Identification of phosphorylation sites in the PKD1-encoded protein C-terminal domain.
The PKD1-encoded protein, "polycystin-1", has a large N-terminal extracellular portion, multiple transmembrane domains, and a short intracellular C-terminal tail with four tyrosine residues and two putative sites for serine phosphorylation. Its function in kidney development and autosomal dominant polycystic kidney disease (ADPKD) is still unknown. We have subcloned the cDNA encoding the polycystin-1 C-terminal domain (PKD1-CTD) into a prokaryotic expression vector, and site-directed mutagenesis was performed to target the four tyrosine residues and four serine residues in two putative phosphorylation sites. In vitro phosphorylation assays were conducted on both wild type and mutant PKD1-CTD fusion proteins. It was found that the wild type PKD1-CTD and all mutant fusion proteins, except S4251G/S4252G, could be phosphorylated by lysates from cultured normal human renal collecting tubule (NHCT) cells, as well as by commercially purified cAMP-dependent protein kinase (PKA). The phosphorylation of the PKD1-CTD fusion protein by NHCT lysates was greatly enhanced by cAMP and its analog 8-Br-cAMP, and inhibited by the specific PKA inhibitors PKI(6-22) and H-89. Activators and inhibitors of protein kinase C (PKC) had no effects on the phosphorylation of the PKD1-CTD fusion protein. Using commercially purified pp60(c-src) (c-src) it was also shown that the PKD1-CTD fusion protein could be phosphorylated by c-src in vitro, and that this phosphorylation could be abolished by a mutation Y4237F. By comparing the amino acid sequence at 4249-4253 (RRSSR) with the consensus sequence for PKA phosphorylation (RRXSX), we suggest that the serine residue at 4252 is the target of phosphorylation by a cAMP-dependent protein kinase in NHCT cell lysates. In addition, we suggest that Y4237 might be phosphorylated by c-src in living cells.
Li
H P
HP
Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029, USA. Hsi-Ping_Li@mtplink.mssm.edu
Geng
L
L
Burrow
C R
CR
Wilson
P D
PD
D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007684:Kidney Tubules / Q000378:metabolism; D010766:Phosphorylation; D016891:Polycystic Kidney, Autosomal Dominant / Q000235:genetics; D011506:Proteins / Q000737:chemistry*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D050396:TRPP Cation Channels
D017868:Cyclic AMP-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D011506:Proteins; D016392:Proto-Oncogene Proteins pp60(c-src); D011993:Recombinant Fusion Proteins; D050396:TRPP Cation Channels