A novel phosphoprotein inhibitor of protein type-1 phosphatase holoenzymes.
Control of protein phosphatases is now understood to depend on binding to a variety of regulatory or targeting subunits to form holoenzymes with restricted localization and substrate specificity. In addition, the catalytic subunits of both type-1 and type-2 phosphatases bind specific inhibitor proteins. Here, we report discovery of a new inhibitor protein called PHI-1 that is specific for type-1 protein phosphatase (PP1). Recombinant tagged PHI-1 was phosphorylated by protein kinase C at two sites, one a Ser and one a Thr; phosphorylation enhanced inhibitory potency 50-fold. Mutation of Thr57 to Ala gave a protein phosphorylated only on Ser, without change in inhibitory activity, indicating that phosphorylation of Thr57 was required for full activity. Immunoblotting showed that PHI-1 was expressed in most animal tissues and several cell lines, and a second larger protein called PHI-2 was present in different muscles, especially cardiac muscle. Unlike any other known inhibitor, PHI-1 inhibited the myosin- and glycogen-associated holoenzyme versions of PP1 as well as the monomeric catalytic subunit of PP1. Discovery of PHI-1 and PHI-2 opens new possibilities for regulation of PP1 via phosphorylation-dependent signaling pathways.
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Center for Cell Signaling, University of Virginia School of Medicine, Charlottesville 22908, USA.
Karginov
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Brautigan
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D000818:Animals; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D020035:Holoenzymes / Q000737:chemistry; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010750:Phosphoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D014176:Protein Biosynthesis; D054645:Protein Phosphatase 1; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D013552:Swine
D004791:Enzyme Inhibitors; D020035:Holoenzymes; D047908:Intracellular Signaling Peptides and Proteins; D009124:Muscle Proteins; C107507:PPP1R14A protein, human; D010750:Phosphoproteins; C491520:Ppp1r14a protein, mouse; C491519:Ppp1r14a protein, rat; C099933:Ppp1r14b protein, mouse; D011506:Proteins; D011993:Recombinant Fusion Proteins; C073882:phosphoprotein phosphatase inhibitor 1; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1