The PI(3)P interactome from a colon cancer cell

Abstract

A comprehensive analysis of the phosphoinositide interactome has been performed using an ω-amino analogue of phosphatidylinositol 3-phosphate (PI(3)P immobilised onto Affi-10 beads for use as an affinity absorbent for cytosolic, membrane and nuclear extracts from the LIM1215 colonic carcinoma cell line. Affinity/LC/MS/MS experiments allowed the identification of 681 proteins/protein complexes which interact with PI(3)P. Protein domain enrichment analysis identified proteins possessing PI(3)P (e.g., FYVE, PX, PH), PIP and PIP/phospholipid binding domains along with small GTPases, GTPase regulators, kinases and SH2/SH3 containing proteins. Functional and pathway enrichment analyses highlighted the major role of PI(3)P in endocytosis dynamics and vesicular trafficking, intracellular cell signalling regulation, cell division and cytokinesis.

Biological significance

This study provides an initial detailed assessment of the phosphatidylinositol 3-phosphate (PI(3)P) interactome, highlights the major role of PI(3)P in endocytosis dynamics and vesicular trafficking, cell intracellular regulation, signalling and cytokinesis and suggests potential PI(3)P specificity for further biochemical and biological characterisation.

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.