Abstract
Class I phosphoinositide 3-kinases exert important cellular effects through their two primary lipid products, phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P(2)). As few molecular targets for PtdIns(3,4)P(2) have yet been identified, a screen for PI 3-kinase-responsive proteins that is selective for these is described. This features a tertiary approach incorporating a unique, primary recruitment of target proteins in intact cells to membranes selectively enriched in PtdIns(3,4)P(2). A secondary purification of these proteins, optimized using tandem pleckstrin homology domain containing protein-1 (TAPP-1), an established PtdIns(3,4)P(2) selective ligand, yields a fraction enriched in proteins of potentially similar lipid binding character that are identified by liquid chromatography-tandem MS. Thirdly, this approach is coupled to stable isotope labeling with amino acids in cell culture using differential isotope labeling of cells stimulated in the absence and presence of the PI 3-kinase inhibitor wortmannin. This provides a ratio-metric readout that distinguishes authentically responsive components from copurifying background proteins. Enriched fractions thus obtained from astrocytoma cells revealed a subset of proteins that exhibited ratios indicative of their initial, cellular responsiveness to PI 3-kinase activation. The inclusion among these of tandem pleckstrin homology domain containing protein-1, three isoforms of Akt, switch associated protein-70, early endosome antigen-1 and of additional proteins expressing recognized lipid binding domains demonstrates the utility of this strategy and lends credibility to the novel candidate proteins identified. The latter encompass a broad set of proteins that include the gene product of TBC1D2A, a putative Rab guanine nucleotide triphosphatase activating protein (GAP) and IQ motif containing GAP1, a potential tumor promoter. A sequence comparison of the former protein indicates the presence of a pleckstrin homology domain whose lipid binding character remains to be established. IQ motif containing GAP1 lacks known lipid interacting components and a preliminary analysis here indicates that this may exemplify a novel class of atypical phosphoinositide (aPI) binding domain.
Highlights
The cell-surface receptor-regulated, class I phosphoinositide (PI)1 3-kinases play a central role in the intracellular mechanisms that regulate important biological processes including cell division, growth, motility, and metabolism (1, 2)
PtdIns(3,4)P2 may exert effects through other target proteins such as tandem pleckstrin homology domain containing protein-1 (TAPP-1) (23) and lamellipodin (24), which bind this lipid with high selectivity, or DAPP-1 (25) and Irgm-1 (26) that interact preferentially with both PtdIns(3,4)P2 and PtdIns(3,4,5)P3
The small proportion of TAPP-1 remaining following digitonin treatment probably represents trapped protein as this was not diminished by pretreatment of cells with the PI 3-kinase inhibitor, wortmannin
Summary
Materials—1321N1 Astrocytoma cells were from the European Collection of Animal Cell Cultures. Proteins bound to 3-PIs synthesized in response to prior cell stimulation, were either recovered selectively from the remaining, insoluble cell material (membrane) by inositol phosphate-specific elution in buffer A (10 mM Hepes, pH 7.2, 0.1 mM EDTA, 0.1 mM EGTA) as indicated or more crudely by extraction into a suitable volume (0.5–5.0 ml) of ice-cold lysis buffer (50 mM Tris, pH 7.5, 50 mM NaF, 1 mM EDTA, 1 mM EGTA, 5 mM sodium pyrophosphate, 10 mM glycerol 2-phosphate, 0.5% (w/v) Triton X-100, supplemented freshly with 0.1% (v/v) 2-mercaptoethanol, 0.1 mM PMSF, 0.1 mM benzamidine and 1 mM sodium vanadate) These extracts were analyzed by SDS-PAGE and mass spectrometry either directly or following further chromatographic enrichment. Protein-lipid interaction was assessed using protein-lipid overlay assays performed as described previously (23, 25) using the GST-tagged PH domains of GRP1, TAPP1 and phospholipase (PLC) ␦, and GST-tagged test protein (see “Results”) each at 0.2 g/ml
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