The PI(3)P interactome from a colon cancer cell
Graphical abstract
Highlights
► An amino analogue of PI(3)P was synthesized and immobilised onto Affi-10 beads. ► A proteomic analysis of PI(3)P proteome using affinity/LC/MS/MS was performed. ► 681 PI(3)P interacting proteins were identified from colonic carcinoma cells. ► Enrichment analysis identified proteins having phosphoinositide binding domains. ► Functional and pathway enrichment analyses highlighted PI(3)P cellular processes.
Introduction
Phosphatidylinositol phosphates (phosphoinositide phosphates, PtdInsPs, PI(x,y,z)Pn) or simply PIPs) are an important class of phospholipids that affect the spatio-temporal organization of key intracellular signalling networks [1], [2], [3]. This is achieved by functioning as precursors of secondary messengers (inositol phosphates or diacylglycerol) as well as directly interacting with intracellular proteins through the inositol headgroup to specific domains [2], [3]. Phosphoinositide regulated proteins are involved in a variety of biological functions for a range of cell based activities, including vesicle trafficking, proliferation, apoptosis, exocytosis and secretion, membrane trafficking, cell metabolism, adhesion and actin skeletal reorganization [4]. In particular, one network that has recently emerged as being important for the regulation of proliferation, growth, apoptosis, cell migration and morphology is the phosphatidylinositol 3-kinase (PI3K) pathway [2], [5]. Aberrant activations of this pathway have been linked to the development of cancers including breast, pancreatic and colon cancer and are frequently detected in malignancies [6], [7], [8], [9], [10], [11].
The majority of studies on the PI3K pathway have focussed upon members of Class I PI3K and the production of PI(3,4,5)P3 from PI(4,5)P2 [2]. Class IA PI3Ks are activated by a wide variety of cell surface receptors including receptor tyrosine kinases, growth factors, hormones and neurotransmitters, while Class IB are activated by cell surface receptors utilizing heteromeric G-proteins as their proximal transduction partners [3], [12]. Class II PI3Ks (PI3K-C2α, β and γ) act upon both phosphatidylinositol (PI) and PI(4)P in vitro to form PI(3)P and PI(3,4)P2 respectively [12]. It has also been shown recently that Class II PI3Ks can also be activated by external stimuli (e.g., insulin, monocyte chemotactic peptide-1 (MCP1)) and are involved in the agonist-mediated regulation of cellular functions [12], [13]. At present the human homologue of the yeast vesicular-protein-sorting protein, Vps34, is the only member of Class III PI3K. It phosphorylates PI to form PI(3)P [12]. The role of PI(3)P in cell signalling has been largely neglected, but has generally been considered as a constitutive cellular component of endosomes playing a role in membrane trafficking, endocytosis and phagocytosis [14], [15]. Key effector proteins involved in these functions are recruited by PI(3)P binding domains (such as FYVE zinc finger domain (for Fab1, YOTB, Vac1 and EEA1) [16], [17], PX (Phagocyte oxidase homology) [18], [19] and PH (Pleckstrin like homology domains) [20]. Several FYVE domain-containing effector proteins (e.g., early-endosomal antigen 1 (EEA1), Vac1/Rabenosyn-5, Rabankiyin-5) have been identified in endocytic membranes, linking endosome fusion and endocytic membrane trafficking to PI(3)P [15]. These proteins bind to Rab5 and the resulting Rab5/PI(3)P effector protein complex controls endocytic membrane fusion via the formation of Snare complexes [15]. Ligand induced endocytosis of signalling receptors is recognised as a negative regulating mechanism of cell surface signalling such as tyrosine kinase signalling and G protein coupled receptors [21], [22], [23], [24]. However, there is growing evidence that the role of endocytosis extends beyond the control of signalling at the cell surface [22], [23], [24], [25], with the endosome membranes themselves important sites of receptor initiated signal transduction [22], [23], [24], [25].
