PHOSPHORYLATED NITRATE REDUCTASE FROM SPINACH LEAVES IS INHIBITED BY 14-3-3-PROTEINS AND ACTIVATED BY FUSICOCCIN

Background: Nitrate reductase (NR) in leaves is rapidly inactivated in the dark by a two-step mechanism in which phosphorylation of NR on th e serine at position 543 (Ser543) promotes binding to nitrate reductas e inhibitor protein (NIP). The eukaryotic 14-3-3 proteins bind to many mammalian signalling components (Raf-1, Bcr, phosphoinositide 3-kinas e, protein kinase C, polyomavirus middle-T antigen and Cdc25), and are implicated in the timing of mitosis, DNA-damage checkpoint control, e xocytosis, and activation of the plant plasma-membrane H+-ATPase by fu sicoccin. Their dimeric, saddle-shaped structures support the proposal that 14-3-3 proteins, are 'adaptors' linking different signalling pro teins, but their precise functions are still a mystery. Results: We pu rified NIP to homogeneity and established by means of amino-acid seque ncing that it is a mixture of several 14-3-3 isoforms. Mammalian and y east 14-3-3 proteins were just as effective as NIP at inhibiting phosp horylated NR. The sequence around Ser543, the phosphorylation site in NR, is strikingly similar to the sequences around the phosphoserine re sidues (Ser259 and Ser621) of mammalian Raf-1 that interact with 14-3- 3 proteins. We found that NIP activity was blocked by a synthetic phos phopeptide corresponding to residues 251-266 of Raf. Fusicoccin also b locked NIP activity, and plant plasma-membrane H+-ATPases were activat ed by either fusicoccin,the phosphoserine259-Raf-1 peptide, or protein phosphatase 2A. Conclusions: Our findings establish that the mechanis m of inactivation of NR involves the phosphorylation of Ser 543 follow ed by interaction with one or more plant 14-3-3 proteins. These result s support the idea of a common mechanism for binding of 14-3-3 to its targets in all eukaryotes, and suggest that the phosphoserine259-Raf-1 peptide and fusicoccin may be of general use for disrupting the inter action of 14-3-3 with its target proteins. We propose that the plant p lasma-membrane H+-ATPase is regulated in an analogous manner to NR-NIP , and speculate that 14-3-3 proteins provide a link between 'sensing' the activity state of NR and signalling to other cellular processes in plants.