TMK-based cell-surface auxin signalling activates cell-wall acidification

The phytohormone auxin controls many processes in plants, at least in part through its regulation of cell expansion1. The acid growth hypothesis has been proposed to explain auxin-stimulated cell expansion for five decades, but the mechanism that underlies auxin-induced cell-wall acidification is poorly characterized. Auxin induces the phosphorylation and activation of the plasma membrane H+-ATPase that pumps protons into the apoplast2, yet how auxin activates its phosphorylation remains unclear. Here we show that the transmembrane kinase (TMK) auxin-signalling proteins interact with plasma membrane H+-ATPases, inducing their phosphorylation, and thereby promoting cell-wall acidification and hypocotyl cell elongation in Arabidopsis. Auxin induced interactions between TMKs and H+-ATPases in the plasma membrane within seconds, as well as TMK-dependent phosphorylation of the penultimate threonine residue on the H+-ATPases. Our genetic, biochemical and molecular evidence demonstrates that TMKs directly phosphorylate plasma membrane H+-ATPase and are required for auxin-induced H+-ATPase activation, apoplastic acidification and cell expansion. Thus, our findings reveal a crucial connection between auxin and plasma membrane H+-ATPase activation in regulating apoplastic pH changes and cell expansion through TMK-based cell surface auxin signalling.

The flavin transferase ApbE flavinylates the ferredoxin:NAD+-oxidoreductase Rnf required for N2 fixation in Azotobacter vinelandii

Azotobacter vinelandii, the model microbe in nitrogen fixation studies, uses the ferredoxin:NAD+-oxidoreductase Rnf to regenerate ferredoxin (flavodoxin) acting as an electron donor for nitrogenase. However, the relative contribution of Rnf into nitrogenase functioning is unknown because this bacterium contains another ferredoxin reductase, FixABCX. Furthermore, Rnf is flavinylated in the cell, but the importance and pathway of this modification reaction also remain largely unknown. We have constructed A. vinelandii cells with impaired activities of FixABCX and/or putative flavin transferase ApbE. The ApbE-deficient mutant could not produce covalently flavinylated membrane proteins and demonstrated a markedly decreased flavodoxin:NAD+ oxidoreductase activity and significant growth defect under diazotrophic conditions. The double ΔFix/ΔApbE mutation abolished the flavodoxin:NAD+ oxidoreductase activity and the ability of A. vinelandii to grow in the absence of fixed nitrogen source. ApbE flavinylated a truncated RnfG subunit of Rnf1 by forming a phosphoester bond between FMN and a threonine residue. These findings indicate that Rnf (presumably its Rnf1 form) is the major ferredoxin-reducing enzyme in the nitrogen fixation system and that the activity of Rnf depends on its covalent flavinylation by the flavin transferase ApbE.

pH modulates interaction of 14-3-3 proteins with pollen plasma membrane H+ ATPases independently from phosphorylation

Pollen grains transport the sperm cells through the style tissue via a fast-growing pollen tube to the ovaries where fertilization takes place. Pollen tube growth requires a precisely regulated network of cellular as well as molecular events including the activity of the plasma membrane H+ ATPase, which is known to be regulated by reversible protein phosphorylation and subsequent binding of 14-3-3 isoforms. Immunodetection of the phosphorylated penultimate threonine residue of the pollen plasma membrane H+ ATPase (LilHA1) of Lilium longiflorum pollen revealed a sudden increase in phosphorylation with the start of pollen tube growth. In addition to phosphorylation, pH modulated the binding of 14-3-3 isoforms to the regulatory domain of the H+ ATPase, whereas metabolic components had only small effects on 14-3-3 binding, as tested with in vitro assays using recombinant 14-3-3 isoforms and phosphomimicking substitutions of the threonine residue. Consequently, local H+ influxes and effluxes as well as pH gradients in the pollen tube tip are generated by localized regulation of the H+ ATPase activity rather than by heterogeneous localized distribution in the plasma membrane.

Threonine phosphorylation regulates the molecular assembly and signaling of EGFR in cooperation with membrane lipids

The cytoplasmic domain of the receptor tyrosine kinases (RTKs) plays roles as a phosphorylation enzyme and a protein scaffold but the regulation of these two functions is not fully understood. We here analyzed assembly of the transmembrane (TM)-juxtamembrane (JM) region of EGFR, one of the best studied species of RTKs, by combining single-pair FRET imaging and a nanodisc technique. The JM domain of EGFR contains a threonine residue that is phosphorylated after ligand association. We observed that the TM-JM peptides of EGFR form anionic lipid-induced dimers and cholesterol-induced oligomers. The two forms involve distinct molecular interactions, with a bias towards oligomer formation upon threonine phosphorylation. We further analyzed the functions of whole EGFR molecules, with or without a threonine to alanine substitution in the JM domain, in living cells. The results suggested an autoregulatory mechanism in which threonine phosphorylation of the JM domain causes a switch from kinase activation dimers to scaffolding oligomers.

First person – Panagiota Giardoglou and Despina Bournele

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Biology Open, helping early-career researchers promote themselves alongside their papers. Panagiota Giardoglou and Despina Bournele are co-first authors on ‘A zebrafish forward genetic screen identifies an indispensable threonine residue in the kinase domain of PRKD2’, published in BiO. Panagiota is a PhD student in the lab of Dr Dimitris Beis at the Biomedical Research Foundation Academy of Athens, Greece, investigating modelling human cardiovascular diseases and underlying the mechanisms involved in their pathophysiology. Despina is a toxicologist in the lab of Dr Kyriaki Machera at the Benaki Phytopathological Institute, Kifissia, Athens, Greece, investigating molecular and developmental biology, and zebrafish toxicology.

