Phosphatidyl Inositol 4-Kinases

In recent decades, phosphoinositides (or PIs) have emerged as essential signaling molecules. Despite their low cellular abundance, PIs are found to be involved in various cellular processes, including cell migration, vesicular trafficking, cell cycle regulation, metabolism, cytoskeletal remodeling, autophagy, aging, apoptosis, and cell signaling. Recent studies have shown that aberrant activity of either lipid kinases or phosphatases leads to various medical implications like cancer, diabetes


Introduction
PI4-Kinase (or phosphatidylinositol 4-kinase) is a vital lipid kinase in cells.It phosphorylates at the fourth hydroxyl position of the inositol ring of PtdIns (phosphatidylinositol), thereby generating PtdIns 4P (phosphatidylinositol 4 phosphate) or PtdIns 4,5 bis phosphate (phosphatidylinositol 4,5 bis phosphate) or PtdIns 3,4,5 triphosphate (phosphatidylinositol 3,4,5 tri phosphate), depending upon substrates [1].These three products act as important cell-signaling molecules by profoundly affecting cell physiology.Based on biochemical studies and sequence analysis, this enzyme is classified into two families: type-II PtdIns 4-kinases and type-III PtdIns 4-kinases.Both families have two isoforms, known as alpha and beta.PtdIns 4-kinases are found at the plasma membrane (as membrane-bound) in the cytoplasm, Golgi complex, endosomes, endoplasmic reticulum, cellular vesicles, and nucleolus.

Phosphatidylinositol
PtdIns are lipid molecules that act as an essential constituent of cellular membranes.PtdIns are present in plasma membranes, membranes of the Golgi complex, endoplasmic reticulum, and synaptic vesicles.PtdIns account for a small percentage of total lipids present in membranes.Although present in minuscule amounts (when compared to other lipids in cell), they play an essential role in the cells.The parent compound from which PtdIns are derived is phosphatidic acid (PA).Various phosphatidylinositol derivatives are generated due to phosphorylation at different positions of the inositol ring by different PtdIns kinases.PtdIns consist of a glycerol backbone, which is esterified at its sn-1 and sn-2 positions to two fatty acid molecules, with phosphate at its sn-3 position.The phosphate group is also esterified with an inositol ring at the D1 position [1].PtdIns are synthesized in the endoplasmic reticulum [2].Many proteins interact with phosphatidylinositol or its phosphorylated derivatives.These phosphoinositides have pleiotropic functions in cells.The balance of the PtdIns kinases and phosphatase activities can thus be important in different cell compartments [3].A detailed description of enzymes involved in PtdIns metabolism is beyond this entry's scope and can be found elsewhere [4].The structure of Ptdins is shown in Figure 1.
metabolism is beyond this entry's scope and can be found elsewhere [4].The structure of Ptdins is shown in Figure 1.

Type-II PtdIns 4-kinases
Type-II phosphatidylinositol 4-kinases are inhibited by adenosine but not by wortmannin.Type-II PtdIns 4-kinase is also inhibited by 4C5G monoclonal antibody [11].Type-II PtdIns α was cloned from rat brain and A431 cell lines (Homo sapiens epithelial carcinoma) and the β isoform from EST of neuroblastoma.Both isoforms phosphorylate PtdIns only.Type-II phosphatidylinositol 4-kinase α has 479 amino acid residues and a molecular mass of 54 kDa.Type-II phosphatidylinositol 4-kinase β has 481 amino acid residues [12][13][14].The km value of PtdIns and ATP for α isoforms is 28 µM and 54 µM, respectively.The km value of PtdIns and ATP for β isoforms is 21 µM and 65 µM, respectively.

Type-III PtdIns 4-kinases
Type-III PtdIns 4-kinase isolated from a fraction of bovine brain and bovine uterus have molecular masses of 230 kDa and 200 kDa, respectively [15].Type-III PtdIns 4-kinase with a molecular mass of 76 kDa has been reported from cytoplasm of rat brains cells.Type-III PtdIns 4-kinases have also been reported in the cytoplasm of rat brain and bovine adrenal cortical cells with molecular mass ranging from 92 to 125 kDa [16][17][18].PtdIns 4kinase 230 shows insensitivity towards adenosine.Its Km value for ATP is about 300 µM.The activity of this enzyme is reduced to 50% of the maximum value by 200 nM wortmannin [19].

