Phosphorylation and 14-3-3 binding of Arabidopsis trehalose-phosphate synthase 5 in response to 2-deoxyglucose

Plant J. 2006 Jul;47(2):211-23. doi: 10.1111/j.1365-313X.2006.02780.x. Epub 2006 Jun 8.

Abstract

Trehalose-6-phosphate is a 'sugar signal' that regulates plant metabolism and development. The Arabidopsis genome encodes trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphatase (TPP) enzymes. It also encodes class II proteins (TPS isoforms 5-11) that contain both TPS-like and TPP-like domains, although whether these have enzymatic activity is unknown. In this paper, we show that TPS5, 6 and 7 are phosphoproteins that bind to 14-3-3 proteins, by using 14-3-3 affinity chromatography, 14-3-3 overlay assays, and by co-immunoprecipitating TPS5 and 14-3-3 isoforms from cell extracts. GST-TPS5 bound to 14-3-3s after in vitro phosphorylation at Ser22 and Thr49 by either mammalian AMP-activated protein kinase (AMPK) or partially purified plant Snf1-related protein kinase 1 (SnRK1s). Dephosphorylation of TPS5, or mutation of either Ser22 or Thr49, abolished binding to 14-3-3s. Ser22 and Thr49 are both conserved in TPS5, 7, 9 and 10. When GST-TPS5 was expressed in human HEK293 cells, Thr49 was phosphorylated in response to 2-deoxyglucose or phenformin, stimuli that activate the AMPK via the upstream kinase LKB1. 2-deoxyglucose stimulated Thr49 phosphorylation of endogenous TPS5 in Arabidopsis cells, whereas phenformin did not. Moreover, extractable SnRK1 activity was increased in Arabidopsis cells in response to 2-deoxyglucose. The plant kinase was inactivated by dephosphorylation and reactivated by phosphorylation with human LKB1, indicating that elements of the SnRK1/AMPK pathway are conserved in Arabidopsis and human cells. We hypothesize that coordinated phosphorylation and 14-3-3 binding of nitrate reductase (NR), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (F2KP) and class II TPS isoforms mediate responses to signals that activate SnRK1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 14-3-3 Proteins / metabolism*
  • AMP-Activated Protein Kinases
  • Alanine / metabolism
  • Arabidopsis / drug effects
  • Arabidopsis / metabolism*
  • Arabidopsis Proteins / metabolism
  • Binding Sites
  • Cells, Cultured
  • Chromatography, Affinity
  • Deoxyglucose / pharmacology*
  • Glucosyltransferases / metabolism*
  • Humans
  • Immunoprecipitation
  • Multienzyme Complexes / metabolism
  • Mutation, Missense
  • Phenformin / pharmacology
  • Phosphorylation / drug effects
  • Protein Isoforms / metabolism
  • Protein-Serine-Threonine Kinases / metabolism
  • Recombinant Fusion Proteins / metabolism
  • Serine / metabolism
  • Threonine / metabolism

Substances

  • 14-3-3 Proteins
  • Arabidopsis Proteins
  • Multienzyme Complexes
  • Protein Isoforms
  • Recombinant Fusion Proteins
  • Threonine
  • Serine
  • Deoxyglucose
  • Phenformin
  • Glucosyltransferases
  • TPS6 protein, Arabidopsis
  • TPS7 protein, Arabidopsis
  • trehalose-6-phosphate synthase
  • SNF1-related protein kinases
  • STK11 protein, human
  • Protein-Serine-Threonine Kinases
  • AMP-Activated Protein Kinases
  • Alanine