Integration of Inositol Phosphate Signaling Pathways via Human ITPK1

Abstract

Inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) is a reversible, poly-specific inositol phosphate kinase that has been implicated as a modifier gene in cystic fibrosis. Upon activation of phospholipase C at the plasma membrane, inositol 1,4,5-trisphosphate enters the cytosol and is inter-converted by an array of kinases and phosphatases into other inositol phosphates with diverse and critical cellular activities. In mammals it has been established that inositol 1,3,4-trisphosphate, produced from inositol 1,4,5-trisphosphate, lies in a branch of the metabolic pathway that is separate from inositol 3,4,5,6-tetrakisphosphate, which inhibits plasma membrane chloride channels. We have determined the molecular mechanism for communication between these two pathways, showing that phosphate is transferred between inositol phosphates via ITPK1-bound nucleotide. Intersubstrate phosphate transfer explains how competing substrates are able to stimulate each others' catalysis by ITPK1. We further show that these features occur in the human protein, but not in plant or protozoan homologues. The high resolution structure of human ITPK1 identifies novel secondary structural features able to impart substrate selectivity and enhance nucleotide binding, thereby promoting intersubstrate phosphate transfer. Our work describes a novel mode of substrate regulation and provides insight into the enzyme evolution of a signaling mechanism from a metabolic role.Cellular inositol phosphate metabolism is an intricate web of kinase and phosphatase reactions that produces a number of important signaling molecules (for review see Ref. 1). A now classic example of these signaling activities is the release of Ca 2ϩ into the cytoplasm through an intracellular channel that is gated by inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ) 4 (2). Additional roles are continually being discovered: inositol phosphates have recently been shown to be critical to the activity of RNA-editing enzymes (3), to participate in telomere maintenance (4), and to be the phosphate donors in certain protein phosphorylation events (5). It is of critical interest, therefore, to establish the regulatory mechanisms that govern the metabolism of inositol phosphates.An interesting feature of inositol phosphate metabolism is the promiscuity with which several key kinases phosphorylate multiple substrates (6). For example, ITPK1 (also known as inositol 1,3,4-trisphosphate 5/6-kinase) adds either a 5-or 6-phosphate to Ins(1,3,4)P 3 and also attaches a 1-phosphate to Ins(3,4,5,6)P 4 (6, 7) (inositol phosphate structures shown in Fig. 2). These reactions have been demonstrated to be reversible: ITPK1 can also dephosphorylate Ins(1,3,4,5,6)P 5 back to Ins(3,4,5,6)P 4 (8). An especially puzzling aspect of this phenomenon is that dephosphorylation of Ins(1,3,4,5,6)P 5 by human ITPK1 is stimulated, rather than competitively inhibited, by one of its alternate substrates, Ins(1,3,4)P 3 (8).The fact that mammalian ITPK1 reversibly phosphorylates both Ins(3,4,5,6)P 4 and Ins(1,3,4)P 3 takes on...

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

“…The genes were cloned into the protein expression vector pGEX4T-1 (GE Healthcare, Waukesha, WI), and the proteins were expressed in Escherichia coli and purified using GST-sepharose beads (GE Healthcare, Waukesha, WI) as previously described [16]. The soybean Ipk2 gene (accession EU033957) was identified based on a search of the soybean database using known Arabidopsis sequences [17].…”

“…Recombinant protein was prepared as described above, and showed canonical Ins(1,3,4,5)P 4 6-kinase activity (data not shown). 3,4,5,6)P 5 were prepared as previously described [16]. Non-radioactive Ins(1,3,4)P 3 , Ins(3,4,6)P 3 and Ins(3,4,5,6)P 4 were purchased from CellSignals (Columbus, OH).…”

“…Reactions were quenched and neutralized as previously described and analyzed by either gravity-fed ion-exchange columns, or by HPLC (using either a 4.6 · 125 mm Partisphere SAX column, or a 250 · 4.6 mm Q100 column) (Thompson Instruments, Clear Brook, VA) [16]. The assays containing [ 32 P] were analyzed using an in-line scintillation counter.…”

“…As well as exhibiting Ins(3,4,5,6)P 4 1-kinase activity, mammalian Itpk1 has been shown to dephosphorylate Ins(1,3,4,5,6)P 5 to Ins(3,4,5,6)P 4 [16,22]. We recently demonstrated that GmItpk2 can also dephosphorylate Ins(1,3,4,5,6)P 5 to Ins(3,4,5,6)P 4 (2932 ± 50 lmol/mg protein/min) [16].…”

“…We recently demonstrated that GmItpk2 can also dephosphorylate Ins(1,3,4,5,6)P 5 to Ins(3,4,5,6)P 4 (2932 ± 50 lmol/mg protein/min) [16]. We now report that the three other GmItpk isoforms also show this activity: type 1 = 403 ± 4, type 3 = 487 ± 47, type 4 = 88 ± 16 (lmol/ mg protein/min), albeit at rates 6-to 33-fold lower than that of the type 2 enzyme.…”