Crystallographic and kinetic analyses of human IPMK reveal disordered domains modulate ATP binding and kinase activity

Abstract

Inositol polyphosphate multikinase (IPMK) is a member of the IPK-superfamily of kinases, catalyzing phosphorylation of several soluble inositols and the signaling phospholipid PI(4,5)P2 (PIP2). IPMK also has critical non-catalytic roles in p53, mTOR/Raptor, TRAF6 and AMPK signaling mediated partly by two disordered domains. Although IPMK non-catalytic functions are well established, it is less clear if the disordered domains are important for IPMK kinase activity or ATP binding. Here, kinetic and structural analyses of an engineered human IPMK lacking all disordered domains (ΔIPMK) are presented. Although the KM for PIP2 is identical between ΔIPMK and wild type, ΔIPMK has a 1.8-fold increase in kcat for PIP2, indicating the native IPMK disordered domains decrease IPMK activity in vitro. The 2.5 Å crystal structure of ΔIPMK is reported, confirming the conserved ATP-grasp fold. A comparison with other IPK-superfamily structures revealed a putative “ATP-clamp” in the disordered N-terminus, we predicted would stabilize ATP binding. Consistent with this observation, removal of the ATP clamp sequence increases the KM for ATP 4.9-fold, indicating the N-terminus enhances ATP binding to IPMK. Together, these structural and kinetic studies suggest in addition to mediating protein-protein interactions, the disordered domains of IPMK impart modulatory capacity to IPMK kinase activity through multiple kinetic mechanisms.

The acyl chains of phosphoinositide PIP3 alter the structure and function of nuclear receptor steroidogenic factor-1

“…The worm ortholog of SF-1 binds PIP3 in vitro and regulates phospholipid metabolism in vivo ( 25 ). Genetically or chemically inhibiting the nuclear inositol polyphosphate multikinase ( 26 , 27 , 28 ) which generates PIP3 regulates SF-1 function in human cells, whereas simply decreasing total cellular PIP3 by inhibiting the p110-class of PI3 kinases does not have similar effects ( 29 , 30 ). Dipalmitoyl PIP3 has nanomolar affinity for SF-1, among the highest affinity of all phospholipids tested in previous work ( 21 ).…”

TNP Analogues Inhibit the Virulence Promoting IP3-4 Kinase Arg1 in the Fungal Pathogen Cryptococcus neoformans

“…Reduced Hs IPMK purity could be due to limited expression levels and contamination with bacterial heat shock proteins. Hs IPMK was previously shown to be monomeric [ 21 , 22 , 40 , 41 ]. The purification yielded ~3.6 mg per litre of Cn Arg1, ~8.8 mg per litre of Ca Ipk2 and ~0.3 mg per litre of Hs IPMK proteins.…”

“…In contrast to fungi, the product of IP 3-4 K, IP 5 , can be generated by more than one route in human and involves 4 different enzymes: inositol polyphosphate multikinase ( Hs IPMK), IP3K, INPP5 and ITPK1 (reviewed in [ 10 , 11 ]). In addition to having IP 3-4 K activity, Hs IPMK functions as a phosphoinositide 3-kinase [ 20 , 21 , 22 ] and as a scaffold protein in the target of rapamycin (TOR) where its enzymatic activity is not required [ 23 ] (reviewed in [ 24 ]). Hs IP6K has been implicated in various health conditions including cancer [ 25 , 26 ], aging [ 27 ] and diabetes and obesity [ 28 ].…”

Inositol Polyphosphate Multikinase Signaling: Multifaceted Functions in Health and Disease

“…Vilazodone treatment of fibroblasts and cancer cells reduced the activities of IPMK as well as Akt phosphorylation, indicating the selective activities of vilazodone against IPMK-dependent catalytic steps in the IP biosynthesis pathway and Akt activation. More studies on the structure of human IPMK and its catalysis (Gu et al, 2019;Seacrist and Blind, 2018) will be useful to design IPMK inhibitors and modulate the IPMK signaling pathway quantitatively and dynamically, with the ultimate goal of targeting IPMK-dependent pathological conditions.…”