The AKAP220 signaling complex regulates renal aquaporin-2 localization

Abstract

A kinase anchoring proteins (AKAPs) localize signaling molecules such as kinases and phosphatases in close proximity to their substrates to control the scope and duration of second messenger responses. AKAP220 is a ubiquitously expressed, 220-kilodalton protein that anchors protein kinase A (PKA), glycogen synthase kinase 3β (GSK3β) and protein phosphatase 1 (PP1). Although AKAP220 has been implicated in promoting cortical actin polymerization, little is currently known about the biological function of this signaling complex. To gain insight toward this question, we used biochemical and cell based assays to explore the kinase anchoring dynamics of AKAP220. We discovered that AKAP220 preferentially anchors catalytically active GSK3β within a 17-amino acid linear region containing a required GSK3β phosphosite. We also identified a novel PKA anchoring helix in the amino terminus of AKAP220 and characterize the isoform-binding properties of both PKA sites. Within the complex, activation of anchored PKA inhibits GSK3β activity and opposes its binding to AKAP220. Using Cre-lox technology, we produced a global AKAP220 knockout mouse model to study the functional role of AKAP220 in vivo. We find that loss of AKAP220 causes a urine-diluting deficit in response to acute water loading. Based on immunostaining experiments, we propose that this phenotype is caused by the abnormal accumulation of aquaporin-2 (AQP2) at the apical plasma membrane of renal principal cells. This mislocalization is consistent with our pulldown assays detecting a decrease in GTP-bound (active) RhoA in AKAP220 knockout kidneys. RhoA activity promotes the formation of an apical actin barrier in renal principal cells. This actin meshwork prevents basal fusion of AQP2-containing vesicles in the absence of vasopressin stimulation. Previous studies have shown that AKAP220 promotes polymerization of cortical actin, so we investigated whether this complex may influence actin barrier formation in the kidney. Using CRISPR-Cas9 genome editing technology we generated mIMCD3 cells that do not express AKAP220. These cells were grown in 3D culture to produce polarized spheroids. Imaging analysis of these spheroids revealed that AKAP220-/- cells had decreased apical actin thickness compared to wild-type. Together, these findings indicate that AKAP220 anchors signaling elements that influence AQP2 localization in the collecting duct of mammalian kidneys.