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. 2009 Oct 30;284(44):30159-66.
doi: 10.1074/jbc.M109.027995. Epub 2009 Sep 9.

Analysis of PTEN complex assembly and identification of heterogeneous nuclear ribonucleoprotein C as a component of the PTEN-associated complex

Sherly Mosessian  1 , Nuraly K Avliyakulov, David J Mulholland, Pinmanee Boontheung, Joseph A Loo, Hong Wu
Affiliations

Analysis of PTEN complex assembly and identification of heterogeneous nuclear ribonucleoprotein C as a component of the PTEN-associated complex

Sherly Mosessian et al. J Biol Chem. .

Abstract

PTEN (phosphatase and tensin homolog deleted on chromosome 10) is well characterized for its role in antagonizing the phosphoinositide 3-kinase pathway. Previous studies using size-exclusion chromatography demonstrated PTEN recruitment into high molecular mass complexes and hypothesized that PTEN phosphorylation status and PDZ binding domain may be required for such complex formation. In this study, we set out to test the structural requirements for PTEN complex assembly and identify the component(s) of the PTEN complex(es). Our results demonstrated that the PTEN catalytic function and PDZ binding domain are not absolutely required for its complex formation. On the other hand, PTEN phosphorylation status has a significant impact on its complex assembly. Our results further demonstrate enrichment of the PTEN complex in nuclear lysates, suggesting a mechanism through which PTEN phosphorylation may regulate its complex assembly. These results prompted further characterization of other protein components within the PTEN complex(es). Using size-exclusion chromatography and two-dimensional difference gel electrophoresis followed by mass spectrometry analysis, we identified heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a novel protein recruited to higher molecular mass fractions in the presence of PTEN. Further analysis indicates that endogenous hnRNP C and PTEN interact and co-localize within the nucleus, suggesting a potential role for PTEN, alongside hnRNP C, in RNA regulation.

