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Open Access Highly Accessed Research article

Inhibition of receptor tyrosine kinase signalling by small molecule agonist of T-cell protein tyrosine phosphatase

Elina Mattila12, Heidi Marttila12, Niko Sahlberg1, Pekka Kohonen1, Siri Tähtinen3, Pasi Halonen1, Merja Perälä1 and Johanna Ivaska123*

Author Affiliations

1 VTT Technical Research Centre of Finland, Medical Biotechnology, Itainen Pitkakatu 4B, FIN-20520 Turku, Finland

2 Centre for Biotechnology, University of Turku, Tykistokatu 6, FIN-20520 Turku, Finland

3 Department of Biochemistry and Food Chemistry, University of Turku, Vatselankatu 2, FIN-20014 Turku, Finland

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BMC Cancer 2010, 10:7  doi:10.1186/1471-2407-10-7

Published: 7 January 2010

Abstract

Background

T-cell protein tyrosine phosphatase (TCPTP/TC45) is a ubiquitously expressed intra-cellular non-receptor protein tyrosine phosphatase involved in the negative regulation of several cancer relevant cellular signalling pathways. We have previously shown that interaction between the α-cytoplasmic tail of α1β1 integrin and TCPTP activates TCPTP by disrupting an inhibitory intra-molecular bond in TCPTP. Thus, inhibition of the regulatory interaction in TCPTP is a desirable strategy for TCPTP activation and attenuation of oncogenic RTK signalling. However, this is challenging with low molecular weight compounds.

Methods

We developed a high-throughput compatible assay to analyse activity of recombinant TCPTP in vitro. Using this assay we have screened 64280 small molecules to identify novel agonists for TCPTP. Dose-dependent response to TCPTP agonist was performed using the in vitro assay. Inhibition effects and specificity of TCPTP agonists were evaluated using TCPTP expressing and null mouse embryonic fibroblasts. Western blot analysis was used to evaluate attenuation of PDGFRβ and EGFR phosphorylation. Inhibition of VEGF signalling was analysed with VEGF-induced endothelial cell sprouting assays.

Results

From the screen we identified six TCPTP agonists. Two compounds competed with α1-cytoplasmic domain for binding to TCPTP, suggesting that they activate TCPTP similar to α1-cyt by disrupting the intra-molecular bond in TCPTP. Importantly, one of the compounds (spermidine) displayed specificity towards TCPTP in cells, since TCPTP -/- cells were 43-fold more resistant to the compound than TCPTP expressing cells. This compound attenuates PDGFRβ and VEGFR2 signalling in cells in a TCPTP-dependent manner and functions as a negative regulator of EGFR phosphorylation in cancer cells.

Conclusions

In this study we showed that small molecules mimicking TCPTP-α1 interaction can be used as TCPTP agonists. These data provide the first proof-of-concept description of the use of high-throughput screening to identify small molecule PTP activators that could function as RTK antagonists in cells.