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.
Wishart
M J
MJ
Department of Physiology, University of Michigan, Ann Arbor 48109-0606, USA.
Denu
J M
JM
Williams
J A
JA
Dixon
J E
JE
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D002474:Cell-Free System; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D019000:Phosphotyrosine / Q000378:metabolism*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D017027:Protein Tyrosine Phosphatases / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014018:Tissue Distribution
D002352:Carrier Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D017027:Protein Tyrosine Phosphatases