The N-terminal Membrane Occupation and Recognition Nexus Domain of Arabidopsis Phosphatidylinositol Phosphate Kinase 1 Regulates Enzyme Activity

Abstract

The type I B family of phosphatidylinositol phosphate kinases (PIPKs) contain a characteristic region of Membrane Occupation and Recognition Nexus (MORN) motifs at the N terminus. These MORN motifs are not found in PIPKs from other eukaryotes. To understand the impact of the additional N-terminal domain on protein function and subcellular distribution, we expressed truncated and full-length versions of AtPIPK1, one member of this family of PIPKs, in Escherichia coli and in tobacco cells grown in suspension culture. Deletion of the Nterminal MORN domain (amino acids 1-251) of AtPIPK1 increased the specific activity of the remaining C-terminal peptide (⌬MORN) >4-fold and eliminated activation by phosphatidic acid (PtdOH). PtdOH activation could also be eliminated by mutating Pro 396 to Ala (P396A) in the predicted linker region between the MORN and the kinase homology domains. AtPIPK1 is product-activated and the MORN domain binds PtdIns(4,5)P 2 . Adding back the MORN peptide to ⌬MORN or to the PtdOH-activated full-length protein increased activity ϳ2-fold. Furthermore, expressing the MORN domain in vivo increased the plasma membrane PtdInsP kinase activity. When cells were exposed to hyperosmotic stress, the MORN peptide redistributed from the plasma membrane to a lower phase or endomembrane fraction. In addition, endogenous PtdInsP kinase activity increased in the endomembrane fraction of hyperosmotically stressed cells. We conclude that the MORN peptide can regulate both the function and distribution of the enzyme in a manner that is sensitive to the lipid environment. Phosphatidylinositol-4-phosphate (PtdInsP)2 5-kinases catalyze the synthesis of phosphatidylinositol-(4,5)-bisphosphate (PtdIns(4,5)P 2 ), a key component in phosphoinositide (PI) signaling that regulates many cellular processes. In Arabidopsis, there are 11 isoforms of PtdInsP 5-kinases that have been divided into 2 subfamilies depending on the size of the proteins (1). Subfamily A consists of AtPIPK10 and AtPIPK11, which contain 401 and 421 amino acids, respectively. The subfamily B proteins (AtPIPK1-9) are larger, from 705 to 859 amino acids, and contain repeated 23-amino acid Membrane Occupation and Recognition Nexus (MORN) motifs in the N terminus. There are 7 MORN motifs in AtPIPK1-3 and 8 motifs in AtPIPK4 -9. The predicted B subfamily PIPKs from rice (2), tomato, and maize also have multiple MORN motifs in the N terminus based on data base analysis; however, these motifs are not found in PIPKs from other eukaryotes including mammals, Caenorhabditis elegans or yeast. In an attempt to understand what advantage there might be for a majority of the plant PIPKs to have this novel N-terminal extension we investigated the effect of the N-terminal MORN domain of AtPIPK1 on enzyme activity and subcellular distribution. MORN motifs were first described in junctophilin (3) and have since been reported to be present in several proteins involved in membrane fission (4, 5). Junctophilin is an integral endoplasmic reticulum (ER) protein that is a compon...

A Dibasic Amino Acid Pair Conserved in the Activation Loop Directs Plasma Membrane Localization and Is Necessary for Activity of Plant Type I/II Phosphatidylinositol Phosphate Kinase

“…1), of which the MD is thought to be a plasma membranelocalizing module (Ma et al, 2006;Im et al, 2007;Kusano et al, 2008). Our demonstration that the two moss PIPKs, GFP-PpPIPK1 and GFP-PpPIPK2, localized to plasma membranes (Saavedra et al, 2009) raised the obvious question of whether the MD truly regulates plasma membrane localization of plant PIPKs.…”

“…Moreover, the MORN domain may play a role in the plasma membrane localization of OsPIPK1 from rice and AtPIP5K1 and AtPIP5K3 from Arabidopsis (Ma et al, 2006;Im et al, 2007;Kusano et al, 2008). However, stable transformation of tobacco (Nicotiana tabacum) cells to express an AtPIP5K1 MORN domain-GFP fusion did not allow visualization of the plasma membrane localization of this protein (Im et al, 2007). Thus, it is not clear if the MORN domain functions as a plasma membrane-targeting module.…”

“…Thus, in animals, type I and II PIPKs are involved in the generation of PtdIns(4,5)P 2 via different pathways. Molecular biological analysis of plant PIPKs was initiated with AtPIP5K1 from Arabidopsis (Mikami et al, 1998), which phosphorylates PtdIns3P, PtdIns4P, and PtdIns(4,5)P 2 to produce PtdIns(3,4)P 2 , PtdIns(4,5)P 2 , and PtdIns (3,4,5)P 3 , respectively, with D-4-and D-5-kinase activity (Elge et al, 2001;Westergren et al, 2001;Im et al, 2007). Similar enzymatic activity was also reported for other PIPKs from Arabidopsis Kusano et al, 2008;Stenzel et al, 2008).…”

“…Indeed, a regulatory role of the MORN domain was reported in the enzymatic activation of AtPIP5K1 (Im et al, 2007) and in root hair formation, but not in enzymatic activation, of AtPIP5K3 . Moreover, the MORN domain may play a role in the plasma membrane localization of OsPIPK1 from rice and AtPIP5K1 and AtPIP5K3 from Arabidopsis (Ma et al, 2006;Im et al, 2007;Kusano et al, 2008). However, stable transformation of tobacco (Nicotiana tabacum) cells to express an AtPIP5K1 MORN domain-GFP fusion did not allow visualization of the plasma membrane localization of this protein (Im et al, 2007).…”

“…Since MORN motifs are not found in PIPKs from nonplant organisms, a plant-specific mode of PIPK activation is speculated. Indeed, a regulatory role of the MORN domain was reported in the enzymatic activation of AtPIP5K1 (Im et al, 2007) and in root hair formation, but not in enzymatic activation, of AtPIP5K3 . Moreover, the MORN domain may play a role in the plasma membrane localization of OsPIPK1 from rice and AtPIP5K1 and AtPIP5K3 from Arabidopsis (Ma et al, 2006;Im et al, 2007;Kusano et al, 2008).…”