In addition to its role in endosome signalling, PI(3)P is emerging as another dynamic secondary messenger. A number of stimuli activate Class II PI3Ks, TNF, leptin [26], MCP1 [27], lysophosphatidic acid (LPA) [13] and insulin [28], [29]. Recent evidence also suggests that an insulin-dependent plasma membrane PI(3)P pool might play a critical role in the translocation of the glucose transporter protein GLUT4 [30], [31], [32]. An amino acid dependent pool that is mediated by the Class III Vps34 PI3K has been shown to be involved in the nutrient signalling pathway by activating mTOR and regulating p70 S6 kinase [33], [34], [35].
It has also become apparent that PI(3)P is essential for cytokinesis and abscission [36]. Local production and accumulation of PI(3)P at the midbody have been reported to serve as a docking platform for regulators of cytokinesis and constitutes a novel regulatory mechanism for cell division [37], [38]. These discoveries indicate that PI(3)P has a more extensive role in cellular processes than anticipated previously. The emergence of PI(3)P as a potential secondary messenger in PIP signalling needs further clarification. A systematic analysis of PI(3)P-binding proteins has the potential to identify candidate downstream targets of PI(3)P signalling. It will also provide insights about the function and regulation of signalling pathways involving these PIP interacting proteins. Studies using phosphoinositide affinity matrices have been used successfully to identify PI(3,4,5)P3, PI(3,4)P2, P(3,5)P2, PI(3)P, Ins(1,3,4,5)P4 and Ins(1,4,5)P3 binding proteins [39], [40], [41], [42], [43]. We have recently reported the use of a proteomics approach which combines phosphoinositide affinity-based techniques coupled to nanoRP-HPLC ESI MS/MS analysis [39], [44] to identify the PI(3,5)P2, PI(4,5)P2 and PI(3,4,5)P3 interactomes in cytosolic extracts from a colorectal carcinoma cell line LIM1215 [45]. We have now expanded this investigation to identify the proteins/protein complexes in cytosolic, membrane and nucleus extracts from LIM1215 cells that bind specifically to PI(3)P and are therefore potential candidates for cell signalling regulation by PI(3)P.
Section snippets
Synthesis and characterization of PI(3)P phosphatidylinositol phosphate
The ω-amino phosphatidylinositol-3-phosphate analogue 1 was synthesized from myo-inositol orthoformate 2 following the procedure we previously described (Fig. 1) [46], [47]. The key differentiation of the hydroxyl groups is realised by DIBAL-mediated reduction of the orthoformate [48]. Thus the intermediate 4 was obtained in nine steps. The lipid sidechain phosphoramidite 5 was synthesized in 6 steps from readily available (+)-1,2-O-isopropylidene-glycerol 3 and was subsequently coupled to the O
Synthesis and characterisation of PI(3)P analogues
The protocol used for the synthesis of the ω-amino PI3P derivative is summarised in Fig. 1. The final product NH2-PI(3)P analogue 1 and was fully characterised and agreed with published literature values: Mp 177–181 °C dec. (lyophilised from water); (c 1.09 in D2O); υmax(neat)/cm− 1 3266br (OH), 2919 s, 2851, 1741 s (CO), 1205, 1159, 1090, 1043vs, 1013, 864, 722; δH(500 MHz; D2O) 5.39–5.32 (1 H, m), 4.58–4.51 (1 H, m), 4.49–4.43 (1 H, m), 4.31–4.23 (1 H, m), 4.18–4.08 (2 H, m), 4.08–3.98 (2 H,
Discussion
Phosphoinositides play significant roles in the regulation of a broad range of intracellular processes, including signal transduction pathways, cell motility, cytokinesis, in both exocytosis and endocytosis, vesicular trafficking as well as controlling the activity of ion channels, pumps, and transporters [2], [3], [73], [5], [74], [75]. The rapid and reversible transformation of the eight known phosphoinositide species is controlled, with high selectivity, by a range of phosphoinositide
Conclusions
We have performed a comprehensive proteomic analysis of PI(3)P interacting proteins using affinity/LC/MS/MS experiments with a synthetic analogue of the PI(3)P phosphatidyl phospholipid immobilised on solid support. Cytosolic, membrane and nuclear extracts of LIM1215 colonic carcinoma cells were probed using an ω-amino (PI(3)P derivative covalently linked to beads, resulting in the purification of 681 PI(3)P interacting protein/protein complexes. Protein domain enrichment analysis identified
Acknowledgments
This work was supported by the Australian Research Council, Discovery Project, grant DP1094497, the NHMRC Program grant 487922 and by funds from the Operational Infrastructure Support Program provided by the Victorian Government, Australia.
