins, as evidenced by co-elution during gel filtration and co-immunoprecipitation. The content of 14-3-3s associated with an SPS immunoprecipitate was inversely related to activity, and was specifically reduced when tissue was pretreated with 5-aminoimidazole-4-carboxamide riboside, suggesting metabolite control in vivo. A synthetic phosphopeptide based on Ser-229 was shown by surface plasmon resonance to bind a recombinant plant 14-3-3, and addition of the phosphorylated SPS-229 peptide was found to stimulate the SPS activity of an SPS:14-3-3 complex. Taken together, the results suggest a regulatory interaction of 14-3-3 proteins with Ser-229 of SPS.}, number={1}, journal={FEBS LETTERS}, author={Toroser, D and Athwal, GS and Huber, SC}, year={1998}, month={Sep}, pages={110–114} }
 @article{athwal_pearson_laurie_1997, title={Regulation of glutamate dehydrogenase activity by manipulation of nucleotide supply in Daucus carota suspension cultures}, volume={101}, ISSN={["0031-9317"]}, DOI={10.1034/j.1399-3054.1997.1010308.x}, number={3}, journal={PHYSIOLOGIA PLANTARUM}, author={Athwal, GS and Pearson, J and Laurie, S}, year={1997}, month={Nov}, pages={503–509} }