Multiple structural elements define the specificity of recombinant human inhibitor-1 as a protein phosphatase-1 inhibitor.
The cDNA encoding human brain protein phosphatase inhibitor-1 (I-1) was expressed in Escherichia coli. Following PKA phosphorylation at a threonine, recombinant human I-1 was indistinguishable from rabbit skeletal muscle I-1 as a potent and specific inhibitor of the type-1 protein serine/threonine phosphatase (PP1). N-Terminal phosphopeptides of I-1 that retained the selectivity of intact human I-1 highlighted a functional domain that mediates PP1 inhibition. Substituting alanine in place of threonine-36 eliminated I-1 phosphorylation by PKA and its phosphatase inhibitor activity. An acidic residue was substituted in place of the phosphoacceptor to produce I-1(T35D), a constitutive phosphate inhibitor. I-1(T35D) was an equally effective inhibitor of PP1 and the type-2 phosphatase, PP2A. However, CNbr digestion of I-1(T35D) yielded an N-terminal peptide that showed 100-fold increased specificity as a PP1 inhibitor. This provided new insight into a unique conformation of the phosphorylated I-1 that accounts for selective inhibition of PP1 activity. Truncation of an active I-1 phosphopeptide identified an N-terminal sequence that was reduced in addition to threonine-35 phosphorylation to inhibit PP1 activity. Biosensor studies demonstrated that PP1 bound to both Phosphorylated and dephosphorylated I-1 and suggested that distinct elements of I-1 structure accounted for PP1 binding and inhibition. Our data point to multiple interactions between the I-1 functional domain. and the PP1 catalytic subunit that define this phosphoprotein as a physiological regulator of the type-1 protein phosphatase.
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Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27708, USA.
Zhou
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Connor
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Wang
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Shenolikar
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001923:Brain Chemistry / Q000235:genetics*; D002352:Carrier Proteins; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004722:Endoribonucleases; D004791:Enzyme Inhibitors; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000235:genetics*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D011487:Protein Conformation; D054645:Protein Phosphatase 1; D011506:Proteins / Q000235:genetics*; D016601:RNA-Binding Proteins; D011817:Rabbits; D011994:Recombinant Proteins; D017384:Sequence Deletion; D013045:Species Specificity
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C021042:protein phosphatase inhibitor-1; D004722:Endoribonucleases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; C097081:PPP1R8 protein, human