Type-II PtdIns 4-Kinases
Type-II phosphatidylinositol 4-kinases are inhibited by adenosine but not by wortmannin.Type-II PtdIns 4-kinase is also inhibited by 4C5G monoclonal antibody [11].Type-II PtdIns α was cloned from rat brain and A431 cell lines (Homo sapiens epithelial carcinoma) and the β isoform from EST of neuroblastoma.Both isoforms phosphorylate PtdIns only.Type-II phosphatidylinositol 4-kinase α has 479 amino acid residues and a molecular mass of 54 kDa.Type-II phosphatidylinositol 4-kinase β has 481 amino acid residues [12][13][14].The km value of PtdIns and ATP for α isoforms is 28 µM and 54 µM, respectively.The km value of PtdIns and ATP for β isoforms is 21 µM and 65 µM, respectively.

Type-III PtdIns 4-Kinases
Type-III PtdIns 4-kinase isolated from a fraction of bovine brain and bovine uterus have molecular masses of 230 kDa and 200 kDa, respectively [15].Type-III PtdIns 4-kinase with a molecular mass of 76 kDa has been reported from cytoplasm of rat brains cells.Type-III PtdIns 4-kinases have also been reported in the cytoplasm of rat brain and bovine adrenal cortical cells with molecular mass ranging from 92 to 125 kDa [16][17][18].PtdIns 4-kinase 230 shows insensitivity towards adenosine.Its Km value for ATP is about 300 µM.The activity of this enzyme is reduced to 50% of the maximum value by 200 nM wortmannin [19].

Structure and Domains of PtdIns 4-Kinases
Stt4p, type-III PtdIns 4-kinase alpha from rat brain have pleckstrin homology (PH) domain.PtdIns 4-kinase alpha also shows ankyrin repeats, which are implicated in the localization of proteins in the cytosol.
Sequence analysis of alpha and beta forms of type-II PtdIns 4-kinase shows that these two isoforms differ in their first 90 amino acid residues.However, the C-terminal is highly conserved in both forms (about 58% identical and 75% homologous).Both proteins lack conspicuous transmembrane regions.Both forms have cysteine-rich palmitoylation motifs known as the CCPC motif that helps in membrane localization.One proline-rich motif, PXXP, is present only in type-II PtdIns 4-kinase beta.Deletions of the first 90 amino acid residues do not affect the ability of alpha form to associate with the membrane.Deleting the CCPC motif reduces the ability of alpha isoforms to associate with the membrane and reduces catalytic activity.The 96 N-terminal amino acid residues are vital for the membrane association of beta isoforms [20].
Our research has meticulously characterized the minimum catalytic domain of mammalian type-II phosphatidylinositol 4-kinase alpha.We have identified critical amino acid residues that play a pivotal role in catalysis and the enzyme's specificity for its substrate.A 43-45 kDa fragment at the C-terminal, lacking 92 amino acid residues of the N-terminal region, forms the minimum catalytic core of the alpha form of type-II PtdIns 4-kinase.Even the removal of a few amino acid residues from the C-terminal dramatically reduces catalytic activity.Notably, Lys -152 is involved in the binding of nucleotides, while Asp-307 is believed to function as a catalytic base.Asn-312 and Asp-345 are shown to bind Mg 2+ .Additionally, Glu-155, Pro-163, and Asp-300 are conserved in the type-II PtdIns 4-kinase class [12].It is important to mention that the crystal structure of several PtdIns 4-kinases is now available which will make drug discovery more likely [21-24].

Localization of Different PtdIns 4-Kinases
Expression and localization of different PtdIns 4-kinases are cell-and tissue-specific.Even within the cell, the localization of various enzyme forms is specific.

Localization of Type-II Phosphatidylinositol 4-Kinase
The diversity of PtdIns 4-kinase localization is striking.For instance, type-II PtdIns 4-kinase α is associated with membranes of the endoplasmic reticulum (ER), trans-Golgi network (TGN), and endosomes [25,26].Similarly, type-II PtdIns 4-kinase β is found in membranes of ER, Golgi complex, in the cytoplasm, and with the plasma membrane.This diversity in localization underscores the complexity and variety of their functions.