MS-based proteomics for comprehensive investigation of protein O-GlcNAcylation

Protein O-GlcNAcylation refers to the covalent binding of a single N-acetylglucosamine (GlcNAc) to the serine or threonine residue. This modification primarily occurs on proteins in the nucleus and the cytosol,...

Contrasting the Conformational Effects of α-O-GalNAc and α-O-Man Glycan Protein Modifications and their Impact on the Mucin-Like Region of alpha-Dystroglycan

We have carried out a comparative study of the conformational impact of modifications to threonine residues of either α-O-Man or α-O-GalNAc in the context of a sequence from the mucin-like region of α-dystroglycan. Both such modifications can coexist in this domain of the glycoprotein. Solution NMR experiments and molecular dynamics calculations were employed. Comparing the results for a unmodified peptide Ac- PPTTTTKKP-NH2 sequence from α-dystroglycan, and glycoconjugates with either modification on the Ts, we find that the impact of the α-O-Man modification on the peptide scaffold is quite limited, while that of the α-O-GalNAc is more profound. The results for the α-O-GalNAc glycoconjugate are consistent with what has been seen earlier in other systems. Further examination of the NMR-based structure and the MD results suggest a more extensive network of hydrogen bond interactions within the α-O-GalNAc-threonine residue than has been previously appreciated, which influence the properties of the protein backbone. The conformational effects are relevant to the mechanical properties of α-dystroglycan.

Structural evidence for a proline-specific glycopeptide recognition domain in an O-glycopeptidase

The glycosylation of proteins is typically considered as a stabilizing modification, including resistance to proteolysis. A class of peptidases, referred to as glycopeptidases or O-glycopeptidases, circumvent the protective effect of glycans against proteolysis by accommodating the glycans in their active sites as specific features of substrate recognition. IMPa from Pseudomonas aeruginosa is such an O-glycopeptidase that cleaves the peptide bond immediately preceding a site of O-glycosylation, and through this glycoprotein-degrading function contributes to the host-pathogen interaction. IMPa, however, is a relatively large multidomain protein and how its additional domains may contribute to its function remains unknown. Here, through the determination of a crystal structure of IMPa in complex with an O-glycopeptide, we reveal that the N-terminal domain of IMPa, which is classified in Pfam as IMPa_N_2, is a proline recognition domain that also shows the properties of recognizing an O-linked glycan on the serine/threonine residue following the proline. The proline is bound in the center of a bowl formed by four functionally conserved aromatic amino acid side chains while the glycan wraps around one of the tyrosine residues in the bowl to make classic aromatic ring-carbohydrate CH-π interactions. This structural evidence provides unprecedented insight into how the ancillary domains in glycoprotein-specific peptidases can noncatalytically recognize specific glycosylated motifs that are common in mucin and mucin-like molecules.

T606-phosphorylated Kaiso interacting with 14-3-3 and p120ctn is sequestered in the cytoplasm of human cells

Kaiso is a transcription factor in the nucleus and p120ctn-binding protein in the cytoplasm. Although it is known that p120ctn is involved in Kaiso cytoplasmic-nuclear transportation, regulatory mechanisms of Kaiso transportation remain to be explored. We firstly found that Kaiso could directly interact with 14-3-3 family proteins, depending on the phosphorylation at the 606 threonine residue (T606) within the RSSTIP motif of Kaiso. AKT1 could phosphorylate Kaiso at T606. T606A mutation abolished most Kaiso-14-3-3 interaction. Notably, we found that the phosphorylated Kaiso (pT606-Kaiso) could also bind to p120ctn in the cytoplasm and block the cytoplasmic-nuclear transportation of Kaiso. The present study indicates, for the first time, that Kaiso can be phosphorylated by AKT1 at T606 and that pT606-Kaiso can bind both 14-3-3 and p120ctn proteins in the cytoplasm. The pT606-Kaiso-p120ctn (and 14-3-3) complexes cannot shift to the nucleus and accumulate in the cytoplasm. T606 phosphorylation regulates intracellular transportation of Kaiso.

Autosomal Recessive Non-syndromic Keratoconus: Homozygous Frameshift Variant in the Candidate Novel Gene GALNT14

Background: Keratoconus (KC) is usually bilateral, noninflammatory progressive corneal ectasia in which the cornea becomes progressively thin and conical. Despite the strong evidence of genetic contribution in KC, the etiology of KC is not understood in most cases. Methods: In this study, we used whole-exome sequencing to identify the genetic cause of KC in two sibs in a consanguineous family. The Homozygous frameshift variant NM_001253826.1:c.60delC;p.Leu21Cysfs*6 was identified in the gene Nacetylgalactosaminyltransferase 14 (GALNT14). The variant does not exist in all public databases neither in our internal exome database. Moreover, no database harbours homozygous loss of function variants in the candidate gene. Result: GALNT14 catalyses the initial reaction in O-linked oligosaccharide biosynthesis, the transfer of an N-acetyl-D- galactosamine residue to a serine or threonine residue on target proteins especially Mucins. Conclusion: As alterations of mucin’s glycosylation are linked to a number of eye diseases, we demonstrate in this study an association between the truncated protein GALNT14 and KC.