Phosphatidylinositol 4-Kinase Type II Alpha
Phosphatidylinositol 4-kinase type II α, also commonly referred to as PI4K2A or PI4KIIα, is a 54 kDa lipid kinase that catalyzes the phosphorylation of the membrane lipid phosphatidylinositol on the D4 position of its inositol headgroup to produce phosphatidylinositol 4-phosphate (PI4P). There are three other phosphatidylinositol 4-kinases expressed in most mammalian cells, PI4KB, PI4KA, and PI4KB. PI4K2A is highly homologous to the PI4KB isozyme, and together they are classified as the type II phosphatidylinositol 4-kinases (reviewed in Boura and Nencka 2015; Clayton et al. 2013). The type II phosphatidylinositol kinases differ from the type III PI4KA and PI4KB isozymes in that they are insensitive to the phosphoinositide 3-kinase inhibitors wortmannin and LY-294002, but are inhibited by low micromolar concentrations of adenosine. Furthermore, PI4K2A activity is also characteristically activated by nonionic detergents such as Triton X-100 and inhibited by Ca2+.
Monophosphorylated inositol phospholipids and the lipid kinases that catalyze their formation were first discovered and described in the 1950s and 1960s by the Hokins and later pioneers such as Michell, Hawthorne, and Harwood. However, it was not until much later in 1987 that the Cantley laboratory biochemically subclassified these enzymatic activities into type I, type II, and type III phosphatidylinositol kinases (Endemann et al. 1987; Whitman et al. 1987). The type 1 phosphatidylinositol kinase activity was later found to be a phosphatidylinositol 3-kinase. However, the type II and type III subclassifications for the phosphatidylinositol 4-kinases remain in use; the subsequent molecular cloning of these enzymes has confirmed that as well as forming biochemically distinguishable groupings, these enzymes also fall into two distinct structural families, with the type III phosphatidylinositol 4-kinases being much more homologous with the phosphatidylinositol 3-kinases. This is a property which underlies their shared sensitivities to wortmannin and LY-294002 inhibition.
Although PI4K2A has a very robust enzymatic activity, accounting for over 80% of the measurable phosphatidylinositol kinase activity in some cells, it proved to be a very difficult protein to purify and clone. Whilst much progress was made in the late 1980s and throughout the 1990s in cloning and characterizing other phosphoinositide kinases, it was not until 2001 that two different research groups independently reported the purification and primary structure of PI4K2A (Barylko et al. 2001; Minogue et al. 2001). The enzyme was found to be a 54 kDa protein encoded by a gene localized to chromosome 10 in the human genome. The enzyme was shown to be constitutively palmitoylated and tightly membrane bound; these properties may explain why it evaded biochemical purification for such a long time. The availability of recombinant PI4K2A heralded a new era in research and facilitated a much deeper understanding of its roles in post-Golgi vesicle trafficking and the regulation of receptor signalling. Finally, in 2014, in a manner reminiscent of the events surrounding the cloning of the enzyme, two independent groups published the X-ray crystal structure of PI4K2A (Baumlova et al. 2014; Zhou et al. 2014). These new structural insights have transformed our understanding of how its enzymatic activity is regulated through interactions with lipids at the membrane interface and have also revealed possible avenues for the rationale design of isoform-specific inhibitors of PI4K2A.
Assay for PI4K2A Activity
A radiometric assay using micellar phosphatidylinositol lipid substrate and radioactive ATP is the most common method for measuring PI4K2A. This assay measures the formation of 32P-PI4P resulting from the transfer of the β-emitting γ-phosphate from [γ32P]ATP to the D4 position of the phosphatidylinositol head group.
PI + [32P]-ATP → [32P]-PI4P + ADP
The [32P]-PI4P phospholipid product is then recovered by organic phase extraction and resolved by thin layer chromatography. [32P]-PI4P is detected by X-ray film visualisation and radioisotope incorporation is subsequently quantified through excision of the [32P]-PI4P spots and liquid scintillation counting. Whilst this is a commonly used method, the spot visualisation and signal quantification steps are increasingly being accomplished through phosphor imaging technologies.
