Phosphorylation and 14-3-3 binding of Arabidopsis trehalose-phosphate synthase 5 in response to 2-deoxyglucose.
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.
Harthill
Jean E
JE
MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK.
Meek
Sarah E M
SE
Morrice
Nick
N
Peggie
Mark W
MW
Borch
Jonas
J
Wong
Barry H C
BH
Mackintosh
Carol
C
D048948:14-3-3 Proteins / Q000378:metabolism*; D000091162:AMP-Activated Protein Kinase Kinases; D055372:AMP-Activated Protein Kinases; D000409:Alanine / Q000378:metabolism; D017360:Arabidopsis / Q000187:drug effects / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000378:metabolism; D001665:Binding Sites; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D003847:Deoxyglucose / Q000494:pharmacology*; D005964:Glucosyltransferases / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D009097:Multienzyme Complexes / Q000378:metabolism; D020125:Mutation, Missense; D010629:Phenformin / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D020033:Protein Isoforms / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism
D048948:14-3-3 Proteins; D029681:Arabidopsis Proteins; D009097:Multienzyme Complexes; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D013912:Threonine; D012694:Serine; D003847:Deoxyglucose; D010629:Phenformin; D005964:Glucosyltransferases; C513675:TPS6 protein, Arabidopsis; C513676:TPS7 protein, Arabidopsis; C023180:trehalose-6-phosphate synthase; C071570:SNF1-related protein kinases; D017346:Protein Serine-Threonine Kinases; C109790:STK11 protein, human; D000091162:AMP-Activated Protein Kinase Kinases; D055372:AMP-Activated Protein Kinases; D000409:Alanine