A Single Mutation Converts a Novel Phosphotyrosine Binding Domain into a Dual-specificity Phosphatase

Abstract

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 Gly 120 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. Dual-specificity phosphatases (dsPTPase)1 hydrolyze phosphoserine/threonine/tyrosine-containing substrates in vitro and exhibit a substrate preference in vitro and in vivo for diphosphorylated (Thr(P)/Tyr(P)) mitogen-activated protein kinase (MAPK) homologues (reviewed in Ref. 1). Most dsPTPases are localized to the nucleus (2-4), and it has been suggested that they are responsible for the nuclear dephosphorylation and inactivation of MAPKs seen in vivo (5). All dsPTPases contain the sequence, HCXXGXXR(S/T), which has been shown to correspond to the active site of PTPases (6). The essential cysteine forms a thiophosphate intermediate during dsPTPasecatalyzed dephosphorylation (7,8). Several investigators have shown that substitution of this Cys, by Ser, in the dsPTPases, abolishes hydrolytic activity in vitro (7, 9 -12) and function in vivo (10,13,14). Interestingly, transient expression of the Cys to Ser mutant prolongs MAPK activation in vivo (12), suggesting that it may compete with native phosphatases for binding to phosphorylated MAPK. Moreover, the mutant dsPTPase co-immunoprecipitates with phosphorylated MAPK (10), indicating that the catalytic cysteine is not essential for substrate binding. This conclusion is also supported by PTPase crystallographic studies showing that Cys to Ser mutants bind sulfate (15) and tyrosine-phosphorylated peptide (16) in a manner identical with native enzymes. This paper describes the identification and isolation of a novel phosphoserine/threonine/tyrosine-binding protein (STYX) that is related in amino acid sequence to dsPTPases, but contains a naturally occurring Gly residue in...

The N‐terminal domains of tensin and auxilin are phosphatase homologues

“…We propose that they may possess phosphopeptide-binding functions. This view is consistent with two literature reports: Jia et al (1999, which shows that replacement of Cys 215 by serine in the PTPIB crystal structure eliminates phosphatase activity yet preserves phosphotyrosine binding, and secondly, Wishart et al (1995), which demonstrates that substitution of Gly for Cys in the phosphatase signature motif in STYX (a non-catalytic DSP homologue) transforms it from a phosphotyrosinebinding domain to an active phosphatase. Furthermore, it is notable that the non-polar side chains at positions 217 and 219, which contribute to the PTPlB phosphotyrosine-binding pocket and are invariant among eukaryotic PTPs, are substituted in auxilin, tensin, and the yeast putative PTP (to Asp, Gly, and Met, respectively, at 217, and Lys or Arg at 219).…”

Cellular prostatic acid phosphatase: a protein tyrosine phosphatase involved in androgen-independent proliferation of prostate cancer

“…The decreased expression of cPAcP is thus implicated as a crucial step of carcinogenesis and/or the progression of human prostate cancer towards androgen independence. Additionally, the polymorphisms in the PAcP gene, heterogeneity of the mRNA and the biochemical differences of the protein in prostate cancer versus normal prostate epithelial cells may lead to differences in substrate specificity and kinetics of the enzyme (Wishart et al 1995, Cirri et al 1996, which have led to the hypothesis of possible linkage of PAcP isoforms with prostate carcinogenesis. Future studies, however, are required to determine whether these differences could serve as a surrogate marker to distinguish between indolent and aggressive prostate cancers.…”

“…Composite c-HER-2 expression in prostate cancer cells www.endocrinology-journals.org differences between cPAcP in cancerous versus noncancerous prostate cells, which can lead to differences in specificity and kinetics of the enzyme as reported for other PTPs (Wishart et al 1995, Cirri et al 1996, may make a critical contribution to the development of prostate cancer. Secondly, among different prostate cancer cell lines, cPAcP level inversely correlates with tumorigenicity and growth rate, both of which are the cellular correlates of cancer and indispensable for tumor progression.…”

A role for MKP3 in axial patterning of the zebrafish embryo

“…Both the MAPK/ERK binding and the phosphatase domains are highly conserved with the other vertebrate homologs (Farooq et al, 2001). The phosphatase domain is located within amino acid residues 288-301 and contains the invariant phosphatase motif VXVHCXXGXSRSXT (Wishart et al, 1995). mkp3 expression and its regulation by FGF signaling Expression of mkp3 was not detected in the embryo prior to zygotic transcription, but appeared very soon after the midblastula transition (MBT) at the high stage (3.3 hpf) within a localized region that we identified as the prospective dorsal or pre-organizer region by comparison with the ventral gene vent/vega2 ( Fig.…”