FIGURE 1:
The PI4KIIα interactome enriches actin-regulatory proteins. (A) PI4KIIα was immunoaffinity purified from DSP–cross-linked SHSY-5Y neuroblastoma cell homogenates. PI4KIIα and coisolating proteins were resolved by SDS–PAGE and silver stained (lane 5). Some polypeptides are absent from controls, which include antibody-coated beads without lysate (lane 3) or incubations in which an excess of antigenic PI4KIIα peptide was included (lane 4). Bead elution was performed with Laemmli sample buffer (lanes 3–5). Lanes 6 and 7 depict antigenic peptide eluates from beads similar to those in lanes 4 and 5. Immunoaffinity purification allowed for the selective elution of PI4KIIα-coisolating proteins against low background (compare lanes 6 and 7; lanes 6′ and 7′ depict contrast-enhanced lanes 6 and 7). An arrow marks the band containing PI4KIIα. Silver stain is a representative image of two independent experiments. Bottom, immunoblot of PI4KIIα in a parallel set of assays as those in the silver-stained SDS–PAGE. Immunoglobulin G chains are marked by asterisks. Input represents 1 and 0.33% for immunoprecipitation and immunoaffinity purifications, respectively. (B) Plots represent PI4KIIα and copurifying proteins (see Supplemental Table S1 for details). The x- and y-axes show SILAC fold of enrichment and the total number of spectral counts used for protein identification, respectively. Reference dots highlight PI4KIIα (green) and Nedd4-1. (C) Teal and red dots highlight actin-related proteins coisolated with PI4KIIα. These include GDP exchange factors RhoGEF1, DOCK7, and GEF-H1. Red dots indicate the actin-related proteins that are subunits or interactors of the WASH complex: strumpellin (KIAA0196), SWIP (KIAA1033), Fam21B, FKBP15, capping protein α, and capping protein β. (D) Blue dots highlight membrane-trafficking related proteins coisolated with PI4KIIα, including clathrin heavy chain (CLTC), dynamin-2 (DNM2), and β3A subunit of the AP-3 complex (AP3B1). (E) Functional annotation analysis of the identified proteins using Gene Ontology annotations revealed a preponderance of actin-related proteins. Enrichment of categories such as cytoskeleton (30/124 interactors) and actin cytoskeleton (13/124) were significant at p = 1.62 × 10−7 and 7.67 × 10−7, respectively. See Supplemental Table S2 for details. (F) Network analysis of putative PI4KIIα interactome components and BLOC-1 subunits. PI4KIIα (green), the BLOC-1 subunits (blue), and WASH complex subunits (red) are highlighted.