Localization of Type-III Phosphatidylinositol 4-Kinase
Studies carried out with type-III PtdIns 4-kinase α have shown that this enzyme localizes in various cellular organelles like nuclei from rat neuronal cells [27], membranes of ER, mitochondria, and multivesicular bodies [28].Type-III PtdIns 4-kinase β is present in early Golgi compartments and nuclei.The cellular localization of PtdIns 4-kinases highly depends on the protein's phosphorylation of various amino acid residues.For example, phosphorylation of type-III PtdIns 4-kinase β on Ser-294 results in its recruitment into Golgi, whereas phosphorylation on Ser-496 or Thr-504 leads to localization of this form inside the nucleus [29].Type-III PtdIns 4-kinase was found to be localized in the Golgi apparatus and vacuoles [30].

Effect of Phosphorylation
Adding IL-1 to human fibroblast and orthovanadate to rat liver membranes increases the PtdIns 4-kinase activity [40].Overexpression of the HER/neu (human epidermal growth factor receptor) gene in breast cancer cells also increases PtdIns 4-kinase activity [41].Adding epidermal growth factor (EGF) to A431 cells increases PtdIns 4-kinase activity [42].
Phosphorylation of GSK3β by activated Akt inactivates GSK3β by phosphorylating it.In its phosphorylated form, GSK3β cannot phosphorylate β-catenin [58].Hence, the concentration of unphosphorylated β catenin builds up in the cytoplasm.As the concentration of ß catenin builds up in the cytoplasm, the translocation of β-catenin into the nucleus starts.As a result, the level of unphosphorylated β-catenin inside the nucleus increases.Increased levels of unphosphorylated β-catenin inside the nucleus cause the induction of many genes [59] like WISP-1 (WNT1-inducible-signaling pathway protein 1) [60], c-myc [61], TCF [62], c-jun, fra [63], etc.In the presence of inhibitors against Akt or PtdIns 3-kinase, these pathways are affected.Unphosphorylated active GSK3β causes phosphorylation of β-catenin, and phosphorylated β-catenin is degraded in the ubiquitin-proteasome [64].
An increased level of unphosphorylated β-catenin inside the cytoplasm causes a rise in the level of p53 protein.This protein causes the induction of several genes, such as BAX, 14-3-3σ, p21, GADD45 (Growth Arrest and DNA Damage), and FAS [65,66].The increased level of p53 causes the activation of GSK3β.As a result, βcatenin is phosphorylated, and phosphorylated β-catenin is degraded in the ubiquitin-proteasome, so the level of β-catenin decreases.
Another pathway from PtdIns 4, 5-P2 involves PLC (phospholipase C), which occurs in different isoforms.The action of PLC on PtdIns 4, 5-P2 generates Ins 1, 4, 5-P3, and DAG (diacylglycerol).Ins 1, 4, 5-P3, so formed, causes an increase in the cytoplasmic calcium level (release of calcium from ER lumen).Increased Ca 2+ in the cytoplasm causes reorganization of the actin cytoskeleton, which leads to cell migration.Increased calcium levels and DAG cause the activation of another kinase known as protein kinase C (PKC) [67].PKC phosphorylates Ser or Thr residues of specific target proteins, changing their catalytic activities.Protein kinase C occurs in different isoforms inside the cell.Elevated activity of PKC is found to be associated with several types of malignancies [68,69].

Proteins Interacting with Phosphoinositides 11.1. Gelsolin
Gelsolin's interaction with PtdIns 4,5P2 at the barbed end of the actin filament is pivotal.As a crucial capping protein of actin filaments, this interaction leads to the disassembly of the actin-gelsolin complex, a process that has significant implications for actin filament dynamics and cellular functions [70].

CapZ
It is also an essential capping protein, like Gelsolin.The binding of PtdIns 4,5P2 to capZ inhibits its capping activity [71].

α-Actinin
It is a crucial actin cross-linking protein.It is present at different cell locations, including cell-cell and cell-matrix contact sites, lamellipodia, and the stress-fiber-dense region [72].

Profilin
It is an actin-binding protein that regulates the polymerization and depolymerization of actin filaments.Apart from binding to actin, profilin also binds to other cytoskeletal proteins, which link actin to extracellular membrane proteins or other molecules [73].

