Depletion of a Putatively Druggable Class of Phosphatidylinositol Kinases Inhibits Growth of p53-Null Tumors

Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are enigmatic lipid kinases with physiological functions that are incompletely understood, not the least because genetic deletion and cell transfection have led to contradictory data. Here, we used the genetic tractability of DT40 cells to create cell lines in which endogenous PI5P4Kα was removed, either stably by genetic deletion or transiently (within 1 h) by tagging the endogenous protein genomically with the auxin degron. In both cases, removal impacted Akt phosphorylation, and by leaving one PI5P4Kα allele present but mutating it to be kinase-dead or have PI4P 5-kinase activity, we show that all of the effects on Akt phosphorylation were dependent on the ability of PI5P4Kα to synthesize phosphatidylinositol (4,5)-bisphosphate [PI(4,5) P 2 ] rather than to remove PI5P. Although stable removal of PI5P4Kα resulted in a pronounced decrease in Akt phosphorylation at Thr308 and Ser473, in part because of reduced plasma membrane PIP 3 , its acute removal led to an increase in Akt phosphorylation only at Ser473. This process invokes activation primarily of mammalian target of rapamycin complex 2 (mTORC2), which was confirmed by increased phosphorylation of other mTORC2 substrates. These findings establish PI5P4Kα as a kinase that synthesizes a physiologically relevant pool of PI(4,5)P 2 and as a regulator of mTORC2, and show a phenomenon similar to the "butterfly effect" described for phosphatidylinositol 3-kinase Iα [Hart JR, et al. (2015) Proc Natl Acad Sci USA 112(4):1131-1136], whereby through apparently the same underlying mechanism, the removal of a protein's activity from a cell can have widely divergent effects depending on the time course of that removal.phosphatidylinositol 5-phosphate 4-kinase | phosphatidylinositol 5-phosphate | phosphatidylinositol (4,5)-bisphosphate | Akt | mTOR T he phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are an enigmatic family of three (PI5P4Kα, -β, and -γ) with cellular functions that remain poorly understood (1-3). In general, their principal activity is believed to be to remove and thus, regulate the levels of their substrate phosphatidylinositol 5-phosphate (PI5P). Phenotypes from knockout mice have highlighted that PI5P4Ks have important roles to play in physiology and pathology, including links between PI5P4Ks and the Akt signaling pathway, and other studies have pointed to roles in the generation of cancer (3). A number of key questions, however, remain unanswered. Knocking proteins out or down (by RNAi) is a long-term strategy that may lead to indirect changes, such as, for example, those highlighted in the recent study by Hart et al. (4) concerning the indirect long-term consequences of a point mutation in phosphatidylinositol 3-kinase-α (PI3Kα). Another issue still unresolved for PI5P4Ks is whether removal of PI5P is their only function or whether their ability to synthesize phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P 2 ] may also be important (5, 6).We have recently used the high homologous recombi...

Section: Discussionmentioning

confidence: 99%

In B cells, phosphatidylinositol 5-phosphate 4-kinase–α synthesizes PI(4,5)P₂to impact mTORC2 and Akt signaling

Bulley

Droubi

Clarke

et al. 2016

“…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).…”

mentioning

confidence: 99%

Mechanism of substrate specificity of phosphatidylinositol phosphate kinases

Muftuoglu

Xue

Gao

et al. 2016

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...

“…Among the PIPKs, PI3Ks are the best-characterized enzymes that commonly are targets for anticancer drugs (8). Less is known about PI-4-phosphate 5 kinases (PIP5Ks), enzymes that are upstream of PI3K (9,10). The PIP5K family of lipid kinases consists of the three isozymes α, β, and γ (11)(12)(13).…”

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confidence: 99%

The role of PI3K/AKT-related PIP5K1α and the discovery of its selective inhibitor for treatment of advanced prostate cancer

Semenas

Hedblom

Miftakhova

et al. 2014

130

Significance Prostate cancer is the most common malignancy and the third leading cancer-related cause of death among men of the Western world. Treatment options at advanced stages of the disease are scarce, and better therapies are in urgent need. In our study, we show that the clinically relevant lipid kinase phosphatidylinositol-4-phosphate 5-kinase-α (PIP5Kα) plays an important role in cancer cell invasion and survival by regulating the PI3K/AKT/androgen receptor pathways. Elevated levels of PIP5K1α contribute to cancer cell proliferation, survival, and invasion. In this context we introduce a newly developed compound, ISA-2011B, with promising anticancer effects by inhibiting the PIP5K1α-associated AKT pathways. Conclusively, we propose that PIP5K1α may be used as a potential therapeutic target for treatment of advanced prostate cancer.