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Identification of protein kinases that phosphorylate the urea channel protein UT‐A1 (1137.11)
Author(s) -
Hwang Gloria,
Chou ChungLin,
Hoffert Jason,
Knepper Mark
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.1137.11
Subject(s) - phosphorylation , kinase , protein phosphorylation , biochemistry , immunoprecipitation , chemistry , protein kinase a , biology , microbiology and biotechnology , gene
Vasopressin regulates the urea channel protein UT‐A1 by phosphorylation at Ser84 and Ser486. What kinases are responsible for these phosphorylation events remains an open question. To identify protein kinases that interact with UT‐A1 in IMCD, we employed crosslinking with BSOCOES, UT‐A1 immunoprecipitation, SDS‐PAGE separation, and quantitative mass spectrometry of trypsin digests of gel pieces. The peptides passing stringent spectral quality thresholds were quantified (label‐free) to identify those with high signal:noise (UTA1 antibody/preimmune IgG>4). This identified four protein kinases bound to the UT‐A1 (Phosphorylase Kinase [PhK], Calmodulin‐Dependent Kinase 2δ□[CAMK2], CDC42‐Binding Protein Kinase β□[CDC42BPK] and mTOR). In vitro phosphorylation assays were carried out by incubating these kinases or Protein Kinase A (purified, active recombinant) with synthetic peptides containing the known phosphorylation sites in UT‐A1. Phosphorylation was determined by immunoblotting using phospho‐specific antibodies. Four of the five tested kinases phosphorylated Ser486 (PKA > CDC42BPK > PhK > CAMK2). Kinetic studies of Ser486 phosphorylation (1 min‐24 hr) also showed PKA > CDC42BPK > PhK. In contrast, only CDC42BPK phosphorylated Ser84‐UT‐A1. mTOR did not phosphorylate either site. These results provide important data for modeling vasopressin signaling pathways. Grant Funding Source : Supported by NHLBI