Dynamic Changes in ABCC4 Protein-Protein Interactions during PKA Signaling: Role of the ABCC4 PDZ Motif.

We recently demonstrated that a protein interaction between the PDZ motif of ABCC4 and a single PDZ-domain-containing protein, MPP1 had an important role in ABCC4 function in acute myeloid leukemia (Pitre et al. Nature Comm. 2017). To gain a better understanding into the role of the highly conserved ABCC4 PDZ-motif, we performed live cell imaging using total internal reflectance fluorescence, TIRF, of ABCC4-GFP expression constructs with or without the PDZ motif. The expression constructs were introduced into HEK293 cells where ABCC4 had been deleted by CRISPR-Cas9 technology. ABCC4-GFP was static at the membrane showing little movement, while deletion of the PDZ motif promoted rapid movement. Activation of PKA with forskolin and the phosphodiesterase inhibitor, iso-butyl-methyl xanthine increased intracellular cAMP and promoted membrane movement of ABCC4 harboring the PDZ motif. To get an unbiased understanding of the proteins interacting with ABCC4 both in cis- and trans-, and under PKA activation conditions, we developed stable cell lines in the ABCC4-null HEK293 cells. These stable cell lines harbored either an APEX2-ABCC4 or an APEX2-ABCC4 lacking a PDZ motif. APEX2-derived ascorbate oxidase and biotin phenol, coupled with Avidin-beads was used to interrogate the neighborhood of proteins within ~10 nm of ABCC4. We also developed stable cells harboring expression constructs for either Avi-tagged ABCC4 or Avi-ABCC4 without the PDZ motif to determine proteins that directly interacted with ABCC4 through the PDZ motif. Each Avi-expression construct harbored a biotin ligase, thus enabling ABCC4 to be tagged by biotin and readily "pulled down" by avidin-beads. The interacting proteins were identified by quantitative proteomics. Several PDZ specific interacting proteins were identified in these cell lines. By using analytical density gradient ultracentrifugation, we identified distinct PDZ specific ABCC4-complexes that formed upon PKA activation. The actin-binding cytoskeletal protein, Moesin (MSN) associated with ABCC4, requiring the PDZ-motif. We used quantitative proteomics and APEX2 ABCC4 constructs to show that the neighborhood surrounding ABCC4 lacking the PDZ-motif had fewer proteins, especially cytoskeletal proteins interacting with it. We propose that the ABCC4-MSN interaction is important for tethering ABCC4 to the cytoskeletal actin network. Importantly, siRNA knockdown of MSN disrupted the association between ABCC4 and its interactors indicating the key role of MSN in the dynamic protein interactions occurring between ABCC4 and its interactors.