fostriecin and microcystin

Topics: Alkenes; Animals; Antifungal Agents; Cantharidin; Marine Toxins; Microcystins; Okadaic Acid; Oxazoles; Peptides, Cyclic; Phosphoprotein Phosphatases; Polyenes; Pyrans; Pyrones; Spiro Compounds

DNA-dependent protein kinase (DNA-PK) is a complex of DNA-PK catalytic subunit (DNA-PKcs) and the DNA end-binding Ku70/Ku80 heterodimer. DNA-PK is required for DNA double strand break repair by the process of nonhomologous end joining. Nonhomologous end joining is a major mechanism for the repair of DNA double strand breaks in mammalian cells. As such, DNA-PK plays essential roles in the cellular response to ionizing radiation and in V(D)J recombination. In vitro, DNA-PK undergoes phosphorylation of all three protein subunits (DNA-PK catalytic subunit, Ku70 and Ku80) and phosphorylation correlates with inactivation of the serine/threonine protein kinase activity of DNA-PK. Here we show that phosphorylation-induced loss of the protein kinase activity of DNA-PK is restored by the addition of the purified catalytic subunit of either protein phosphatase 1 or protein phosphatase 2A (PP2A) and that this reactivation is blocked by the potent protein phosphatase inhibitor, microcystin. We also show that treating human lymphoblastoid cells with either okadaic acid or fostriecin, at PP2A-selective concentrations, causes a 50-60% decrease in DNA-PK protein kinase activity, although the protein phosphatase 1 activity in these cells was unaffected. In vivo phosphorylation of DNA-PKcs, Ku70, and Ku80 was observed when cells were labeled with [(32)P]inorganic phosphate in the presence of the protein phosphatase inhibitor, okadaic acid. Together, our data suggest that reversible protein phosphorylation is an important mechanism for the regulation of DNA-PK protein kinase activity and that the protein phosphatase responsible for reactivation in vivo is a PP2A-like enzyme.

Topics: Alkenes; Antigens, Nuclear; Catalysis; Catalytic Domain; Cell Line; Cells, Cultured; DNA Damage; DNA Helicases; DNA Repair; DNA-Activated Protein Kinase; DNA-Binding Proteins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Humans; Ku Autoantigen; Microcystins; Nuclear Proteins; Okadaic Acid; Peptides, Cyclic; Phosphates; Phosphoprotein Phosphatases; Phosphorylation; Polyenes; Precipitin Tests; Protein Binding; Protein Kinases; Protein Phosphatase 1; Protein Phosphatase 2; Protein Serine-Threonine Kinases; Pyrones; Recombinant Proteins; Time Factors

Type-1 protein serine/threonine phosphatases (PP1) are uniquely inhibited by the mammalian proteins, inhibitor-1 (I-1), inhibitor-2 (I-2), and nuclear inhibitor of PP1 (NIPP-1). In addition, several natural compounds inhibit both PP1 and the type-2 phosphatase, PP2A. Deletion of C-terminal sequences that included the beta12-beta13 loop attenuated the inhibition of the resulting PP1alpha catalytic core by I-1, I-2, NIPP-1, and several toxins, including tautomycin, microcystin-LR, calyculin A, and okadaic acid. Substitution of C-terminal sequences from the PP2A catalytic subunit produced a chimeric enzyme, CRHM2, that was inhibited by toxins with dose-response characteristics of PP1 and not PP2A. However, CRHM2 was insensitive to the PP1-specific inhibitors, I-1, I-2, and NIPP-1. The anticancer compound, fostriecin, differed from other phosphatase inhibitors in that it inhibited wild-type PP1alpha, the PP1alpha catalytic core, and CRHM2 with identical IC(50). Binding of wild-type and mutant phosphatases to immobilized microcystin-LR, NIPP-1, and I-2 established that the beta12-beta13 loop was essential for the association of PP1 with toxins and the protein inhibitors. These studies point to the importance of the beta12-beta13 loop structure and conformation for the control of PP1 functions by toxins and endogenous proteins.

Topics: Alkenes; Amino Acid Sequence; Antifungal Agents; Enzyme Activation; Enzyme Inhibitors; Humans; Microcystins; Models, Molecular; Molecular Sequence Data; Peptides, Cyclic; Phosphoprotein Phosphatases; Polyenes; Protein Binding; Protein Phosphatase 1; Protein Structure, Secondary; Proteins; Pyrans; Pyrones; Sequence Homology, Amino Acid; Spiro Compounds