Molecular and Biochemical Characterization of Two Plant Inositol Polyphosphate 6-/3-/5-Kinases

Abstract

Despite the high deposition of inositol hexakisphosphate (IP 6 ), also known as phytate or phytin, in certain plant tissues little is known at the molecular level about the pathway(s) involved in its production. In budding yeast, IP 6 synthesis occurs through the sequential phosphorylation of I(1,4,5)P 3 by two gene products, Ipk2 and Ipk1, a IP 3 /IP 4 dual-specificity 6-/3-kinase and an inositol 1,3,4,5,6-pentakisphosphate 2-kinase, respectively. Here we report the identification and characterization of two inositol polyphosphate kinases from Arabidopsis thaliana, designated AtIpk2␣ and AtIpk2␤ that are encoded by distinct genes on chromosome 5 and that are ubiquitously expressed in mature tissue. The primary structures of AtIpk2␣ and AtIpk2␤ are 70% identical to each other and 12-18% identical to Ipk2s from yeast and mammals. Similar to yeast Ipk2, purified recombinant AtIpk2␣ and AtIpk2␤ have 6-/3-kinase activities that sequentially phosphorylate I(1,4,5)P 3 to generate I(1,3,4,5,6)P 5 predominantly via an I(1,4,5,6)P 4 intermediate. While I(1,3,4,5)P 4 is a substrate for the plant Ipk2s, it does not appear to be a detectable product of the IP 3 reaction. Additionally, we report that the plant and yeast Ipk2 have a novel 5-kinase activity toward I(1,3,4,6)P 4 and I(1,2,3,4,6)P 5 , which would allow these proteins to participate in at least two proposed pathways in the synthesis of IP 6 . Heterologous expression of either plant isoform in an ipk2 mutant yeast strain restores IP 4 and IP 5 production in vivo and rescues its temperature-sensitive growth defects. Collectively our results provide a molecular basis for the synthesis of higher inositol polyphosphates in plants through multiple routes and indicate that the 6-/3-/5-kinase activities found in plant extracts may be encoded by the IPK2 gene class.Phosphorylation events are a vital part of intracellular communication and enable specific responses to various stimuli. As an asymmetric cyclitol harboring six hydroxyls, myo-inositol is a scaffold that has the potential to transduce a wealth of information through the combinatorial addition of phosphates.

Metabolic and signaling properties of an Itpk gene family in Glycine max

“…3B). Additionally, we noted that Arabidopsis Ipk2 has previously been shown to convert Ins(1,3,4,5)P 4 and Ins(1,3,4,6)P 4 to Ins(1,3,4,5,6)P 5 [17]. Therefore, to further confirm the nature of the two InsP 4 products made by the soybean Itpks, the InsP 4 s were incubated with recombinant GmIpk2 and were both found to be completely phosphorylated to Ins(1,3,4,5,6)P 5 (data not shown).…”

“…The soybean Ipk2 gene (accession EU033957) was identified based on a search of the soybean database using known Arabidopsis sequences [17]. GmIpk2 was amplified and cloned into pGEX4T-1 using the following primer pair: 5 0 -GACTGGATCCATGCTCAAGATCCCGGAGC-3 0 and 5 0 -AT GATCGCGGCCGCTCAGTCACTTGTGAAGACATGCTAC-3 0 .…”

MORN motifs in plant PIPKs are involved in the regulation of subcellular localization and phospholipid binding

“…Now most phosphoinositide molecules presented in mammalian cells have been identified in plant cells, and plant genes encoding the PI metabolism enzymes have been cloned in succession, including those for PI synthase [16], PI 3-kinases [17,18], PI4K [19], PIPK [20], PI-PLCs [21,22], inositol polyphosphate phosphatases [23,24], inositol polyphosphate kinases [25,26], and others. In 1998, a cDNA of plant PIPK (AtPIP5K1) from Arabidopsis was cloned, which was the first report of plant PIPK [20].…”

Generation of phytate-free seeds in Arabidopsis through disruption of inositol polyphosphate kinases

“…Pathways (I) and (II) are inositol lipid-dependent, whereas pathway (III) does not require lipid synthesis. Pathway (I) initiates through phospholipase C and subsequent sequential phosphorylation of inositol 1,4,5-trisphosphate [I(1,4,5)P 3 ] by IP kinases (IPKs), IPK2 (7)(8)(9)(10)(11)(12) and IPK1 (13)(14)(15). Pathway (II) is also a phospholipasedependent pathway whereby I(1,4,5)P 3 is phosphorylated to inositol 1,3,4,5-tetrakisphosphate [I(1,3,4,5)P 4 ] by either Ipk2 or I(1,4,5)P 3 3-kinase, dephosphorylated to inositol 1,3,4-trisphosphate [I(1,3,4)P 3 ], and phosphorylated by a I(1,3,4)P 3 5͞6-kinase, Ipk2, and Ipk1, respectively, through the indicated intermediates (14,(16)(17)(18).…”