Abstract
Polycystin signaling is likely to be regulated by phosphorylation. While a number of potential protein kinases and their target phosphorylation sites on polycystin-1 have been identified, the corresponding phosphatases have not been extensively studied. We have now determined that polycystin-1 is a regulatory subunit for protein phosphatase-1α (PP1α). Sequence analysis has revealed the presence of a highly conserved PP1-interaction motif in the cytosolic, C-terminal tail of polycystin-1; and we have shown that transfected PP1α specifically co-immunoprecipitates with a polycystin-1 C-tail construct. To determine whether PP1α dephosphorylates polycystin-1, a PKA-phosphorylated GST-polycystin-1 fusion protein was shown to be dephosphorylated by PP1α but not by PP2B (calcineurin). Mutations within the PP1-binding motif of polycystin-1, including an autosomal dominant polycystic kidney disease (ADPKD)-associated mutation, significantly reduced PP1α-mediated dephosphorylation of polycystin-1. The results suggest that polycystin-1 forms a holoenzyme complex with PP1α via a conserved PP1-binding motif within the polycystin-1 C-tail, and that PKA-phosphorylated polycystin-1 serves as a substrate for the holoenzyme.
Highlights
Phosphorylation is a common post-translational modification of proteins that affects their structure and ability to interact with other proteins, and their function
As such, understanding the functions of protein kinases and phosphatases is of critical importance to cellular biology
The broad substrate specificity of PP1 is dictated by its interactions with a wide range of regulatory proteins [3]
Summary
Phosphorylation is a common post-translational modification of proteins that affects their structure and ability to interact with other proteins, and their function. The broad substrate specificity of PP1 is dictated by its interactions with a wide range of regulatory proteins [3] These ‘‘holoenzyme’’ complexes can dephosphorylate single or multiple substrates and are themselves subject to being regulated by events such as post-translational modification or binding of additional accessory proteins [1,2,4]. The mammalian genome contains three genes encoding four isoforms of PP1 (a, b, c1, and c2) that are approximately 90% identical. Differences within their N- and C-termini affect binding to regulatory proteins and are important for enzyme specificity [2]
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