Catalytic activation of the membrane distal domain of protein tyrosine phosphatase ϵ, but not CD45, by two point mutations

Abstract

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 PTPα, 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 PTPα-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 PTPϵ-D2, closely related to PTPα-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 PTPϵ-D2 and PTPα-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.