AQP2 is a substrate for endogenous PP2B activity within an inner medullary AKAP-signaling complex.
We have demonstrated that inner medullary collecting duct (IMCD) heavy endosomes purified from rat kidney IMCD contain the type II protein kinase A (PKA) regulatory subunit (RII), protein phosphatase (PP)2B, PKCzeta, and an RII-binding protein (relative molecular mass ~90 kDa) representing a putative A kinase anchoring protein (AKAP). Affinity chromatography of detergent-solubilized endosomes on cAMP-agarose permits recovery of a protein complex consisting of the 90-kDa AKAP, RII, PP2B, and PKCzeta. With the use of small-particle flow cytometry, RII and PKCzeta were localized to an identical population of endosomes, suggesting that these proteins are components of an endosomal multiprotein complex. (32)P-labeled aquaporin-2 (AQP2) present in these PKA-phosphorylated endosomes was dephosphorylated in vitro by either addition of exogenous PP2B or by an endogenous endosomal phosphatase that was inhibited by the PP2B inhibitors EDTA and the cyclophilin-cyclosporin A complex. We conclude that IMCD heavy endosomes possess an AKAP multiprotein-signaling complex similar to that described previously in hippocampal neurons. This signaling complex potentially mediates the phosphorylation of AQP2 to regulate its trafficking into the IMCD apical membrane. In addition, the PP2B component of the AKAP-signaling complex could also dephosphorylate AQP2 in vivo.
Jo
I
I
Division of Nephrology, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Ward
D T
DT
Baum
M A
MA
Scott
J D
JD
Coghlan
V M
VM
Hammond
T G
TG
Harris
H W
HW
D000818:Animals; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins / Q000378:metabolism*; D001345:Autoradiography; D019703:Calcineurin / Q000032:analysis / Q000378:metabolism*; D002352:Carrier Proteins / Q000032:analysis / Q000378:metabolism*; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D011992:Endosomes / Q000737:chemistry / Q000201:enzymology; D005434:Flow Cytometry; D007163:Immunosorbent Techniques; D007679:Kidney Medulla / Q000201:enzymology* / Q000648:ultrastructure; D007685:Kidney Tubules, Collecting / Q000201:enzymology / Q000648:ultrastructure; D008297:Male; D010766:Phosphorylation; D011493:Protein Kinase C / Q000032:analysis / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D013379:Substrate Specificity
C496025:Aqp2 protein, rat; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins; D002352:Carrier Proteins; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D019703:Calcineurin