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 p rotein 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 transmem brane domain suggests that DdPTPa is a nonreceptor type cytoplasmic pr otein tyrosine phosphatase. Southern blot analysis of genomic DNA indi cates 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 develo pmentally regulated. The entire coding sequence of DdPTPa was subclone d into the pET15-b vector and expressed in Escherichia coli. Affinity- purified DdPTPa protein efficiently dephosphorylates both p-nitropheny l phosphate and tyrosine-phosphorylated reduced, carboxyamidomethylate d, and maleylated lysozyme. A Dictyostelium transformant overexpressin g DdPTPa does not develop normally. The overexpresser fails to aggrega te, in contrast to the control transformant containing vector alone, a nd after 24 h gives rise to only a few abnormal slugs and small fruiti ng bodies.