The use of radioisotopes can give rise to legal and safety issues and this has spurred the search for nonradioactive methods to detect PI4K2A activity. The Balla laboratory has developed a nonradioactive, bioluminescence-based phosphatidylinositol 4-kinase assay (Tai et al. 2011). This method may be particularly suitable for high-throughput screening using purified or recombinant PI4K2A preparations.
Subcellular Localization and Functions
In nonneuronal cells, PI4K2A is localized mainly to late endosomes and the trans-Golgi network. Smaller pools of the enzyme have also been reported at the plasma membrane, Glut4 trafficking vesicles, and the endoplasmic reticulum (reviewed in (Clayton et al. 2013). In neurons, PI4K2A has been found on vesicles in the secretory pathway. It is important to note that each of the four mammalian phosphatidylinositol 4-kinases have unique and non-overlapping subcellular distributions that relate to their isozyme-specific functions in signalling and vesicle trafficking. The pool of PI4P generated by PI4K2A has been identified at the trans-Golgi network (Wang et al. 2003) and on endosomes (Henmi et al. 2016). At the trans-Golgi network, PI4K2A activity has been implicated in the recruitment of the clathrin adaptors AP-1 (Wang et al. 2003) and GGA (Wang et al. 2007), although more recent work has implicated the highly homologous PI4K2B isoform as the isoform responsible for AP-1 recruitment (Wieffer et al. 2013). At the trans-Golgi network, PI4K2A is also required for the PI4P-dependent recruitment of the oxysterol-binding protein OSBP1 (Banerji et al. 2010) which functions in the nonvesicular transfer of ceramide, required for sphingomyelin synthesis, from the endoplasmic reticulum.
It is important to note that the functions of PI4K2A in intracellular trafficking are also mediated independently of its catalytic function, through its participation in large, membrane-associated protein-protein interaction networks, such the multiprotein BLOC-1 complex implicated in lysosomal biogenesis, and the dysbindin-BLOC-1 complex, which is important for axonal trafficking in the CNS (Larimore et al. 2011). Moreover, accumulating evidence indicates that many of the isozyme-specific functions of the phosphatidylinositol 4-kinases are due to their interactions with particular protein-binding partners at different subcellular sites, rather than their common PI4P synthetic activities.
PI4K2A in Cell Signalling and Disease
Loss of function studies with PI4K2A gene-knockout mice revealed important functions for this enzyme in mediating the survival of spinal cord axons and cerebellar Purkinje cells. Over time, homozygous knockout animals develop a number of neurodegenerative symptoms, including late onset tremor, hind-leg clasping, urinary incontinence, and ultimately premature death (Simons et al. 2009). However, the molecular basis of this phenotype has not yet been established.
Knockdown of PI4K2A expression in cultured cells using RNAi gene silencing techniques induces an enlarged late-endosomal phenotype and associated Golgi-endosomal trafficking defects, such as delayed degradation of activated EGF receptors (Minogue et al. 2006) or impaired Golgi-lysosomal trafficking of lysosomal enzymes such as β-glucocerebrosidase (Jovic et al. 2012). In some cell lines, RNAi-ablated PI4K2A expression results in decreased EGF activation of the important prosurvival and promigratory Akt enzyme (Chu et al. 2010). However, this effect has been attributed to aberrant endosomal trafficking as opposed to decreased PI4P synthesis impacting on downstream receptor-signalling pools of PI(4,5)P2 and PI(3,4,5)P3, where the latter lipid is required for the membrane recruitment and subsequent activation of Akt during receptor-activated phosphoinositide 3-kinase signalling. Furthermore, it is noteworthy that the most recent work indicates that the main cellular pool of PI4P required for agonist-stimulated phospholipase C signalling is thought to be generated by PI4KA and not the PI4K2A isozyme.
PI4K2A is required for the trafficking and signalling of human oncoproteins such as EGFR, and alterations to PI4K2A expression have been associated with some cancers. Augmented PI4K2A expression in some breast cancers has been shown to lead to upregulated HER2 signalling and hypoxia-induced angiogenesis (Li et al. 2010). On the other hand, decreased PI4K2A expression has been noted in glioblastoma multiforme; but this may relate to an altered karyotype in advanced tumors rather than a tumor suppressor role for the PI4K2A gene (Waugh 2016).