Vinculin
The binding of vinculin to PtdIns 4, 5-P2 triggers a significant conformational change in vinculin.This change exposes the binding sites for actin and talin, leading to the stabilization and promotion of actin binding to the membrane.This clear cause-and-effect relationship underscores the importance of these interactions in cellular processes [74,75].

Protein Interactors of Phosphatidylinositol 4-Kinases
It is important to mention that PtdIns 4-kinase interacts with several proteins, including 14-3-3 proteins involved in several processes [76][77][78][79][80]. Identification of PI4K-interacting proteins will be important as this will help us to better understand the biology of this enzyme.For example, in yeast, Stt4 interact with more than 30 proteins physically [81].Similarly, another yeast PI4K, PIk1 also interact physically with around 30 proteins [82].
Interaction of PI4K with proteins showed some degree of specificity.Studies involved checking human PI4K protein interactome is missing but may be possible in future.

Functions of Phosphatidylinositol 4-Kinases
Phosphatidylinositol 4-kinases, in addition to generating second messengers, actively perform diverse roles within cells.These roles are closely tied to their specific cellular locations.For instance, PtdIns 4P and PtdIns 4,5P2 actively participate in actin cytoskeletal reorganization and maintain the integrity of yeast cell walls [83].The downregulation of PtdIns 4-kinase III α, in conjunction with PtdIns 3-kinase, actively leads to developmental defects in the pectoral fin of zebrafish [84].PtdIns 4-kinase III α actively maintains the phosphoinositide pool at the plasma membrane [85].Furthermore, it actively participates in the replication of the hepatitis C virus (HCV) [86].This enzyme is also actively involved in endocytosis, exocytosis, vesicular trafficking, and cell secretion, demonstrating its active and diverse roles.
Loss of phosphatidylinositol 4-kinase II alpha is associated with the degeneration of axons in aged mice [87].PtdIns 4-kinase II α is implicated in tumor growth and angiogenesis by regulating HIF-1α (Hypoxia-inducible factor) [88].It is observed that type-II PtdIns 4-kinase is important in wnt signaling in Xenopus embryos, and it is also believed to interact with Dvl [89,90].Some studies also pointed towards the association of PtdIns 4-kinase II α with cellugyrin-positive Glut 4 vesicles [91].Phosphatidylinositol 4-kinase type-II alpha is also believed to be necessary for endosomal trafficking and degradation of activated epidermal growth factor receptors [92].PtdIns 4-kinase is also associated with the adaptor protein (AP) complex, which plays a vital role in the trafficking of cellular vesicles [93].PtdIns 4-kinase also plays a crucial role in activating phospholipase D [94].In yeast type-II, PtdIns 4-kinase lsb6 deletion impairs endosome motility and sorting, supporting their role in endocytic pathways [95].Inhibition of type-II phosphatidylinositol 4-kinase activities by resveratrol correlates with reducing Jurkat cell adhesion to fibronectin or matrigel-coated surfaces [96].Type-II phosphatidylinositol 4-kinase also enters Listeria monocytogenes in mammalian cells [97].Inhibition by monoclonal antibodies' type-II phosphatidylinositol 4-kinase activity inhibits phagocytosis and neutrophil respiratory burst [98].Inhibition of type-II phosphatidylinositol 4-kinase in RBL2H3 cells (Rat Basophilic Leukemia) is associated with inhibition of β hexosaminidase release [99].Overexpression of type-II phosphatidylinositol 4-kinase α enhances FcεRI-mediated degranulation in RBL2H3 cells [100].Inhibition of chromaffin granule-associated phosphatidylinositol 4-kinase activity impairs stimulated secretion [101].Diverse roles or functions of phosphatidylinositol 4-kinase are highlighted in Table 1.

Conclusions
From the perspectives discussed above, despite their low abundance, phospholipids and the enzymes involved in their metabolism profoundly impact cell physiology.These enzymes and lipids are involved in diverse cellular pathways and functions.Apart from their involvement in normal cellular physiology, these enzymes are now found to be involved or associated with diverse clinical or medical conditions, ranging from cancer to neurological diseases to microbial infection.Targeting these enzymes can be the right strategy in developing drugs.