Dictyostelium discoideum protein phosphatase-1 catalytic subunit exhibits distinct biochemical properties.
Protein phosphatase-1 (PP1) is expressed ubiquitously and is involved in many eukaryotic cellular functions, although PP1 enzyme activity could not be detected in the social amoeba Dictyostelium discoideum cell extracts. In the present paper, we show that D. discoideum has a single copy gene that codes for the catalytic subunit of PP1 (DdPP1c). DdPP1c is expressed throughout the D. discoideum life cycle with constant levels of mRNA, and its protein and amino acid sequence show a mean identity of 80% with other PP1c enzymes. However, it has a distinctive difference: the substitution of a phenylalanine residue (Phe(269) in the DdPP1c) for a highly conserved cysteine residue (Cys(273) in rabbit PP1c) in a region that was shown to have a critical role in the interaction of rabbit PP1c with toxin inhibitors. Wild-type DdPP1c and an engineered mutant form in which Phe(269) was replaced by a cysteine residue were expressed in Escherichia coli. Both recombinant activities were similarly inhibited by okadaic acid, tautomycin and microcystin. However, the Phe(269)-->Cys mutation resulted in a large increase in enzyme activity towards phosphorylase a and a higher sensitivity to calyculin A. These results, together with the molecular modelling of DdPP1c structure, indicate that the Phe(269) residue, which occurs naturally in D. discoideum, confers distinct biochemical properties on this enzyme.
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Avenida Professor Lineu Prestes 748, 05508-900, São Paulo, SP, Brazil.
Luiz P M
LP
Andrioli
Paulo A
PA
Zaini
Wladia
W
Viviani
Aline M
AM
Da Silva
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain; D017931:DNA Primers; D018076:DNA, Complementary; D004023:Dictyostelium / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000378:metabolism*; D054645:Protein Phosphatase 1; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1