Identification of the Interactome of a Palmitoylated Membrane Protein, Phosphatidylinositol 4-Kinase Type II Alpha

Abstract

Phosphatidylinositol 4-kinases (PI4K) are enzymes responsible for the production of phosphatidylinositol 4-phosphates, important intermediates in several cell signaling pathways. PI4KIIα is the most abundant membrane-associated kinase in mammalian cells and is involved in a variety of essential cellular functions. However, the precise role(s) of PI4KIIα in the cell is not yet completely deciphered. Here we present an experimental protocol that uses a chemical cross-linker, DSP, combined with immunoprecipitation and immunoaffinity purification to identify novel PI4KIIα interactors. As predicted, PI4KIIα participates in transient, low-affinity interactions that are stabilized by the use of DSP. Using this optimized protocol we have successfully identified actin cytoskeleton regulators-the WASH complex and RhoGEF1, as major novel interactors of PI4KIIα. While this chapter focuses on the PI4KIIα interactome, this protocol can and has been used to generate other membrane interactome networks.

Keywords: Cross-linking; Endosome; Interactome; Mass spectrometry; Phosphatidylinositol; Phosphatidylinositol kinase; Phosphoinositide; Phospholipid.

Figure 1. Silver stain of Immunoprecipitation and elution (Immunoaffinity precipitation) using PI4KIIα –specific reagents (PI4KIIα antigenic peptide and antibody)

Lane 1: Molecular weight standard (Biorad). Lane 2: Crosslinked soluble homogenate from SH-SY5Y (ATCC) neuroblastoma cells. Lane 3: Immunomagnetic beads incubated only with the PI4KIIα antibody. This negative control predominantly depicts background IgG heavy (*) and light (**) bands eluted of the beads when heated with the Laemmli buffer. Lane 4–5: Immunoprecipitation with the PI4KIIα antibody. Lane 4: Immunoprecipitation with the PI4KIIα antibody in the presence of the excess PI4KIIα peptide. This is an outcompetition control that represents a profile of non-specific peptides that may bind to the magnetic beads. Lane 5: Immunoprecipitation with the PI4KIIα antibody. Note the presence of a prominent band at ~100kD in Lane 5 that is absent from the control lanes 3 and 4 depicting a putative PI4KIIα specific interactor. Samples in lane 4 and 5 and prepared by elution with the Laemmli buffer. Lane 6–7: Immunoaffinity purification of PI4KIIα interactors. Lane 6: Immunoprecipitation with PI4KIIα with the PI4KIIα antibody in the presence of antigenic peptide for outcompetition followed by elution with the excess PI4KIIα peptide. Note the absence of bands in this control. Lane 7: Immunoprecipitation followed by elution with the PI4KIIα peptide allowing for selective elution of putative PI4KIIα interacting proteins with low background (compare lanes 6 and 7 with lanes 4 and 5). Note the absence of heavy (*) and light (**) IgG chains in lanes 6 and 7. MS/MS analysis of the sample in lane 7 identified highly enriched polypeptides that were absent in all the control samples. The two notable peptides shown here include (a) the PI4KIIα peptide at ~55kDa as expected and (b) a completely novel PI4KIIα interactor RhoGEF1 migrating at ~100kDa seen prominently in lanes 7 as well as lane 5.