Phosphatidic Acid Binds and Stimulates Arabidopsis Sphingosine Kinases

Abstract

Phosphatidic acid (PA) and phytosphingosine-1-phosphate (phyto-S1P) have both been identified as lipid messengers mediating plant response to abscisic acid (ABA). To determine the relationship of these messengers, we investigated the direct interaction of PA with Arabidopsis sphingosine kinases (SPHKs) that phosphorylate phytosphingosine to generate phyto-S1P. Two unique SPHK cDNAs were cloned from the annotated At4g21540 locus of Arabidopsis, and the two transcripts are differentially expressed in Arabidopsis tissues. Both SPHKs are catalytically active, phosphorylating various longchain sphingoid bases (LCBs) and are associated with the tonoplast. Phosphatidic acid (PA) 4 has emerged as a class of pivotal lipid messengers in cell growth, development, and stress responses, and the regulatory functions of PA are being established in plants, animals, and fungi (1-3). PA is a minor membrane lipid, constituting less than 1% of total phospholipids in most plant tissues (4). However, the cellular level of PA in plants is dynamic, increasing rapidly under various conditions, including chilling, freezing, wounding, pathogen elicitation, dehydration, salt, nutrient starvation, nodule induction, and oxidative stress (1, 2, 5, 6). The functional significance of PA has been indicated by characterization of various phospholipase Ds (PLD) that produce regulatory PA and by measurements of PA changes under different stress conditions (1, 2). Characterization of genetic ablations, together with biochemical analyses, has shown that different PLDs have unique functions (1, 7). The differential activation, expression, and cellular locales, as well as substrate preferences of PLDs, indicate that the cellular location and timing of PA production are important determinants of PA function.A series of recent results have provided mechanistic insights into how specific PLD and PA mediate the abscisic acid (ABA) promotion of stomatal closure in Arabidopsis (8, 9). Recently, PLD␣1 and PA were found to regulate NADPH oxidase activity and the production of reactive oxygen species (ROS) in ABAmediated stomatal closure (10). In addition to PA, another lipid messenger, long-chain base-1-phosphate (LCBP) including sphingosine-1-phosphate (S1P) and phyto-S1P has been found to promote the ABA effect on stomatal closure (11-13). Arabidopsis sphingosine kinase (SPHK) activity was mainly associated with the membrane fraction (13). Recent study suggests that sphingosine and S1P are not detectable in Arabidopsis leaves due to the lack of expression of sphingolipid ⌬4-desaturase, indicating that sphingosine and S1P are unlikely to play a significant role in ABA-mediated stomatal closure (14). However, knock-out of Arabidopsis SPHK1 rendered the stomatal closure less sensitive to ABA, whereas overexpression of SPHK1 increased stomatal closure and ABA sensitivity (15). These results suggest that other LCBPs are involved in ABA signaling in Arabidopsis (16). Phytosphingosine is one of such LCBs in Arabidopsis leaves and its phosphorylated form, phyto-S1...

Phosphatidic Acid Binds to Cytosolic Glyceraldehyde-3-phosphate Dehydrogenase and Promotes Its Cleavage in Arabidopsis

“…4C). We tested the SPR response using a mutated Myb protein as a negative control and SPHK1 as a positive control (22). When the same amounts of proteins were used as that of GAPCs, SPHK1 displayed the binding response to PAϩPC liposomes as reported previously (22), whereas a negligible response was obtained from the negative control protein (Fig.…”

“…No binding was detected in the empty vector control. SPHK1, a sphingosine kinase known to bind to PA (22), was included as a positive control for the PA binding assay. In addition, GAPCs did not bind to either of two common polyphosphoinositides PI(4)P or PI(4,5)P 2 (Fig.…”

“…Surface Plasmon Resonance (SPR) Analysis-SPR assay was performed as described previously (22). Briefly, a sensor chip preimmobilized with nitrilotriacetic acid was used to capture purified, His-tagged GAPC1 or GAPC2.…”

“…An NADdependent GAPDH activity assay was performed by spectrophotometric quantification (340 nm) of NADH produced in the reaction according to the method described previously (32). SPHK1 was expressed in Escherichia coli and purified as described previously (22).…”

“…PA was found to interact with abscisic acid-insensitive 1 (ABI1), a protein phosphatase, constitutive triple response 1 (CTR1), a protein kinase, and phosphoethanolamine N-methyltransferase (PEAMT) and inhibit their phosphatase or kinase activities (18 -20). PA stimulates the catalytic activity of a phosphoinositide-dependent protein kinase (PDK1), sphingosine kinases (SPHK1/2), NADPH oxidases (RbohD/F), a mitogenactivated protein kinase (MPK6), and a SNF1-related kinase (SnRK2) by direct interaction (21)(22)(23)(24). In addition to regulating the catalytic activity, PA may tether its binding proteins to membranes.…”