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

Summary 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.