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Figures

FIGURE 1.
FIGURE 1.
PTEN catalytic activity and PDZ binding domain are not absolutely required for complex assembly. A, PC3 WT- and CS (inactive)-PTEN-inducible cells were induced with Dox and run through a gel filtration column. Fractions were collected and immunoblotted with anti-PTEN antibody. Higher molecular mass and monomeric fractions are depicted as II and I, respectively. B, PC3 WT- and CS-PTEN-inducible cells were treated with 2 μg/ml Dox for 36 h, and cell lysates were immunoblotted (IB) with antibodies against PTEN, V5 tag and the PTEN downstream target, P-AKT, and total AKT, using anti-actin as a loading control. C, PC3 cells were transfected with WT- or ΔPDZ-PTEN (PTEN lacking the PDZ binding domain) and run on a gel filtration column. Complex (19–21) and monomer (33–36) fractions were collected and immunoblotted with PTEN antibody. D, complex fractions (19–21) from PC3 WT-PTEN-induced (+Dox) and uninduced (−Dox) were immunoblotted with an antibody against hDLG.
FIGURE 2.
FIGURE 2.
Dephosphorylation of PTEN on Ser380, Thr382, Thr383, and Ser385 is critical for complex assembly. A, PC3 WT-PTEN-induced cells were run through a gel filtration column, and fractions were collected and immunoblotted (IB) with anti-phospho-PTEN (P380) and total PTEN antibodies. B, relative proportion of P380PTEN to unphosphorylated PTEN (calculated by subtracting P380PTEN from total PTEN) was quantified from A for each fraction by densitometry analysis. C, PC3 cells were transfected with E4 (4 Ser/Thr amino acids being mutated to glutamic acid) and A4 (4 Ser/Thr amino acids being mutated to alanine) PTEN mutant constructs. After gel filtration, complex (19–22) and monomer (34–37) fractions were collected and immunoblotted with PTEN antibody.
FIGURE 3.
FIGURE 3.
PTEN complex is enriched in the nucleus. A, WT-PTEN and NLS-PTEN were transfected into PC3 cells. Cell lysates were applied to a gel filtration column. Fractions were collected and subjected to immunoblotting (IB) with anti-PTEN antibody. The percentage of PTEN in the complex fractions was calculated by dividing the amount of PTEN in the high molecular mass fractions by total PTEN (complex + monomer). The amount of PTEN in these fractions was determined through densitometry analysis. B, NLS-PTEN was transfected into PC3 cells, the nuclear and cytoplasmic fractions were prepared and run through a gel filtration column, and fractions were collected and immunoblotted with anti-PTEN antibody. C, percentage of NLS-PTEN in the complex fractions was calculated for nuclear (Nuc) and cytosolic (Cyt) extracts as described in A and depicted in a bar graph. D, nuclear and cytosolic fractions (column input) were immunoblotted with anti-PTEN and anti-HDAC (nuclear marker) antibodies.
FIGURE 4.
FIGURE 4.
Schematic outline of isolation and two-dimensional DIGE analysis of the PTEN-associated protein complex. A, proteins from PC3 WT-PTEN-inducible cells under induced (+Dox) and uninduced (−Dox) conditions were extracted and separated using the same parameters through a gel filtration Sephacryl S-300 column. PTEN-associated complex fractions 19 and 20 in the presence of Dox (PTEN-induced) and in the absence of Dox (PTEN uninduced) were combined and labeled with Cy5 and Cy3, respectively, whereas the pool of these two samples was labeled with Cy2. Protein samples were combined, and isoelectric focusing was performed on pH 3–11 nonlinear strips and then separated on 12–16% SDS gradient gels. False-colored Cy5 (red)-labeled PTEN-induced and Cy3 (green)-labeled PTEN uninduced representative gel images are shown. B, three-dimensional profiles and the graphical view of fold changes are shown for hnRNP C protein under PTEN-induced and uninduced conditions.
FIGURE 5.
FIGURE 5.
HnRNP C is a candidate component of the PTEN-associated complex. A, PC3 WT-PTEN-inducible cells were treated with Dox and run through a gel filtration column. Fractions were collected and immunoblotted (IB) with hnRNP C and PTEN antibodies. B, fractions 19 and 20 from the PC3 WT-PTEN-inducible cells in the presence and absence of Dox were immunoblotted with PTEN and hnRNP C. The histogram on the right represents the quantitative densitometry analysis of bands shown on the left. C, fractions 17–26 from PC3 WT-PTEN-inducible cells under induced and uninduced conditions were run on SDS-polyacrylamide gels and immunoblotted for hnRNP C and GRP78 control. D, whole lysates from PC3 WT-PTEN-inducible cells in the presence and absence of Dox were analyzed with the indicated antibodies.
FIGURE 6.
FIGURE 6.
Physical interaction of PTEN and HnRNP C. A, PC3 WT-PTEN-inducible cells treated with Dox were immunoprecipitated (IP) using PTEN and IgG control antibodies and immunoblotted (IB) with PTEN and hnRNP C. B, HeLa cells under control conditions or treated with 1 mm H2O2 for 1 h were immunoprecipitated with PTEN and IgG antibodies and immunoblotted with anti-PTEN and -hnRNP C antibodies. Bottom, quantitation of hnRNP C immunoprecipitated with PTEN antibody in the presence and absence of H2O2, (normalized to IgG controls). C, cytoplasmic (Cyt) and nuclear (Nuc) fractions from HeLa cells untreated and treated with 1 mm H2O2 for 1 h were immunoblotted with the indicated antibodies. D, cytoplasmic and nuclear fractions from HeLa cells were immunoprecipitated using PTEN and IgG control antibodies and immunoblotted with PTEN and hnRNP C antibodies. The nuclear and cytoplasmic inputs were immunoblotted with anti-PTEN, anti-hnRNP C, and anti-HDAC antibodies. E, PTEN and hnRNP C co-localize in the nucleus. PC3 WT-PTEN-inducible cells with and without Dox were co-stained with anti-PTEN and -hnRNP C antibodies and counterstained with DAPI.
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