Structure-function studies of protein phosphatase-1: Toxin and substrate binding sites

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)


Biochemistry and Molecular Biology

First Committee Member

Ernest Y. C. Lee, Committee Chair


Protein phosphatase-1 (PP1) is potently inhibited by several toxins of diverse origins. We have previously identified a region near the C-terminus of PP1 (residues 274-277) that is involved in toxin binding (Zhang et al., (1994) J. Biol. Chem. 269, 16997-17000). In this study, the role of this region in toxin binding was further explored by site-directed mutagenesis between residues 268-278. The IC$\sb{50}$'s of each mutant toward okadaic acid, tautomycin, calyculin A, microcystin-LR, nodularin, inhibitor-2 and cantharidic acid were determined and compared to that of the wild-type enzyme. Tyrosine 272 was identified as a residue whose mutation has drastic effects on the spectrum of inhibitor sensitivity of PP1. The sensitivity of the Tyr-272 mutant toward tautomycin and calyculin A was markedly decreased, by as much as 3 orders of magnitude, with lesser effects on okadaic acid and nodularin, and with microcystin-LR and inhibitor-2 being the least affected. These studies show that Tyr-272 is of specific importance for the binding of these inhibitors and provide strong evidence for the postulate that these toxins all bind to a common inhibitor site of PP1. The Y272 mutants were very useful since it can be used as a tool to differentiate PP1 and PP2A: at the concentration of calyculinA and tautomycin that completely inhibit wild type PP1 and PP2A, this mutant of PP1 is still active and dephosphorylates any given substrate. We also found that cysteine at residue 276 enhanced okadaic acid sensitivity. Cys-273 was involved in forming a covalent adduct when microcystin-LR is bound to PP1, providing the direct evidence that this region is involved in toxin binding.The role of residues that are involved in substrate recognition by PP1 was investigated by site-directed mutagenesis and kinetic analyses using $\sp{32}$P-labeled phosphorylase a, RII peptide, Kemptide and PNPP (p-nitrophenyl phosphate) as substrates. The crystal structure of PP1 has shown that the active site is at the confluence of three shallow grooves, a C-terminal groove, an acidic groove and a hydrophobic groove. We systematically mutated D208, D210, D212, E218, D220, E252, D253, E256 in the acidic groove, R221, W206 and Y134 which have been suggested to be involved in phosphate/phosphoserine binding, and C127, I130 and D197 in the hydrophobic groove. Kinetic analyses of each mutant using four substrates were performed. Our results show that mutations in the acidic groove lead to modest changes in substrate binding, consistent with a role of the acidic residues is forming a negatively charged surface well for binding of peptides with a basic N-terminus. Mutants of R221, D208 and W206 showed severely reduced $k\sb{\rm cat}$ values, consistent with the proposal that R221 plays an important role as a phosphate oxygen ligand which positions the substrate for catalysis. The kinetic behavior of mutants at W206 and D208 can be explained by the observation that together with R221, these residues form a microenvironment which dictates the orientation of the imidazole ring of H248, one of the metal binding ligands, as well as contributing to the orientation of R221 itself.


Biology, Molecular; Health Sciences, Toxicology; Chemistry, Biochemistry

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