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Science 325 (5945): 1261-1265

Copyright © 2009 by the American Association for the Advancement of Science

Activation of the PI3K Pathway in Cancer Through Inhibition of PTEN by Exchange Factor P-REX2a

Barry Fine1, Cindy Hodakoski1, Susan Koujak1, Tao Su1,2, Lao H. Saal1, Matthew Maurer1,4, Benjamin Hopkins1, Megan Keniry1, Maria Luisa Sulis1,3, Sarah Mense1, Hanina Hibshoosh1,2, and Ramon Parsons1,2,4,*

1 Institute for Cancer Genetics and Herbert Irving Comprehensive Cancer Center, Columbia University, 1130 St. Nicholas Avenue, New York, NY 10032, USA.
2 Department of Pathology, Columbia University Medical Center, 630 West 168th Street, New York, NY 10032, USA.
3 Division of Pediatric Oncology, Columbia University Medical Center, 630 West 168th Street, New York, NY 10032, USA.
4 Department of Medicine, Columbia University Medical Center, 630 West 168th Street, New York, NY 10032, USA.

Figure 1
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Fig. 1. P-REX2a as a PTEN-binding protein. (A) Silver stain of 1 M salt elutions of affinity-purified PTEN-binding proteins. GST and GST-PTEN columns were incubated with (left and middle) and without (right) DBTRG-05MG cytoplasmic extract, and proteins eluted with high salt were separated and identified by mass spectrometry (9). P-REX2a and MVP are indicated with arrows. Peptide sequences of P-REX2a are shown. (B) Coimmunoprecipitation of endogenous PTEN and P-REX2a. Immunoprecipitations were performed (8), and proteins were detected by immunoblotting. (C) Box plot of P-REX2a expression in PTEN-positive and -negative breast tumors. Bars above and below represent the maximum and minimum expression, respectively. The box delineates the first to third quartiles of expression, and the central bar represents the median. P-REX2a levels are significantly associated with PTEN status (P = 0.027) by two-tailed t test.


Figure 2
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Fig. 2. Inhibition of PTEN phosphatase activity by P-REX2a. (A) Full-length P-REX2a, or a deletion of the DHPH domain ({Delta}DHPH), or the DHPH domain alone were added in the indicated molar volumes to 40 nM PTEN, purified from HEK293 cells, and phosphatase activity of PTEN was assayed with 20 µM di-C8-PIP3. This is a representative experiment; error bars indicate standard deviation (n = 3), **P < 0.005, *P < 0.05 by ANOVA. (B) P-REX2a (10 nM or 20 nM) was added to the reactions, and the phosphate released was measured over a range of di-C8-PIP3 by using 40 nM PTEN. Regression line (solid) to Michaelis-Menten kinetics is shown along with 95% CI (dotted lines). Vmax values are shown in the table along with standard deviation and 95% CI. A representative experiment is shown, and error bars represent standard deviation (n = 3). (C) Effect of P-REX2a and a GEF dead point mutant of P-REX2a, N212A, on phosphorylation of AKT in the presence and absence of PTEN. (D) Effect of expression of P-REX2a alone in MCF10A cells on abundance of p473AKT. The phosphorylation status of T308AKT was not detectable under normal growth conditions in MCF10A cells. (E) Effect of P-REX2a and DHPH on proliferation of MCF10A cells grown in reduced growth factors (0.1% serum). OD, optical density.


Figure 3
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Fig. 3. P-REX2a and PI3K mutants cooperate. (A) P-REX2a expression levels in PTEN wild-type breast tumors without PIK3CA–activating mutations (left) and with them (right) (P = 0.014; two-tailed t test). (B) Proliferation in the absence of all growth factors and serum in MCF10A cells expressing P-REX2a and PI3K[EK]. (C) Microscopy of branched multilobulated mammospheres in MCF10A cells expressing combinations of PI3K[EK], P-REX2a, or DHPH as indicated when grown in matrigel. (Inset) Amount of phospho-473-AKT (p473AKT) and total AKT (tAKT) are shown. All phase-contrast images are displayed at 100x magnification. (D) Colony formation by MCF10A cells grown in soft agar. Cells expressing P-REX2a and PI3K[EK] as indicated (**P < 0.01; t test).


Figure 4
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Fig. 4. Diminished phosphorylation of AKT and proliferation after depletion of P-REX2a. Phosphorylation of AKT (A) and proliferation (B) after depletion of P-REX2a in PTEN wild-type MCF7 cells or PTEN-null BT549 cells. OD595, absorbance at 595 nm. (C) Effect of P-REX2a depletion in MCF10A cells on pAKT and pGSK3β and amounts of the p21 and p27 cell cycle inhibitors. (D) X-gal staining of indicated cell lines. Phase-contrast images displayed at 40x magnification. (E) MCF10A proliferation after P-REX2a depletion.


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