Original article
PTP-PEST phosphatase variations in human cancer

https://doi.org/10.1016/j.cancergencyto.2006.05.013Get rights and content

Abstract

Signal transduction via tyrosine phosphorylation, normally fine-tuned by the concerted action of both protein tyrosine kinases and protein tyrosine phosphatases (PTPs), is a key mechanism in tumorigenesis. PTP-PEST, a ubiquitously expressed cytoplasmic tyrosine phosphatase, is thought to play an important role in cell adhesion and motility, and may be involved in metastasis. A search for sequence variations within the gene PTPN12 (alias PTP-PEST) was performed in breast cancer cell lines, leading to the identification of three amino acid substitutions at positions 322, 573, and 709. These alterations were also found in squamous cell carcinoma cell lines and could be verified in primary human breast and kidney tumor samples. Analysis of peripheral blood samples confirmed the germline origin of these alterations. Furthermore, functional characterization of the Ile322 and Ala573 PTP-PEST mutants revealed an enhancement of in vitro phosphatase activity, whereas the Lys709 variant showed reduced catalytic activity. These data demonstrate the existence of PTP-PEST variants that might be meaningful for human cancer and underscore the need for further characterizing PTP-PEST and its signaling pathways in context of this disease.

Introduction

Tyrosine phosphorylation, regulated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), is a key step in signal transduction and deregulation of their balanced action has often been implicated in the development of cancer. The protein tyrosine phosphatase PTP-PEST has been shown to be important for cellular migration and may therefore be involved in metastasis. PTP-PEST is a ubiquitously expressed protein that, together with PEP, PTP-HSCF, and BDP-1, forms a subfamily of cytoplasmic protein tyrosine phosphatases [1]. These phosphatases show high homology within their N-terminally located PTP domain and share a similar amino acid motif at the carboxy-terminus, termed CTH domain [2]. Additionally, the long C-terminal tail of PTP-PEST carries several signal transduction motifs, among them two classical proline-rich stretches and a novel nonclassical polyproline region. The proline-rich regions have been implicated in binding with p130Cas, as well as paxillin and Hic-5 [3], [4]. p130Cas thereby represents the major physiologic substrate of PTP-PEST [5], which makes this phosphatase particularly intriguing with respect to cell motility. In this context, two studies are of special interest. On the one hand, overexpression of PTP-PEST in Rat 1 fibroblasts has been shown to reduce tyrosine phosphorylation of p130Cas and to retard cell motility [6]. Conversely, PTPN12−/− fibroblasts exhibit elevated phosphorylation levels of p130Cas and, surprisingly, also reduced motility [7]. Other substrates include the Shc proteins, c-Abl, Grb2, Pyk2, PSTPIP, and FAK [8], [9], [10]. Because of and through its variety of interactions, the PTP-PEST phosphatase product encoded by the PTPN12 gene very likely exerts its effects on motility via multiple pathways.

So far, only a few PTP genes have been implicated in cancer; they are thought to act as tumor suppressors that may be inactivated by genetic alterations [11]. In a recent analysis of PTP genes, point mutations have been identified in cancer samples and were shown to reduce phosphatase activity, which supports the potential tumor suppressor function of protein tyrosine phosphatases [12]. The present study was designed to evaluate whether PTP-PEST is genetically altered in human cancer. To this end, we performed sequence analysis of the PTPN12 gene in 19 breast cancer cell lines. Fragments covering the whole coding region were amplified by polymerase chain reaction (PCR) and directly sequenced. We identified three amino acid substitutions in the C-terminal half of the phosphatase with different allele frequencies. These alterations were also found in a group of 14 squamous cell carcinoma cell lines and could be confirmed in tumor samples from patients with breast and kidney cancers. We demonstrated germline origin of these variations and, furthermore, detected a variant-specific impact on in vitro phosphatase activity of PTP-PEST. To our knowledge, this is the first report of PTPN12 alterations in tumor samples and on their functional impact on catalytic activity. The findings should further stimulate discussion on the potential role of the PTP-PEST phosphatase in cancer disease.

Section snippets

Patients and tumor samples

Samples of primary invasive breast carcinomas were collected from the archives of the Department of Pathology of the Technical University of Munich, Germany (Prof. H. Hoefler) and the Department of Oncology of the University of Chieti, Italy (Dr. S. Iacobelli). An additional group of primary mammary tumors and blood samples was collected from the Breast Cancer Department of the N. N. Petrov Institute of Oncology St. Petersburg, Russia (Prof. P. Knyazev). Kidney tissue materials were collected

Results and discussion

To screen for genetically based alterations of the PTP-PEST protein, we set out to analyze the entire coding region of this gene. Overlapping 900-bp fragments were amplified from cDNA of 19 breast cancer cell lines by PCR and directly sequenced in both directions. Comparison with GenBank sequence data resulted in the identification of three amino acid substitutions. In addition to the exchange of valine by isoleucine at amino acid position 322, we found threonine to be substituted by alanine at

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    Present address: Singapore OncoGenome Laboratory (SOG), Centre for Molecular Medicine, 61 Biopolis Drive, Proteos, Singapore 138673.

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