[Show abstract][Hide abstract] ABSTRACT: Growth factor receptor levels are aberrantly high in diverse cancers, driving the proliferation and survival of tumor cells. Understanding the molecular basis for this aberrant elevation has profound clinical implications. Here we show that the pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) suppresses receptor tyrosine kinase (RTK) signaling output by a previously unidentified epigenetic mechanism unrelated to its previously described function as the hydrophobic motif phosphatase for the protein kinase AKT, protein kinase C, and S6 kinase. Specifically, we show that nuclear-localized PHLPP suppresses histone phosphorylation and acetylation, in turn suppressing the transcription of diverse growth factor receptors, including the EGF receptor. These data uncover a much broader role for PHLPP in regulation of growth factor signaling beyond its direct inactivation of AKT: By suppressing RTK levels, PHLPP dampens the downstream signaling output of two major oncogenic pathways, the PI3 kinase/AKT and the Rat sarcoma (RAS)/ERK pathways. Our data are consistent with a model in which PHLPP modifies the histone code to control the transcription of RTKs.
Proceedings of the National Academy of Sciences 09/2014; 111(38). DOI:10.1073/pnas.1404221111 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The pro-survival kinase Akt requires phosphorylation at two conserved residues, the activation loop site (Thr-308) and the hydrophobic motif site (Ser-473), for maximal activation. Previous reports indicate that mTORC2 is necessary for phosphorylation of the hydrophobic motif and that this site is not phosphorylated in cells lacking components of the mTORC2 complex, such as Sin1. Here we show that Akt can be phosphorylated at the hydrophobic motif site (Ser-473) in the absence of mTORC2. First, increasing the levels of PIP(3) in Sin1(-/-) MEFs by (i) expression of a constitutively active PI3K or (ii) relief of a negative feedback loop on PI3K by prolonged inhibition of mTORC1 or S6K is sufficient to rescue hydrophobic motif phosphorylation of Akt. The resulting accumulation of PIP(3) at the plasma membrane results in Ser-473 phosphorylation. Second, constructs of Akt in which the PH domain is constitutively disengaged from the kinase domain are phosphorylated at the hydrophobic motif site in Sin1(-/-) MEFs; both myristoylated-Akt and Akt lacking the PH domain are phosphorylated at Ser-473. Thus, disruption of the interface between the PH and kinase domains of Akt bypasses the requirement for mTORC2. In summary, these data support a model in which Akt can be phosphorylated at Ser-473 and activated in the absence of mTORC2 by mechanisms that depend on removal of the PH domain from the kinase domain.
[Show abstract][Hide abstract] ABSTRACT: The PH domain leucine-rich repeat protein phosphatase, PHLPP, plays a central role in controlling the amplitude of growth factor signaling by directly dephosphorylating and thereby inactivating Akt. The cellular levels of PHLPP1 have recently been shown to be enhanced by its substrate, activated Akt, via modulation of a phosphodegron recognized by the E3 ligase β-TrCP1, thus providing a negative feedback loop to tightly control cellular Akt output. Here we show that this feedback loop is lost in aggressive glioblastoma but not less aggressive astrocytoma. Overexpression and pharmacological studies reveal that loss of the feedback loop does not result from a defect in PHLPP1 protein or in the upstream kinases that control its phosphodegron. Rather, the defect arises from altered localization of β-TrCP1; in astrocytoma cell lines and in normal brain tissue the E3 ligase is predominantly cytoplasmic, whereas in glioblastoma cell lines and patient-derived tumor neurospheres, the E3 ligase is confined to the nucleus and thus spatially separated from PHLPP1, which is cytoplasmic. Restoring the localization of β-TrCP1 to the cytosol of glioblastoma cells rescues the ability of Akt to regulate PHLPP1 stability. Additionally, we show that the degradation of another β-TrCP1 substrate, β-catenin, is impaired and accumulates in the cytosol of glioblastoma cell lines. Our findings reveal that the cellular localization of β-TrCP1 is altered in glioblastoma, resulting in dysregulation of PHLPP1 and other substrates such as β-catenin.