A SINGLE MUTATION CONVERTS A NOVEL PHOSPHOTYROSINE BINDING DOMAIN INTO A DUAL-SPECIFICITY PHOSPHATASE

Dual-specificity protein-tyrosine phosphatases (dsPTPases) have been i mplicated in the inactivation of mitogen-activated protein kinases (MA PKs), We have identified a novel. phosphoserine/thhreonine/tyrosine-bi nding protein (STYX) that is related in amino acid sequence to dsPTPas es, except for the substitution of Gly for Cys in the conserved dsPTPa se catalytic loop (HCXYGXXR(S/T)), cDNA subcloning and Northern blot a nalysis 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-transc ribed poly(A(+)) RNA revealed an alternatively spliced form of STYX co ntaining a unique carboxyl terminus, Bacterially expressed STYX. is in capable of hydrolyzing Tyr(P)-containing substrates; however, mutation of Gly(120) to Cys (G120C), which structurally mimics the active site of dsPTPases, confers phosphatase activity to this molecule, STYX-G12 0C mutant hydrolyzes p-nitrophenyl phosphate and dephosphorylates both Tyr(P) and Thr(P) residues of peptide sequences of MAPK homologues. T he kinetic parameters of dephosphorylation are similar to human dsPTPa se, 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 structura lly related to dsPTPases.