Cloning and functional expression of a Dictyostelium discoideum protein tyrosine phosphatase.
Using polymerase chain reaction methods, we cloned a 1.7-kilobase cDNA, denoted DdPTPa, that has high homology with other known eukaryotic protein tyrosine phosphatases. DdPTPa possess a 241-amino acid protein tyrosine phosphatase domain located in the C terminus, which exhibits a 39-43% amino acid sequence identity with published protein tyrosine phosphatases. Absence of a characteristic signal sequence and transmembrane domain suggests that DdPTPa is a nonreceptor type cytoplasmic protein tyrosine phosphatase. Southern blot analysis of genomic DNA indicates the presence of a multigene protein tyrosine phosphatase family in Dictyostelium. Northern blot analysis reveals four species of mRNA that hybridize to the DdPTPa probe, at least three of which are developmentally regulated. The entire coding sequence of DdPTPa was subcloned into the pET15-b vector and expressed in Escherichia coli. Affinity-purified DdPTPa protein efficiently dephosphorylates both p-nitrophenyl phosphate and tyrosine-phosphorylated reduced, carboxyamidomethylated, and maleylated lysozyme. A Dictyostelium transformant overexpressing DdPTPa does not develop normally. The overexpresser fails to aggregate, in contrast to the control transformant containing vector alone, and after 24 h gives rise to only a few abnormal slugs and small fruiting bodies.
Ramalingam
R
R
Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110.
Shaw
D R
DR
Ennis
H L
HL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D004023:Dictyostelium / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D017027:Protein Tyrosine Phosphatases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014638:Vanadates / Q000494:pharmacology
D017931:DNA Primers; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014638:Vanadates; D017027:Protein Tyrosine Phosphatases