p85 Associates with unphosphorylated PTEN and the PTEN-associated complex

Abstract

The lipid phosphatase PTEN functions as a tumor suppressor by dephosphorylating the D3 position of phosphoinositide-3,4,5-trisphosphate, thereby negatively regulating the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway. In mammalian cells, PTEN exists either as a monomer or as a part of a >600-kDa complex (the PTEN-associated complex [PAC]). Previous studies suggest that the antagonism of PI3K/AKT signaling by PTEN may be mediated by a nonphosphorylated form of the protein resident within the multiprotein complex. Here we show that PTEN associates with p85, the regulatory subunit of PI3K. Using newly generated antibodies, we demonstrate that this PTEN-p85 association involves the unphosphorylated form of PTEN engaged within the PAC and also includes the p110beta isoform of PI3K. The PTEN-p85 association is enhanced by trastuzumab treatment and linked to a decline in AKT phosphorylation in some ERBB2-amplified breast cancer cell lines. Together, these results suggest that integration of p85 into the PAC may provide a novel means of downregulating the PI3K/AKT pathway.

FIG. 1.

Coimmunoprecipitation of endogenous p85 and PTEN proteins. (A) WCEs from 293-T, ACHN, HeLa (PTEN-positive), and 786-0 (PTEN-negative) cell lines were immunoprecipitated with anti-PTEN (6H2.1) or anti-p85α (U13) antibodies and immunoblotted with independent anti-PTEN (C54) and anti-pan-p85 antibodies. (B) Endogenous p85α was coimmunoprecipitated with endogenous PTEN in 293-T cells using either polyclonal (C54) or monoclonal (11G8 and 6H2.1) anti-PTEN. Immunoblotting was performed with 6H2.1, C54 (anti-PTEN), or U13 (anti-p85α) antibody. IgG, immunoglobulin G. (C) Endogenous p85α and p85β coimmunoprecipitated with endogenous PTEN in MEFs derived from compound homo- and heterozygote p85α and p85β knockout mice. The 6H2.1 (PTEN) antibody was used for immunoprecipitation, and either C54 (PTEN) or polyclonal anti-pan-p85 antibody was used for immunoblotting. (D) Full-length GST-PTEN was incubated with lysates prepared from 293-T and 786-0 cells. p85α was detected by immunoblotting the U13 antibody (p85α). FT, flowthrough.

FIG. 2.

Generation of specific anti-unphosphorylated PTEN antibodies. Antisera recognizing unphosphorylated PTEN were generated using an epitope inclusive of S380 through S385 (top, underlined). The initial immunized sera (upper panel) and the resulting preabsorbed antisera (lower panel) were examined by immunoblotting for their ability to detect recombinant GST-PTEN. Numbers (left) are molecular masses in kilodaltons.

FIG. 3.

Validation of anti-unphosphorylated PTEN antibodies. Recombinant GST-PTEN (1 mg) was immunoblotted with the indicated dilutions of T382/383 (A) or T382 and T383 (B) antibodies, in comparison to the C54 antibody. Alternatively, recombinant GST-PTEN protein (0.1 ng to 1 μg) was separated by gel electrophoresis and immunoblotted with a fixed dilution (1:1,000) of T382/383 (C) or T382 and T383 (D) antibodies, in comparison to the C54 antibody. To evaluate specificity for unphosphorylated PTEN, different quantities of purified GST-PTEN were immunoprecipitated with C54 (E), T382 (F), T383 (G), and anti-T382/383 (H) antibodies, followed by immunoblotting with anti-PTEN (6H2.1) antibodies. Numbers at left of panels A, B, and E to H are molecular masses in kilodaltons.

FIG. 4.

Specificity of anti-unphosphorylated PTEN antibodies. (A) Endogenous PTEN was immunoprecipitated from HeLa (PTEN-positive) cell lysates using anti-PTEN (6H2.1) antibody and immunoblotted with antibodies recognizing unphosphorylated PTEN (T382/383), phosphorylated PTEN (p380), or total PTEN (C54). 786-0 (PTEN-null) cell lysates were used as a negative control. Numbers at left are molecular masses in kilodaltons. (B) pSGL-PTEN was translated in either rabbit reticulocyte or wheat germ in vitro transcription-translation lysate systems in the presence of radioactively ³⁵S-labeled methionine to generate phosphorylated and unphosphorylated PTEN, respectively. Labeled, translated extracts were immunoprecipitated using C54, S380, T382, T383, and S385 antibodies. Bound proteins were separated by gel electrophoresis, and ³⁵S labeling was detected by autoradiography.

FIG. 5.

p85 comigrates with a high-molecular-weight PAC. (A and B) WCEs (A) or cytosolic fractions (B) from 293-T cells were separated by gel filtration. Eluted fractions were separated by electrophoresis and immunoblotted for unphosphorylated PTEN (T382/383), total PTEN (C54), and p85 (anti-p85α [U13] and anti-pan-p85). WCEs were also immunoblotted for p110α; cytosolic extracts were immunoblotted for both p110α and p110β. (C) Gel filtration fractions containing monomeric (“control”) or high-molecular-weight (“complex”) PTEN were pooled, immunoprecipitated with anti-PTEN (6H2.1), and immunoblotted with anti-PTEN (C54) and anti-pan-p85 antibodies.

FIG. 6.

p110β coimmunoprecipitates with PTEN and p85. (A) WCEs from 786-0 (PTEN-negative) and HeLa and 293-T (PTEN-positive) cell lines were immunoprecipitated with anti-PTEN (6H2.1 or C54) and immunoblotted with anti-PTEN (C54), anti-pan-p85, or anti-p11β antibodies. (B) WCEs from 786-0 and 293-T cell lines were immunoprecipitated with anti-PTEN (6H2.1) under more stringent washing conditions (see Materials and Methods) and immunoblotted for PTEN (C54), p85α, or p110β.

FIG. 7.

p85α interacts with unphosphorylated PTEN. WCEs (200 mg) from 293-T cells were loaded over a T382/383 antibody column (see Materials and Methods). Eluted fractions were separated by gel electrophoresis and immunoblotted for p110β, p85α (U13), and PTEN (C54), respectively.

FIG. 8.

Trastuzumab enhances the association between p85α and PTEN. BT474, SKBR3 (ERBB2-amplified), MDA-MB-231, and MCF-7 (ERBB2-wt) cells were treated with trastuzumab for 40, 60, and 120 min and 24 h. WCEs were then immunoprecipitated with anti-PTEN (6H2.1) antibodies and immunoblotted for total PTEN (C54), p85 protein (U13), p110α, and p110β. AKT phosphorylation and steady-state levels of the aforementioned proteins were monitored in WCEs from these experiments using a rabbit monoclonal antibody recognizing phosphorylation at Ser473 (pAKT S473).