Analysis of pten complex assembly and identification of hnrnp c as a component of the pten-associated complex

PTEN is well characterized for antagonizing the PI3K pathway, and although PTEN’s catalytic domain is critical for its biological function, its phosphorylation status and the ability to form protein complexes have also been proposed to be important in regulating its phosphatasedependent and -independent functions. Previous studies using size exclusion chromatography demonstrated PTEN’s recruitment into high molecular weight complexes and hypothesized that PTEN’s phsophorylation 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 PTEN’s catalytic function and PDZ binding domain are not absolutely required for its complex formation. On the other hand, PTEN’s phosphorylation status has a significant impact on its complex assembly. While PTEN with dephosphorylated 4 key residues in its C-terminal tail behaves like wild type protein, the phospho-mimicking mutant significantly diminishes complex formation. Our results further demonstrate enrichment of 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 2D-DIGE followed by mass spectrometry analysis, we identified hnRNP C as a novel protein recruited to higher molecular weight 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.