Cloning and functional expression of a Dictyostelium discoideum protein tyrosine phosphatase.

D.R Shaw,R Ramalingam,H.L Ennis

doi:10.1016/s0021-9258(18)41581-5

Abstract

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.

Highlights

The nucleotide sequence(s)reported in thispaper has been submitted to the GenBankTM/EMBL Data Bank withaccession numberfs) LI 5420
TwocDNAs encoding protein tyrosine kinases in Dictyostelium havebeen cloned (Tan andSpudich, 1990).In thispaper we report the isolation, developmental expression, and possible role in multicellular development of a 1.7-kb PTPase cDNA
372-bp fragment may contain a 112-bp intron embedded in blot of RNA isolated from different stages of the life cycle the sequence encoding the same amino acids as the 260-bp showed that the 1.7-kb DdPTPa mRNA was present in low sequence

Summary

RESULTS

The deduced amino acid sequenocfethe 260-bp fragment was highly homologous to other PTPases in the data bases. Under low stringency hybridization conditions, the DdPTPa probe hybridized strongly to the DdPTPa-specific fragments and weakly to additional bands(Fig. 3B). 372-bp fragment may contain a 112-bp intron embedded in blot of RNA isolated from different stages of the life cycle the sequence encoding the same amino acids as the 260-bp showed that the 1.7-kb DdPTPa mRNA was present in low sequence. Conserved 241-amino acid PTPase domain at theC terminus Bacterially Expressed DdPTPa Protein Possesses PTPase (underlined in Fig. 1).The DdPTPa PTPase domain showed Enzyme Actiuity-To determine whether DdPTPapossessed a high homology (between 39-43% identity) (Fig. 2) to several PTPaseactivity, a fragmentcorrespondingtothe coding published PTPases including PTP-1B (Chernoffale.,t 1990), region encompassing the putative initiator methioninceodon. Amino acid residues conserved in DdPTPa and other PTPases are shown in boldface. An asterisk indicates the conserved cysteine residue thought to be at the active site

DdPTPa DLar PTPlB TCPTP

Phenotype of a Dictyostelium Cell Line That Overexpresses

DISCUSSION