The Phosphatase PHLPP Controls the Cellular Levels of Protein Kinase C

Abstract

The life cycle of protein kinase C (PKC) is controlled by multiple phosphorylation and dephosphorylation steps. The maturation of PKC requires three ordered phosphorylations, one at the activation loop and two at COOH-terminal sites, the turn motif and the hydrophobic motif, to yield a stable and signalingcompetent enzyme. Dephosphorylation of the enzyme leads to protein degradation. We have recently discovered a novel family of protein phosphatases named PH domain leucine-rich repeat protein phosphatase (PHLPP) whose members terminate Akt signaling by dephosphorylating the hydrophobic motif on Akt. Here we show that the two PHLPP isoforms, PHLPP1 and PHLPP2, also dephosphorylate the hydrophobic motif on PKC ␤II, an event that shunts PKC to the detergent-insoluble fraction, effectively terminating its life cycle. Deletion mutagenesis reveals that the PH domain is necessary for the effective dephosphorylation of PKC ␤II by PHLPP in cells, whereas the PDZbinding motif, required for Akt regulation, is dispensable. The phorbol ester-mediated dephosphorylation of the hydrophobic site, but not the turn motif or activation loop, is insensitive to okadaic acid, consistent with PHLPP, a PP2C family member, controlling the hydrophobic site. In addition, knockdown of PHLPP expression reduces the rate of phorbol ester-triggered dephosphorylation of the hydrophobic motif, but not turn motif, of PKC ␣. Last, we show that depletion of PHLPP in colon cancer and normal breast epithelial cells results in an increase in conventional and novel PKC levels. These data reveal that PHLPP controls the cellular levels of PKC by specifically dephosphorylating the hydrophobic motif, thus destabilizing the enzyme and promoting its degradation.Protein phosphorylation defines one of the most important and pervasive regulatory mechanisms in cell signaling. Crucial cellular decisions such as those between death or survival and proliferation or differentiation are made depending on the phosphorylation state of signaling molecules. Thus, precise control of the balance between phosphorylation and dephosphorylation is critical for living organisms to maintain normal physiological functions. Dysregulation of signaling pathways that results in disturbing this balance leads to the development of diseases such as cancer and diabetes. Loss of control of either phosphorylation or dephosphorylation mechanisms leads to pathogenic states, and both kinases and phosphatases have been identified as oncogenes or tumor suppressors (1).We have recently identified a novel family of Ser/Thr phosphatases, which we named PH domain leucine-rich repeat protein phosphatase (PHLPP) 3 based on domain composition (2, 3). PHLPP comprises three isozymes: the alternatively spliced PHLPP1␣ and PHLPP1␤, which differ in an amino-terminal extension on PHLPP1␤, and PHLPP2. PHLPP1 and PHLPP2 share 50% overall identity at the amino acid level. PHLPP1 and PHLPP2 contain a PP2C-like phosphatase domain and they dephosphorylate Akt in vitro in a Mn 2ϩ -dependent manner (2, 3). Althoug...

Pleckstrin homology domain leucine-rich repeat protein phosphatases set the amplitude of receptor tyrosine kinase output

“…1A function of PHLPP, we confirmed that lack of PHLPP1 resulted in increased steady-state levels of PKCα (PKCα panel; see ref. 38, which shows that phosphorylation increases the stability of PKC isozymes); reintroduction of PHLPP1β decreased the steady-state levels of PKCα in a manner that depended on the catalytic activity and presence of the PH domain (which is required for PHLPP to recognize PKC in cells) but not in the presence of the PDZ ligand (which is not required for PHLPP to recognize PKC in cells) (38). These data reveal that PHLPP1 suppresses the steady-state levels of the EGFR by a mechanism depending on its catalytic activity and an intact LRR segment.…”

“…Recent studies have established that PHLPP1 and PHLPP2 suppress oncogenic signaling by at least two mechanisms (reviewed in ref. 37): (i) direct dephosphorylation and inactivation of the prosurvival kinase AKT, PKC (38), and S6 kinase (39), and (ii) direct dephosphorylation and activation of the proapoptotic kinase Mst1 (40). Whether additional mechanisms account for the tumor-suppressive function of PHLPP is largely unexplored.…”

Regulation of Protein Kinase C function by phosphorylation on conserved and non-conserved sites

“…Dephosphorylation of PKCs in cells was initially shown to be sensitive to okadaic acid, which implicated the PP2a-like phosphatases in this process [79,80,75]. More recent studies have identified an additional family of okadaic acidinsensitive phosphatases called PHLPPs that specifically dephosphorylate the HM (but not the TM) of PKCs upon prolonged phorbol ester treatment [81]. It should be noted however that while dephosphorylation serves as an important switch to turn off the activity of these enzymes, dephosphorylation does not necessarily predispose the enzyme to degradation [79].…”

Reduced expression of the tumor suppressor PHLPP1 enhances the antiapoptotic B-cell receptor signal in chronic lymphocytic leukemia B-cells

“…20 In addition, both PHLPP1 and PHLPP2 have been shown to dephosphorylate PKCa and PKCbII, whereas PHLPP1 has also been implicated in the negative regulation of the Ras/Raf/MEK/ERK pathway. 22,23 Recently, several lines of evidence have suggested that PHLPP1 and PHLPP2 are involved in the pathogenesis of various types of cancer. Reduced or absent expression of PHLPP1 and PHLPP2 has been reported in approximately 80% of primary colorectal cancer specimen.…”