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 th e membrane proximal domains (D1), with little to no activity associated wit h the membrane distal domains (D2). Two point mutations in the D2 domain of PTP alpha, which restore invariant amino acids found in the KNRY motif and WPD loop of all active D1 domains, synergistically confer D1-equivalent ki netic properties towards the phosphotyrosine analogue pNPP, and activate PT P alpha-D2 catalysed phosphopeptide hydrolysis (Lim et al., J. Biol. Chem. 273 (1998) 28986-28993; Buist et al., Biochemistry 38 (1999) 914-922). As a ll D2 domains lack these two D1-invariant amino acids, we have investigated whether other D2 domains are activated by such point mutations. Mutant PTP epsilon-D2, closely related to PTP alpha-D2 and belonging to a subgroup of D2 domains with minimal and conservative substitutions of D1-invariant ami no acids, exhibits synergistic activation towards pNPP but not towards a ph osphopeptide substrate. CD45-D2, belonging to another subgroup of D2 domain s with considerable substitutions in D1-invariant amino acids, is not activ ated by these mutations, even in the context of a third mutation which rest ores the minimal essential active site sequence C(X-5)R, indicating that ad ditional defects are sufficient to preclude catalysis. The ability of the K NRY and WPD replacements to activate PTP epsilon-D2 and PTP alpha-D2, but n ot CD45-D2, in conjunction with the extent and nature of their wild-type am ino acid substitutions, suggests that these D2 domains are representative o f two functionally distinct groups of D2 domain. (C) 1999 Elsevier Science B.V. All rights reserved.