Cloning and Characterization of a Saccharomyces cerevisiae Gene Encoding the Low Molecular Weight Protein-tyrosine Phosphatase

Abstract

The low molecular weight protein-tyrosine phosphatase (low M(r) PTPase) is an 18-kDa cytoplasmic enzyme of unknown function that has been previously found in several vertebrates. Using an oligonucleotide probe derived from the active site sequence of the mammalian low M(r) PTPases, a Saccharomyces cerevisiae gene that encodes a homolog of this enzyme was cloned by low stringency hybridization. This gene, LTP1, together with a neighboring gene, TKL1, is shown to be located on the right arm of chromosome XVI. The deduced amino acid sequence of its 161-amino acid residue product shows a 39% average identity with that of the mammalian enzymes. The yeast Ltp1 protein was expressed in Escherichia coli, purified to homogeneity, and shown to possess PTPase activity. The recombinant Ltp1 efficiently hydrolyzes phosphotyrosine and a phosphotyrosine-containing peptide, Tyr531-fyn, but it shows low activity toward phosphoserine and phosphothreonine. The catalytic activity of Ltp1 toward a number of substrates was approximately 30-fold lower than the corresponding values measured for the bovine low M(r) PTPase. However, the yeast enzyme was markedly activated by adenine and some purine nucleosides and nucleotides, including cAMP and cGMP. In the case of adenine, the activity of Ltp1 was increased by approximately 30-fold. The high degree of evolutionary conservation of the low M(r) PTPases implies a significant role for this enzyme. However, neither the disruption of the LTP1 gene nor an approximately 10-fold overexpression of its product in S. cerevisiae caused any apparent phenotypic changes under the conditions tested. No proteins related to Ltp1 could be detected in extracts of the ltp1 null mutant, either by immunoblotting or by gel-filtration analysis accompanied by extended kinetic assays, consistent with the conclusion that LTP1 is the only low M(r) PTPase-encoding gene in S. cerevisiae.

In Saccharomyces cerevisiae an unbalanced level of tyrosine phosphorylation down-regulates the Ras/PKA pathway

“…Moreover, in keeping with previous reports (Ostanin et al, 1995), no effect was detected upon overexpression of Ltp1, the S. cerevisiae homologous of Stp1 (see Section 4).…”

“…Attempts to obtain further information on the physiological role of tyrosine phosphorylation by use of low molecular weight compounds, such as ortho-vanadate that proved to be effective in higher eukaryotes (reviewed in Walton & Dixon, 1993) or by single or multiple deletions of phosphatase-encoding genes (Sakumoto et al, 2002), have been unsatisfactory, because of excessive toxicity (Kanik-Ennulat, Montalvo, & Neff, 1995 and references therein) or lack of detectable phenotypic effects, respectively. Furthermore, overexpression of the S. cerevisiae Ltp1 low molecular weight tyrosine phosphatase did not elicit any significant phenotype (data not shown; Ostanin et al, 1995). On the contrary, overexpression of Stp1, the fission yeast counterpart of Ltp1, has been shown to deeply affect the yeast proteome, as monitored by 2D electrophoresis (Modesti et al, 2001).…”

“…Six of them encode proteins similar to dual-specificity protein phosphatases (acting on both phospho-Tyr and phospho-Thr (Russell, Moreno, & Reed, 1989)), and four Tyr-specific phosphatases: Ptp1, Ptp2, Ptp3 and Ltp1, all belong to the non-receptor PTPs sub-family. Ltp1 (Ostanin, Pokalsky, Wang, & Van Etten, 1995) and Stp1 (Mondesert, Moreno, & Russel, 1994) are the budding and fission yeast counterparts of the low molecular weight PTP present ubiquitously in mammalian tissues. All these proteins possess the active site motif (H/V) C (X) 5R(S/T) and share the same catalytic mechanism of classical PTPs (Cirri et al, 1993).…”

The in vivo tyrosine phosphorylation level of yeast immunophilin Fpr3 is influenced by the LMW-PTP Ltp1

“…This modification may have a consequence for the activity or localization of Fpr3. The LMW-PTP from the yeast S. cerevisiae, Ltp1, shows substrate specificity toward artificial substrates that is generally similar to that exhibited by the homologous mammalian enzymes [9]. However, it is even more activated by adenine, bringing its activity to a level comparable with those of its vertebrate counterparts [18].…”

“…Yeast serves as a simple, but often useful, model system for studying the biological function of many protein kinases and PTPs. Two LMW-PTP from yeast have been cloned, namely, Ltp1 from S. cerevisiae [9] and Stp1 from Schizosaccharomyces pombe [10]. The sequences of both enzymes are relatively similar to those of the mammalian LMW-PTP, with identity of 42% and 39% for the Stp1 and Ltp1, respectively.…”