Monomeric CCTδ Interacts with Dynactin Associated Protein (dynAP)

Abstract

Introduction‐The chaperonin containing tailless complex polypeptide 1 (CCT) is a molecular chaperone consisting of eight distinct protein subunits. In the monomeric form, several CCT subunits possess additional functions distinct from their oligomeric function [1]. Over‐expression of CCTδ, but not the other seven CCT subunits, results in the appearance of numerous protrusions at the cell surface termed a “protrusion phenotype” which is abrogated by mutations in CCTδ apical domain (G357D) and the ATP binding pocket (D104E) [2]. In addition, it is dependent on CCTδ binding the p150Glued of the dynactin complex. Depletion of either p150Glued or the dynactin complex‐associated transmembrane protein, dynAP prevents GFP‐CCTδ localization to the plasma membrane and blocks protrusion phenotype formation [3]. The work of others [4] shows dynAP binds p150Glued. However, biochemical evidence of CCTδ interacting with dynAP has not been shown. We hypothesized either a direct transient interaction between CCTδ and dynAP or a trimer with p150Glued occurring.Materials & Method‐Mouse melanoma B16F1 cells were cultured as described in [2], transiently transfected with CCTδ constructs, and mutants thereof fused to GFP as described in [2], and full‐length human; p150Glued with C‐terminal Myc and dynAP with an N‐terminal FLAG epitope tags as described in [3]. To stabilize transient interactions in‐vivo, confluent cells were crosslinked with 0.5 mM 3, 3′‐Dithiodipropionic acid di (N‐hydroxysuccinimide ester). Post‐nuclear supernatants from lysed cells were applied to GFP‐trap beads (Chromotek), followed by Western Blotting to detect precipitating proteins.Results‐Following the double transfection of B16 cells with Flag‐dynAP and wild type (WT) GFP‐CCTδ, there was a significant interaction between dynAP and WT CCTδ versus control, D104E. In addition, following the triple transfection of GFP‐CCTδ/p150Glued‐Myc/Flag‐dynAP, there was a significant interaction between dynAP and WT CCTδ versus control, D104E. Furthermore, following the double transfection of WT CCTδ apical domain of (AD) and Flag‐dynAP, there was a significant interaction between dynAP and WT AD versus control, G357D.Conclusion‐Here, we show for the first time an interaction between CCTδ and dynAP via immunoprecipitation representing either a direct interaction or as part of a trimeric complex with p150Glued. This provides biochemical evidence for the mechanism underlying the formation of the protrusion phenotype. This work lends more information on monomeric CCTδ function in cells and would contribute to more understanding of CCTδ’ s role in basic cell and cancer biology. More research is needed to understand the molecular effects of these interactions in cells and in‐vivo.References Svanstrom & Grantham (2015) Cell Stress and Chaperones; 21:55–62. Spiess, M. et al (2015) Journal of Molecular Biology; 427:2757‐2764. Echbarthi M. et al (2018) Experimental Cell Research; 370:137‐149. Kunoh T. et al (2010) Molecular Cancer Therapeutics; 9:2934‐2942.