A mutant of protein phosphatase-1 that exhibits altered toxin sensitivity.
A chimeric mutant was constructed in which a 4-amino acid region (GEFD, residues 274-277) of rabbit muscle protein phosphatase-1 was replaced with the sequence YRCG corresponding to residues 267-270 of rabbit protein phosphatase-2A. This was based on the findings of a gene mutation in okadaic acid-resistant cells which results in a Cys-->Gly conversion in protein phosphatase-2A. The YRCG mutant of protein phosphatase-1 was expressed and purified. The properties of the mutant enzyme were investigated in terms of its sensitivity toward several toxin inhibitors (okadaic acid, microcystin, nodularin, calyculin A, and cantharidic acid), as well as inhibitor-2. The mutant enzyme exhibited a gain of function in the form of a 10-fold increased sensitivity toward okadaic acid that suggests this region is involved in toxin binding. Significant changes in sensitivity to inhibitor-2 and several of the other toxins were also observed, indicating that these may have a common binding region.
Zhang
Z
Z
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Florida 33101.
Zhao
S
S
Long
F
F
Zhang
L
L
Bai
G
G
Shima
H
H
Nagao
M
M
Lee
E Y
EY
D000595:Amino Acid Sequence; D000818:Animals; D017931:DNA Primers / Q000737:chemistry; D004988:Ethers, Cyclic / Q000494:pharmacology; D007700:Kinetics; D008387:Marine Toxins; D052998:Microcystins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019319:Okadaic Acid; D010080:Oxazoles / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D011817:Rabbits; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D017931:DNA Primers; D004988:Ethers, Cyclic; D008387:Marine Toxins; D052998:Microcystins; D010080:Oxazoles; D010456:Peptides, Cyclic; D011993:Recombinant Fusion Proteins; C063998:nodularin; D019319:Okadaic Acid; C059041:calyculin A; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; C057862:cyanoginosin LR