Purification and Characterization of the Low Molecular Weight Protein Tyrosine Phosphatase, Stpl, from the Fission Yeast Schizosaccharomyces pombe

Zhong Yin Zhang, Gaochao Zhou, John M. Denu, L. Wu, Xuejun Tang, Odile Mondesert, Paul Russell, Elizabeth Butch, Kun Liang Guan

Research output: Contribution to journalArticle

33 Scopus citations

Abstract

Genetic screening in fission yeast has identified a gene named stpl+ that rescues cdc25-22 [Mondesert et al. (1994) J. Biol. Chem. 269, 27996-27999]. This gene encodes a 17.4 kDa protein that is 42% identical to members of the low molecular weight protein tyrosine phosphatases (low Mr PTPases) previously known to exist only in mammalian species. A simple and efficient purification procedure was developed to obtain the homogeneous recombinant yeast low Mr PTPase, Stpl, in large quantities suitable for kinetic and structural studies. Authentic Stpl was produced as judged by amino terminal protein sequencing and electrospray ionization mass spectrometry analyses. Stpl was shown to possess intrinsic phosphatase activity toward both aryl phosphates (such as phosphotyrosine) and alkyl phosphates (such as phosphoserine). Stpl also dephosphorylated phosphotyrosyl peptide/protein substrates. The yeast enzyme was 6-fold slower than the mammalian enzymes, which made it amenable to pre-steady-state stopped-flow spectroscopic kinetic analysis at 30 °C and pH 6.0. Burst kinetics was observed with Stpl using p-nitrophenyl phosphate as a substrate, suggesting that the rate-limiting step corresponds to the decomposition of the phosphoenzyme intermediate. Interestingly, the bovine heart low Mr PTPase was capable of removing phosphate groups from both phosphotyrosyl and phosphoseryl/threonyl protein substrates with comparable efficiencies. The low Mr PTPases, like the Cdc25 family of phosphatases, may represent a new group of dual specificity phosphatases which may be involved in cell cycle control.

Original languageEnglish (US)
Pages (from-to)10560-10568
Number of pages9
JournalBiochemistry
Volume34
Issue number33
DOIs
StatePublished - Aug 1995

ASJC Scopus subject areas

  • Biochemistry

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