Mechanism of substrate specificity of phosphatidylinositol phosphate kinases

Abstract

The phosphatidylinositol phosphate kinase (PIPK) family of enzymes is primarily responsible for converting singly phosphorylated phosphatidylinositol derivatives to phosphatidylinositol bisphosphates. As such, these kinases are central to many signaling and membrane trafficking processes in the eukaryotic cell. The three types of phosphatidylinositol phosphate kinases are homologous in sequence but differ in catalytic activities and biological functions. Type I and type II kinases generate phosphatidylinositol 4,5-bisphosphate from phosphatidylinositol 4-phosphate and phosphatidylinositol 5-phosphate, respectively, whereas the type III kinase produces phosphatidylinositol 3,5-bisphosphate from phosphatidylinositol 3-phosphate. Based on crystallographic analysis of the zebrafish type I kinase PIP5K α , we identified a structural motif unique to the kinase family that serves to recognize the monophosphate on the substrate. Our data indicate that the complex pattern of substrate recognition and phosphorylation results from the interplay between the monophosphate binding site and the specificity loop: the specificity loop functions to recognize different orientations of the inositol ring, whereas residues flanking the phosphate binding Arg244 determine whether phosphatidylinositol 3-phosphate is exclusively bound and phosphorylated at the 5-position. This work provides a thorough picture of how PIPKs achieve their exquisite substrate specificity.lipid kinases | protein engineering | crystallography | substrate specificity P hosphatidylinositol phosphate (PIP) kinases (PIPKs) are key players in the metabolism of phosphoinositides in eukaryotic cells. PIPKs are primarily responsible for the synthesis of doubly phosphorylated phosphatidylinositol (PI) derivatives from singly phosphorylated PIs (1-4). PIPK catalytic activities are important to the cell in part because they produce essential PI bisphosphates such as PI(4,5)P 2 , which is involved in a wide variety of signaling pathways and membrane trafficking events, and PI(3,5)P 2 , which has a more specialized role in endosomes (5). In addition, these kinases control the level of certain PI monophosphates such as PI(5)P, which appears to function as a stress signal (6). Mutations in PIPKs have been linked to human diseases (7,8), and, in cancerous cells, the activities of PIPKs are often up-regulated, usually as a consequence of overexpression (9, 10).There are three subfamilies of PIPKs that share sequence identity within the kinase domain (2). The type I kinase phosphatidylinositol 4-phosphate 5-kinase (PIP5K), localized mainly to the plasma membrane, is responsible for synthesizing the majority of PI(4,5)P 2 in the cell and accomplishes this process by phosphorylating the 5-hydroxyl group of PI(4)P (Fig. S1A). In vitro, the type I kinase also has a robust activity against PI(3)P, generating not only PI(3,4)P 2 and PI(3,5)P 2 but also the triply phosphorylated PI(3,4,5)P 3 (11, 12). Furthermore, the type I kinase can weakly phosphorylate PI to produce PI(5)P. In con...

Carbohydrate Kinases: A Conserved Mechanism Across Differing Folds

“…This class is mainly responsible for the generation of a large fraction of the PtdIns-3-phosphate, a central phospholipid for membrane trafficking processes [123]. PtdIns kinases appear to strongly prefer ATP as the phosphate donor [119]. The structure forms two domains of approximately equal size (shown in teal and raspberry; PDB ID: 1WUU [36]).…”

Activity-specificity trade-off gives PI5P4Kβ a nucleotide preference to function as a GTP-sensing kinase

“…The cognate ATP kinase PI4P5Kα also interacts with ATP in the same binding mode ( Fig. 2A, middle) (Muftuoglu et al, 2016).…”

Development of an in vitro screening assay for PIP5K1α lipid kinase and identification of potent inhibitors

“…Our assay can be used to identify ATP-non-competitive compounds, like it was shown for the substratecompetitive compound 13. Muftuoglu et al [24] in their study about the mechanism of substrate specificity of PIPKs found out that the substrate binding site is in closed vicinity to the ATP binding site. This finding might be a reason for the uncorrelated docking results with the actual binding mode.…”

“…Compared to natural PI(4)P and PIP 2 , PI(4)P fluorescein and PIP 2 fluorescein have truncated fatty acid chains and an additional fluorescein moiety conjugated to the end of one chain. It was shown for the zebrafish enzyme that only the polar inositol head group of the lipid is recognized by the active site of the enzyme [24]. Due to the fact that a reliable separation method was available, we decided to test whether the artificial phosphoinositide PI(4)P fluorescein is accepted as substrate by hPIP5K1a, leading to the corresponding fluorescent product (Fig.…”