Protein Kinase A Activation Promotes ABCC4 Protein Network Assembly

Abstract ID 95276 Poster Board 431 The ATP-binding cassette transporter ABCC4 regulates cAMP membrane microdomain formation after adenylate cyclase activation but how ABCC4 localizes to these membrane microdomains are still unclear. Herein we demonstrate that the assembly and spatiotemporal organization of ABCC4's protein neighborhood by PKA requires the PDZ motif of ABCC4 and PKA activation. To investigate 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 endogenous ABCC4 had been deleted by CRISPR-Cas9 (ABCC4-null HEK293 cells). Deletion of the PDZ motif untethered ABCC4 promoting rapid movement. Treatment with the adenylate cyclase agonist, forskolin and the phosphodiesterase inhibitor, iso-butyl-methyl xanthine increased intracellular cAMP and promoted the membrane localization of ABCC4 harboring the PDZ motif, which was significantly reduced by treatment with a PKA specific peptide inhibitor, PKI-14-22. Density gradient ultracentrifugation revealed the formation of an ABCC4 PDZ motif–specific, large protein complex upon PKA activation. To elucidate how PKA ushers ABCC4 to form this membrane supercomplex, we identified proteins that closely interact with ABCC4 and proteins in ABCC4's "neighborhood" network using Avi-tagged or APEX-tagged ABCC4 constructs. Cell lines were generated in ABCC4-null HEK293 cells by inserting Avi- and APEX-tagged ABCC4 constructs, and their deletion mutants lacking the PDZ motif. Each Avi-expression construct harbored a biotin ligase, thus enabling ABCC4 to be tagged by biotin. We used a crosslinker, DSP (dithiobis(succinimidyl propionate)) to capture weakly or transiently interacting proteins. Ascorbic acid peroxidase (APEX) - tagged to ABCC4 catalyzes the externally added biotin-phenol molecules in the presence of hydrogen peroxidase into biotin-phenol radicals which bind to tyrosine residues of proteins within ∼10 nm of ABCC4. The biotinylated proteins were isolated by affinity purification using avidin beads and mass spectrometry. Our results from the proteomic analysis indicated that ABCC4 interacts with numerous PDZ domain containing proteins (PDZ proteins), and the PKA-induced enrichment of actin filament-binding proteins in the vicinity of ABCC4 requires the PDZ motif. The assembly of the supercomplex was crucially dependent on the actin-binding protein Moesin (MSN), which also interacts with the PDZ motif. siRNA knockdown of MSN disrupted the ABCC4-SCRIB interaction within the PKA-driven supercomplex, as well as ABCC4 interaction with actin and filamin A. Furthermore, we demonstrated that Ceefourin 2, an ABCC4 specific inhibitor, reduced membrane levels of ABCC4, induced by PKA, by disrupting the actin cytoskeletal network around ABCC4. Altogether, these findings indicate that PKA activation promotes ABCC4 membrane localization by enhancing the assembly of a protein scaffold at its PDZ motif through PDZ proteins, MSN and actin cytoskeletal proteins. This work was supported by NIH and by ALSAC.