Protein Interactions of Phosphatase and Tensin Homologue (PTEN) and Its Cancer-associated G20E Mutant Compared by Using Stable Isotope Labeling by Amino Acids in Cell Culture-based Parallel Affinity Purification

Abstract

The tumor suppressor PTEN (phosphatase and tensin homologue) negatively regulates the PI3K pathway through its lipid phosphatase activity and is one of the most commonly lost tumor suppressors in human cancers. Though the tumor suppressive function involves the lipid phosphatase-dependent and -independent activities of PTEN, the mechanism leading to the phosphatase-independent function of PTEN is understood poorly. Some PTEN mutants have lipid phosphatase activity but fail to suppress cell growth. Here, we use a cancer-associated mutant, G20E, to gain insight into the phosphatase-independent function of PTEN by investigating protein-protein interactions using MS-based stable isotope labeling by amino acids in cell culture (SILAC). A strategy named parallel affinity purification (PAP) and SILAC has been developed to prioritize interactors and to compare the interactions between wild-type and G20E PTEN. Clustering of the prioritized interactors acquired by the PAP-SILAC approach shows three distinct clusters: 1) wild-type-specific interactors, 2) interactors unique to the G20E mutant, and 3) proteins common to wild-type and mutant. These interactors are involved mainly in cell migration and apoptosis pathways. We further demonstrate that the wild-typespecific interactor, NUDTL16L1, is required for the regulatory function of wild-type PTEN in cell migration. These findings contribute to a better understanding of the mechanisms of the phosphatase-dependent and -independent functions of PTEN.PTEN is a tumor suppressor gene that frequently is somatically deleted or mutated in a variety of human cancers, including those of the brain, endometrium, prostate, and lung (1, 2). Germ line mutations of PTEN are the cause of Cowden disease and Bannayan-Riley-Ruvalcaba syndrome, an autosomal dominant hamartoma syndrome with increased risk for the development of tumors in a variety of tissues (1, 2). The PTEN protein consists of N-terminal phosphatase, and C-terminal C2, phosphorylation, and PDZ (PSD-95, DLG1, and ZO-1) binding domains (2). The catalytic domain of PTEN functions to dephosphorylate the 3Ј position of the phospholipids PI(3,4,5)P3 (PIP 3 ) and PI(3,4)P2 (3). As such, PTEN lipid phosphatase activity regulates the Akt serine/threonine kinase pathway through modulation of PIP 3 levels and hence, regulates cell cycle progression, apoptosis, and migration, cell proliferation and motility, which also are critical for tumor development (4). Thus, deregulation of the PI3K/PTEN/Akt pathway has been found in many malignant cancers.More than 20% of all known PTEN mutations in tumors are located outside of the catalytic site, and these mutants exhibit normal phosphatase activity (1), suggesting two possibilities. First, mutations in the interacting domain for the regulatory proteins of PTEN may affect its ability for tumor suppression through PTEN phosphatase activity, which is regulated by PTEN interactors (5-10). Indeed, a PTEN mutation within a binding site of PICT1, which controls stability of PTEN, affects the protein le...

IQGAPs choreograph cellular signaling from the membrane to the nucleus

“…By contrast, neither overexpression nor knockdown of IQGAP3 in HeLa and A549 lung adenocarcinoma cell lines, respectively, alters Akt activation [48]. Interestingly, phosphatase and tensin homolog (PTEN), which terminates Akt signaling by dephosphorylating PtdInsP 3 [49], co-immunoprecipitates with IQGAP1 [55]. Although the function of this interaction has not been identified, it could serve as an additional point of regulation of Akt.…”

IQGAP1 and its binding proteins control diverse biological functions

“…For example, a PTENG20E mutant has been reported in patients with endometrial carcinoma, and in vitro analyses confirmed that this mutation reduces phosphatase activity to ~20% of the activity of wild-type PTEN (53). IQGAP1 co-immunoprecipitates with Flag-tagged PTEN from U87MG human glioma cells and with endogenous PTEN from both HEK293T and LN229 cells (54). Interestingly, mass spectrometry analysis with stable isotope labelling by amino acids in cell culture (SILAC) yielded higher ratios of IQGAP1 with PTENG20E than with wild-type PTEN, implying that this mutant may have a higher propensity to interact with IQGAP1 (54).…”

“…IQGAP1 co-immunoprecipitates with Flag-tagged PTEN from U87MG human glioma cells and with endogenous PTEN from both HEK293T and LN229 cells (54). Interestingly, mass spectrometry analysis with stable isotope labelling by amino acids in cell culture (SILAC) yielded higher ratios of IQGAP1 with PTENG20E than with wild-type PTEN, implying that this mutant may have a higher propensity to interact with IQGAP1 (54). Further work is required to ascertain whether IQGAP1 binds PTEN directly and if the interaction has any effect on PTEN function.…”

“…In addition, the functions of several IQGAP1 binding interactions remain completely unknown (Table 1), implying that IQGAP1 may be involved in other biological processes. For example, analogous to the MAPK cascade, IQGAP1 associates with several components of the PI3K/Akt pathway (47, 51, 54, 98, 99). Scaffold proteins such as β-arrestin are known to have important roles in modulating PI3K/Akt activity (100), introducing the concept that IQGAP1 may be a PI3K/Akt regulator.…”

Mutant p53 interactome identifies nardilysin as a p53R273H‐specific binding partner that promotes invasion

“…To search for new mutant p53R273H-specific binding proteins, we undertook a comparative immunoprecipitation (IP) analysis using stable isotope labelling by amino acids in cell culture (SILAC) and mass spectrometry. SILAC has proved to be a powerful approach to distinguish adventitious from specific interactors, while retaining many weaker or transient binding partners [19][20][21][22][23][24][25]. Through this method we discovered several new mutant p53-specific binding proteins.…”