References (104)
- et al.
Mammalian phosphoinositide kinases and phosphatases
Prog Lipid Res
(2009) - et al.
Phosphatidylinositol 3-phosphate recognition by the FYVE domain
Mol Cell
(1999) - et al.
Crystal structure of a phosphatidylinositol 3-phosphate-specific membrane-targeting motif, the FYVE domain of Vps27p
Cell
(1999) - et al.
Signaling on the endocytic pathway
Curr Opin Cell Biol
(2007) - et al.
Endocytosis conducts the cell signaling orchestra
Cell
(2006) - et al.
Signaling from endosomes: location makes a difference
Exp Cell Res
(2009) - et al.
TNF-alpha and leptin activate the alpha-isoform of class II phosphoinositide 3-kinase
Biochem Biophys Res Commun
(2003) - et al.
The CC chemokine monocyte chemotactic peptide-1 activates both the class I p85/p110 phosphatidylinositol 3-kinase and the class II PI3K-alpha
J Biol Chem
(1998) - et al.
Insulin activates the alpha isoform of class II phosphoinositide 3-kinase
J Biol Chem
(1999) - et al.
hVps34 is a nutrient-regulated lipid kinase required for activation of p70 S6 kinase
J Biol Chem
(2005)
Divide and ProsPer: the emerging role of PtdIns3P in cytokinesis
Trends Cell Biol
Identification of ARAP3, a novel PI3K effector regulating both Arf and Rho GTPases, by selective capture on phosphoinositide affinity matrices
Mol Cell
A chemical proteomics approach to phosphatidylinositol 3-kinase signaling in macrophages
Mol Cell Proteomics
acid-catalysed rearrangements of myo-inositol orthoformate derivatives
Carbohydr Res
Quantitation of intracellular membrane-bound enzymes and receptors in digitonin-permeabilized cells
Anal Biochem
Identification of a Wnt-induced protein complex by affinity proteomics using an antibody that recognizes a sub-population of beta-catenin
Biochim Biophys Acta
Analysis of cellular phosphatidylinositol (3,4,5)-trisphosphate levels and distribution using confocal fluorescent microscopy
Anal Biochem
Multivalent endosome targeting by homodimeric EEA1
Mol Cell
Structural basis for endosomal targeting by FYVE domains
J Biol Chem
Structural basis of 3-phosphoinositide recognition by pleckstrin homology domains
Mol Cell
Phosphoinositide signaling disorders in human diseases
FEBS Lett
Rethinking phosphatidylinositol 3-monophosphate
Biochim Biophys Acta
Endosomal crosstalk: meeting points for signaling pathways
Trends Cell Biol
Use of multidimensional separation protocols for the purification of trace components in complex biological samples for proteomics analysis
J Chromatogr A
The Phox (PX) domain proteins and membrane traffic
Biochim Biophys Acta
SNX-BAR proteins in phosphoinositide-mediated, tubular-based endosomal sorting
Semin Cell Dev Biol
Role of the Phox homology domain and phosphorylation in activation of serum and glucocorticoid-regulated kinase-3
J Biol Chem
Structural and membrane binding analysis of the Phox homology domain of phosphoinositide 3-kinase-C2alpha
J Biol Chem
Genome-wide analysis of membrane targeting by S. cerevisiae pleckstrin homology domains
Mol Cell
Comprehensive identification of PIP3-regulated PH domains from C. elegans to H. sapiens by model prediction and live imaging
Mol Cell
Modular phosphoinositide-binding domains—their role in signalling and membrane trafficking
Curr Biol
Coincidence detection in phosphoinositide signaling
Trends Cell Biol
Inositol trisphosphate and calcium signalling
Nature
The phosphoinositide 3-kinase pathway
Science
Synthesis and function of 3-phosphorylated inositol lipids
Annu Rev Biochem
The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism
Nat Rev Genet