Recently, an unexpected role for PI4K2A in mediating endosome exocytosis in X-linked centronuclear myopathy has been discovered (Ketel et al. 2016). The mutated gene in this inherited condition is a phosphatidylinositol 3-phosphatase MTM1 that normally converts endosomal phosphatidylinositol 3-phosphate produced by the phosphatidylinositol 3-kinase Vps34 to phosphatidylinositol, the PI4K2A lipid substrate. In patient cells, MTM1 phosphatase activity is inactivated and this leads to reduced phosphatidylinositol substrate and consequently decreased PI4K2A-dependent PI4P generation. This inhibition of PI4K2A activity through substrate depletion manifests as reduced endosomal delivery of cell attachment proteins such as β1-integrin to the plasma membrane; this has been proposed as a trafficking-based mechanism to explain the underlying molecular pathology of this particular X-linked disease.
Structure and Regulation of PI4K2A
The three-dimensional crystal structure of the PI4K2A kinase domain has recently been described and was shown to consist of two structural lobes termed N and C, with residues from both lobes contributing to the binding of the ATP substrate (Baumlova et al. 2014; Zhou et al. 2014). A further ATP-binding pocket was identified in the C lobe, although there is a possibility that this hydrophobic structure may bind membrane molecules other than nucleotides under physiological conditions.
PI4K2A is the only phosphatidylinositol 4-kinase that is constitutively associated with biological membranes. Moreover, PI4K2A is unique in that its catalytic rate is highly dependent on the membrane environment to which it is targeted. Palmitoylation of cysteine residues in a 174CCPCC178 motif localized within the kinase domain is absolutely required for PI4K2A activity; recombinant nonpalmitoylated mutant versions of the enzyme are catalytically inactive even though they remain membrane-associated (Lu et al. 2012). PI4K2A is palmitoylated by late-Golgi localized palmitoyl transferase enzymes which require the presence of membrane cholesterol for their activity. Furthermore, both PI4P generation and the membrane diffusion dynamics of PI4K2A are sensitive to cholesterol depletion with methyl-β-cyclodextrin indicating a major role for cholesterol and thus membrane environment in regulating this enzyme (Minogue et al. 2010).
The elucidation of the three-dimensional structure of the enzyme has also revealed the existence of positively charged regions on the membrane-interacting regions of PI4K2A, including arginine and lysine-rich insertions in the catalytic domain, that mediate electrostatic binding with negatively charged membrane lipids, such as phosphatidylinositol, and are crucial for maximal enzymatic activity. Molecular modelling studies have also indicated that at the membrane interface, the protein may be present in an octameric form, composed of two interacting PI4K2A tetramers. Hence, interactions with the target membrane, together with the membrane’s electrostatic charge, are critical determinants of PI4K2A activity (Zhou et al. 2014).
In addition to the influence of membrane environment, PI4K2A activity is negatively regulated through direct binding of the U3 ubiquitin ligase Itch to a PPxY motif located within the disordered N-terminal noncatalytic domain (Mossinger et al. 2012). This interaction with Itch results in multi-ubiquitination of PI4K2A and may represent a key point of regulatory crosstalk between the endosomal phosphoinositide and ubiquitination pathways, as Itch activity is reciprocally increased by binding PI4K2A.
PI4K2A is a 54 kDa lipid kinase that catalyzes the formation of PI4P from ATP and phosphatidylinositol substrates. The enzyme mostly localizes to endosomes and the trans-Golgi network, and it has been ascribed several functions related to intracellular vesicle trafficking and the regulation of signalling by oncoproteins such as the EGFR and HER2. PI4P synthesised by PI4K2A can recruit the clathrin adaptor proteins AP-1 and AP-3, or it can be further phosphorylated to PI(4,5)P2. PI4K2A and the related PI4K2B isoform are structurally distinct from other phosphatidylinositol kinases. PI4K2A catalysis is modulated by the membrane environment and is very dependent on the presence of positively charged amino acid residues on the membrane-interacting surface of the protein and posttranslational palmitoylation of a CCPCC motif within the active site. Alterations to PI4K2A expression may be important for the survival of particular cell types within the CNS and may impact on receptor-activated signalling in some cancers.
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