A single mutation converts a novel phosphotyrosine binding domain into a dual-specificity phosphatase

Dual-specificity protein-tyrosine phosphatases (dsPTPases) have been implicated in the inactivation of mitogen-activated protein kinases (MAPKs). We have identified a novel phosphoserine/threonine/tyrosine-binding protein (STYX) that is related in amino acid sequence to dsPTPases, except for the substitution of Gly for Cys in the conserved dsPTPase catalytic loop (HCXXGXXR(S/T)). cDNA subcloning and Northern blot analysis in mouse shows poly(A+) hybridization bands of 4.6, 2.4, 1.5, and 1.2 kilobases, with highest abundance in skeletal muscle, testis, and heart. Polymerase chain reaction amplification of reverse-transcribed poly(A+) RNA revealed an alternatively spliced form of STYX containing a unique carboxyl terminus. Bacterially expressed STYX is incapable of hydrolyzing Tyr(P)-containing substrates; however, mutation of Gly120 to Cys (G120C), which structurally mimics the active site of dsPTPases, confers phosphatase activity to this molecule. STYX-G120C mutant hydrolyzes p-nitrophenyl phosphate and dephosphorylates both Tyr(P) and Thr(P) residues of peptide sequences of MAPK homologues. The kinetic parameters of dephosphorylation are similar to human dsPTPase, Vaccinia H1-related, including inhibition by vanadate. We believe this is the first example of a naturally occurring "dominant negative" phosphotyrosine/serine/threonine-binding protein which is structurally related to dsPTPases.