Catalysis by the tumor-suppressor enzymes PTEN and PTEN-L.

Sean B Johnston,Ronald T Raines

doi:10.1371/journal.pone.0116898

Abstract

Phosphatase and tensin homologue deleted from chromosome ten (PTEN) is a lipid phosphatase tumor suppressor that is lost or inactivated in most human tumors. The enzyme catalyzes the hydrolysis of phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) to form phosphatidylinositol-(4,5)-bisphosphate (PIP2) and inorganic phosphate. Here, we report on the first continuous assay for the catalytic activity of PTEN. Using this assay, we demonstrate that human PTEN is activated by the reaction product PIP2, as well as in solutions of low salt concentration. This activation is abrogated in the K13A variant, which has a disruption in a putative binding site for PIP2. We also demonstrate that PTEN-L, which derives from alternative translation of the PTEN mRNA, is activated constitutively. These findings have implications for catalysis by PTEN in physiological environments and could expedite the development of PTEN-based chemotherapeutic agents.

Highlights

The PTEN gene located at human chromosome 10q23 encodes the tumor suppressor enzyme PTEN (EC 3.1.3.67) [1,2,3]
Enzyme purity was assessed with SDS–PAGE (Fig. 2A), and typical isolated yields of wild-type PTEN, K13A PTEN, and PTEN-L were 5, 5, and 1 mg per L of E. coli culture, respectively
When we purify PTEN by gel-filtration chromatography at 100 mM NaCl, we observe only one peak corresponding to monomeric PTEN. (Concentrations of PTEN in these purifications approach 50 μM.) As our assays are performed at enzyme concentrations of 250 nM, we believe that our data report on catalysis by monomeric PTEN, replicating the state in cellulo

Summary

Introduction

The PTEN gene located at human chromosome 10q23 encodes the tumor suppressor enzyme PTEN (EC 3.1.3.67) [1,2,3]. PTEN is thought to be mutated in 50–80% of sporadic human cancers [4]. In the 15 years since the discovery of its canonical substrate, phosphatidylinositol(3,4,5)-trisphosphate (PIP3) [5], PTEN has entered center-stage for cancer biologists. By catalyzing the dephosphorylation of PIP3 (Fig. 1A), PTEN antagonizes the phosphatidylinositol-3kinase (PI3K) Akt pathway, mediating cell proliferation and apoptosis. In addition to its phosphatase activity on PIP3, PTEN dephosphorylates protein substrates in the cytosol [6]. PTEN has roles elsewhere in the cell, in the nucleus [7], where it is thought to have effects on gene expression, genomic stability, and cell-cycle regulation [8]

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Conclusion