Abstract
De novo phosphatidylcholine (PC) biosynthesis via the Kennedy pathway involves highly endergonic biochemical reactions that must be fine-tuned with energy homeostasis. Previous studies have shown that CTP:phosphocholine cytidylyltransferase (CCT) is an important regulatory enzyme in this pathway and that its activity can be controlled at both transcriptional and posttranslational levels. Here we identified an important additional mechanism regulating plant CCT1 activity. Comparative analysis revealed that Arabidopsis CCT1 (AtCCT1) contains catalytic and membrane-binding domains that are homologous to those of rat CCT1. In contrast, the C-terminal phosphorylation domain important for stringent regulation of rat CCT1 was apparently missing in AtCCT1. Instead, we found that AtCCT1 contains a putative consensus site (Ser-187) for modification by sucrose nonfermenting 1-related protein kinase 1 (SnRK1 or KIN10/SnRK1.1), involved in energy homeostasis. Phos-tag SDS-PAGE coupled with MS analysis disclosed that SnRK1 indeed phosphorylates AtCCT1 at Ser-187, and we found that AtCCT1 phosphorylation substantially reduces its activity by as much as 70%. An S187A variant exhibited decreased activity, indicating the importance of Ser-187 in catalysis, and this variant was less susceptible to SnRK1-mediated inhibition. Protein truncation and liposome binding studies indicated that SnRK1-mediated AtCCT1 phosphorylation directly affects the catalytic domain rather than interfering with phosphatidate-mediated AtCCT1 activation. Overexpression of the AtCCT1 catalytic domain in Nicotiana benthamiana leaves increased PC content, and SnRK1 co-expression reduced this effect. Taken together, our results suggest that SnRK1 mediates the phosphorylation and concomitant inhibition of AtCCT1, revealing an additional mode of regulation for this key enzyme in plant PC biosynthesis.
Highlights
De novo phosphatidylcholine (PC) biosynthesis via the Kennedy pathway involves highly endergonic biochemical reactions that must be fine-tuned with energy homeostasis
To probe whether Arabidopsis CCT1 has the same property, the cDNAs encoding AtCCT1, AtCCT1 catalytic domain (AtCCT11–197, hereafter referred to as AtCCT1-CD), and AtCCT11–221 were recombinantly expressed in Escherichia coli and partially purified through Ni-NTA (Fig. S2)
Our results indicate that sucrose nonfermenting 1–related protein kinase 1 (SnRK1) regulates the activity of AtCCT1, which provides a mechanistic link between sugar/energy homeostasis and the de novo PC biosynthesis
Summary
De novo phosphatidylcholine (PC) biosynthesis via the Kennedy pathway involves highly endergonic biochemical reactions that must be fine-tuned with energy homeostasis. Our results suggest that SnRK1 mediates the phosphorylation and concomitant inhibition of AtCCT1, revealing an additional mode of regulation for this key enzyme in plant PC biosynthesis. A number of studies have shown that, among the enzymes for de novo PC biosynthesis in plants, CCT catalyzes a key regulatory step [6, 7], and its activity is controlled at the transcriptional [7] and posttranslational levels [6]. As for carbohydrates, SnRK1 regulates two key enzymes, sucrose phosphate synthase and ADP-glucose pyrophosphorylase, in the biosynthesis of sucrose and starch, respectively [11, 12] This kinase appears to modulate the transcription of carbohydrate metabolic genes, the exact mechanisms have not yet been fully elucidated [13, 14]
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