PI3K pathway alterations in cancer: variations on a theme
Oncogene
The phosphatidylinositol 3-kinase AKT pathway in human cancer
Nat Rev Cancer
Exploiting the PI3K/AKT pathway for cancer drug discovery
Nat Rev Drug Discov
Cancer-specific mutations in PIK3CA are oncogenic in vivo
Proc Natl Acad Sci U S A
Protein kinase B/Akt at a glance
J Cell Sci
Class I PI3K in oncogenic cellular transformation
Oncogene
The phosphoinositide (PI) 3-kinase family
J Cell Sci
Class II phosphoinositide 3-kinase defines a novel signaling pathway in cell migration
J Cell Biol
Emerging roles of phosphatidylinositol 3-monophosphate as a dynamic lipid second messenger
Arch Physiol Biochem
Regulation of membrane traffic by phosphoinositide 3-kinases
J Cell Sci
The PX domain: a new phosphoinositide-binding module
J Cell Sci
The PX domains of p47phox and p40phox bind to lipid products of PI(3)K
Nat Cell Biol
Specific and high-affinity binding of inositol phosphates to an isolated pleckstrin homology domain
Proc Natl Acad Sci U S A
Pathways and mechanisms of endocytic recycling
Nat Rev Mol Cell Biol
Cited by (25)
PI3Kα Translocation Mediates Nuclear PtdIns(3,4,5)P<inf>3</inf> Effector Signaling in Colorectal Cancer
2023, Molecular and Cellular ProteomicsPhosphatidylinositol 3-monophosphate: A novel actor in thrombopoiesis and thrombosis
2020, Research and Practice in Thrombosis and HaemostasisPhosphatidylinositol 3 monophosphate metabolizing enzymes in blood platelet production and in thrombosis
2020, Advances in Biological RegulationCitation Excerpt :In mammalian cells, PtdIns3P has the ability to recruit effectors by interacting with specific protein domains such as FYVE (Fab1p, YOTB, Vac1p and EEA1) or PX (Phox) domains (Chandra and Collins, 2018; Gaullier et al., 1999). A study using a combination of affinity isolation and mass spectrometry analysis in a colon cancer cell line has shown that 681 proteins can bind PtdIns3P, while only 69 of them express known PtdIns3P-binding domains (Catimel et al., 2013). Although some of these proteins may bind the lipid indirectly, this study suggests that PtdIns3P could also interact with yet uncharacterized protein domains.
Polyphosphoinositides in the nucleus: Roadmap of their effectors and mechanisms of interaction
2019, Advances in Biological RegulationCitation Excerpt :With the advancement of mass spectrometry, more quantitative methods have been employed. Bead immobilized or liposomes incorporated PPIn have been used to identify PtdIns3P, PtdIns(3,5)P2, PtdIns(4,5)P2 and PtdIns(3,4,5)P3 target proteins in colorectal carcinoma cytosolic extracts (Catimel et al., 2008, 2009, 2013). To discriminate specific PPIn binders from background proteins, quantitative approaches such as stable isotope labelling by amino acids in cell culture (SILAC) have been employed (Dixon et al., 2011; Jungmichel et al., 2014; Lewis et al., 2011).
Endosomes: Emerging Platforms for Integrin-Mediated FAK Signalling
2016, Trends in Cell BiologyThe role of class I, II and III PI 3-kinases in platelet production and activation and their implication in thrombosis
2016, Advances in Biological RegulationCitation Excerpt :How this housekeeping pool of PtdIns3P can regulate membrane skeleton integrity remains an open question. A PtdIns3P interactome study on colorectal cancer cell line has revealed that spectrin, myosin and filamin can interact with PtdIns3P (Catimel et al., 2013). It is thus tempting to speculate that the housekeeping pool of PtdIns3P produced by PI3KC2α is involved in the recruitment of important proteins of the membrane skeleton.