Substitution of two variant residues in the protein tyrosine phosphatase-like PTP35/IA-2 sequence reconstitutes catalytic activity.
The PTP35/IA-2 protein shows high homology to protein tyrosine phosphatases (PTPases) but harbours a few changes in invariant PTPase residues. Accordingly, PTP35/IA-2 has been reported to lack catalytic activity in vitro, and its in vivo biological function remains to be determined. We investigated if reversion of selected amino acids to the PTPase consensus could reconstitute enzymatic activity. Substitution of aspartic acid 911 in the putative active site with alanine resulted in the appearance of low but reproducible activity on pNPP dephosphorylation. Moreover, contemporary replacement of alanine 877 with aspartic acid greatly increased the catalytic efficiency of the D911A mutant. The A877D/D911A double mutant protein was also found to specifically dephosphorylate myelin basic protein phosphorylated on tyrosine. These results suggest that the general scaffold of the PTP35 protein is compatible with a common catalytic mechanism shared by PTPases and argue against an intrinsic enzymatic function of the wild type form.
Magistrelli
G
G
Molecular Biology Department, Pharmacia and Upjohn, Nerviano, Milan, Italy.
Toma
S
S
Isacchi
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001324:Autoantigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D016384:Consensus Sequence; D017931:DNA Primers; D014644:Genetic Variation; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D004676:Myelin Basic Protein / Q000378:metabolism; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry*; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D001324:Autoantigens; D017931:DNA Primers; D008565:Membrane Proteins; D004676:Myelin Basic Protein; D020794:Receptor Protein-Tyrosine Kinases; D017027:Protein Tyrosine Phosphatases; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8