Catalytic activation of the membrane distal domain of protein tyrosine phosphatase epsilon, but not CD45, by two point mutations.
Most, if not all, of the catalytic activity of the tandem catalytic domain-containing receptor-like protein tyrosine phosphatases (PTPs) resides in the membrane proximal domains (D1), with little to no activity associated with the membrane distal domains (D2). Two point mutations in the D2 domain of PTPalpha, which restore invariant amino acids found in the KNRY motif and WPD loop of all active D1 domains, synergistically confer D1-equivalent kinetic properties towards the phosphotyrosine analogue pNPP, and activate PTPalpha-D2 catalysed phosphopeptide hydrolysis (Lim et al., J. Biol. Chem. 273 (1998) 28986-28993; Buist et al., Biochemistry 38 (1999) 914-922). As all D2 domains lack these two D1-invariant amino acids, we have investigated whether other D2 domains are activated by such point mutations. Mutant PTPepsilon-D2, closely related to PTPalpha-D2 and belonging to a subgroup of D2 domains with minimal and conservative substitutions of D1-invariant amino acids, exhibits synergistic activation towards pNPP but not towards a phosphopeptide substrate. CD45-D2, belonging to another subgroup of D2 domains with considerable substitutions in D1-invariant amino acids, is not activated by these mutations, even in the context of a third mutation which restores the minimal essential active site sequence C(X(5))R, indicating that additional defects are sufficient to preclude catalysis. The ability of the KNRY and WPD replacements to activate PTPepsilon-D2 and PTPalpha-D2, but not CD45-D2, in conjunction with the extent and nature of their wild-type amino acid substitutions, suggests that these D2 domains are representative of two functionally distinct groups of D2 domain.
Lim
K L
KL
Cell Regulation Laboratory, Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore, Singapore.
Ng
C H
CH
Pallen
C J
CJ
D000595:Amino Acid Sequence; D000814:Aniline Compounds / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D004789:Enzyme Activation; D015870:Gene Expression; D017493:Leukocyte Common Antigens / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009943:Organophosphorus Compounds / Q000737:chemistry; D010766:Phosphorylation; D010957:Plasmids; D017354:Point Mutation; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000235:genetics*; D016415:Sequence Alignment; D014443:Tyrosine / Q000737:chemistry
D000814:Aniline Compounds; D009943:Organophosphorus Compounds; D001224:Aspartic Acid; D014443:Tyrosine; C068798:4-aminophenylphosphate; D017493:Leukocyte Common Antigens; D017027:Protein Tyrosine Phosphatases