Biochemical characterization of the phosphatase domain of the tumor suppressor PH domain leucine-rich repeat protein phosphatase

Abstract

PH domain leucine-rich repeat protein phosphatase (PHLPP) directly dephosphorylates and inactivates Akt and protein kinase C and is therefore a prime target for pharmacological intervention of two key signaling pathways, the phosphatidylinositol 3-kinase and diacylglycerol signaling pathways. Here we report on the first biochemical characterization of the phosphatase domain of a PHLPP family member. The human PHLPP1 and PHLPP2 phosphatase domains were expressed and purified from bacteria or insect cells and their activities compared to that of full-length proteins immunoprecipitated from mammalian cells. Biochemical analyses reveal that the PHLPP phosphatase domain effectively dephosphorylates synthetic and peptidic substrates, that its activity is modulated by metals and lipophilic compounds, and that it has relatively high thermal stability. Mutational analysis of PHLPP2 reveals an unusual active site architecture compared to the canonical architecture of PP2C phosphatases and identifies key acidic residues (Asp 806, Glu 989, and Asp 1024) and bulky aromatic residues (Phe 783 and Phe 808) whose mutation impairs activity. Consistent with a unique active site architecture, we identify inhibitors that discriminate between PHLPP2 and PP2Cα. These data establish PHLPP as a member of the PP2C family of phosphatases with a unique active site architecture.

Figure 1

PHLPP activity is highly dependent on the substrate. (a) PHLPP1 and PHLPP2 have similar phosphatase activities in vitro. The saturating kinetics of the phosphatase domains of PHLPP1 (empty diamonds) and PHLPP2 (gray squares) with increasing concentrations of the substrate pNPP is shown. The rates were determined by measuring the production of pNP at 405 nm in Tricine buffer (pH 7.5). The data are fit to the Michaelis–Menten equation. The graph shows mean values ± SEM for three separate experiments. (b) Activities of the phosphatase domain of PHLPP2 isolated from bacteria or insect cells are comparable on peptidic substrates. The saturating kinetics of the phosphatase domains of PHLPP2 isolated from Sf21 cells (black circles) or Escherichia coli BL21(DE3)pLysS (gray squares) with increasing concentrations of the substrate peptide is shown. The rates were determined by measuring the liberation of free phosphate by the malachite green assay in a Tricine buffer (pH 7.5). The data are fit to the Michaelis–Menten equation. The graph shows mean values ± SEM for three separate experiments.

Figure 2

Effect of pH on the kinetic parameters. (a) Activity of PHLPP1 (empty diamonds) and PHLPP2 (gray squares) phosphatase domains was evaluated at different pH values. Reactions occurred at 10 mM pNPP and 100 mM pH-appropriate buffer (see Table 1 of the Supporting Information). Graphs show means ± SEM for at least three separate experiments. (b) k_cat and (c) k_cat/K_M for PHLPP1 and PHLPP2 phosphatase domains evaluated at different pH values. Saturating kinetics were determined with increasing concentrations of the substrate pNPP (typically 1–15 mM) in 100 mM buffer, and data were fit to a Michaelis–Menten equation. pH data in panel c are fit to the equation v = C/(1+ H/K_a + H/K_b). Graphs show means ± SEM.

Figure 3

Influence of the cation on PHLPP activity. Relative activity of PHLPP2 phosphatase domains at 10 mM pNPP with increasing concentrations of (a) EDTA (red) or MgCl₂ (yellow), (b) CaCl₂, or (c) CuSO₄ (blue) or NiSO₄ (orange). The activity is given as a percentage of the activity of the protein in the absence of additive. (d) Relative activity of PHLPP2 phosphatase domains isolated from insect cells (black circles) or bacteria (gray squares) and full-length PHLPP1 immunoprecipitated from mammalian cells (white triangles) or the PHLPP1 phosphatase domain from bacteria (white diamonds) at 10 mM pNPP with increasing concentrations of MnCl₂. k values for PHLPP1 (e) and PHLPP2 (f) phosphatase domains purified from BL21(DE3)pLysS cells were evaluated at 10 mM pNPP, at different pH values in the absence (empty symbols) or presence (black symbols) of 10 mM MnCl₂. Graphs represent means ± SEM for three separate experiments.

Figure 4

Activation of PHLPP by DTT and selected lipophilic compounds. (a) Effect of DTT on PHLPP1 and PHLPP2 activity. k values for PHLPP1 (white bars) and PHLPP2 (gray bars) were determined at 10 mM pNPP, in the absence of Mn²⁺ for different concentrations of DTT. (b) Effect of different lipophilic compounds on PHLPP2 phosphatase activity. Reactions were conducted in Tricine (pH 7.5) at 10 mM pNPP and 1 mM MnCl₂ in the presence of 100 μM lipophilic compounds. (c) Relative activity of the PHLPP2 phosphatase domain with increasing concentrations of oleic (circles) and arachidonic (triangles) acid. Reactions were conducted in Tricine (pH 7.5) at 10 mM pNPP and 5 mM MnCl₂. Graphs show means ± SEM for three separate experiments. *P < 0.01, and **P < 0.001 (Student’s t test).

Figure 5

Structural stability of the isolated phosphatase domain of PHLPP. (a) Circular dichroism (CD) spectra for PHLPP1 (■) and PHLPP2 (△) phosphatase domains, isolated from bacteria. (b) The phosphatase domain of PHLPP2 was subjected to increasing temperatures, and variations of the mean residue ellipticity at 222 nm (by CD) were recorded. (c) Activity of the phosphatase domain of PHLPP1 (empty diamonds) or PHLPP2 (gray squares) was measured by fluorescence at different temperatures using DiFMUP as the substrate. (d) Phosphatase domain PHLPP2 treated with EDTA (black squares) and subsequently incubated with MnCl₂ (orange squares), MgCl₂ (yellow squares), or CaCl₂ (pink squares) was subjected to increasing temperatures, and fluorescence emission at 330 nm was recorded. (e) Activity of PHLPP2 incubated with MnCl₂ (orange squares), MgCl₂ (yellow squares), or CaCl₂ (pink squares) was measured by fluorescence at different temperatures using DiFMUP as the substrate.

Figure 6

Unique inhibitor sensitivity of the PHLPP PP2C domain. Relative activity of the PHLPP2 PP2C domain (gray squares) and PP2Cα (white circles) in the presence of different molecules (NCS 270156, NCS 170008, NCS 73101, and NCS 11241). Compounds NCS 270156 and NCS 170008 were selective for PHLPP2 compared to PP2Cα, whereas NCS 73101 inhibits PP2Cα preferentially. Reactions were conducted in Tricine (pH 7.5) at 10 mM pNPP at various inhibitor concentrations. Graphs show means ± SEM for three separate experiments.

Figure 7

Mutational analysis of PHLPP2. (a) Relative activity of point mutants of PHLPP2 using a peptide as the substrate. Point mutants of full-length PHLPP2 were overexpressed as HA-tagged fusion proteins in COS7 cells and immunoprecipitated. They were used to dephosphorylate the threonine-phosphorylated peptide RRA^PTVA in Tricine (pH 7.5) in the presence of 5 mM MnCl₂. Release of inorganic phosphate was monitored by the malachite green assay, and the speed of dephosphorylation was divided by the relative amount of protein, as determined by Western blotting. Activity is given relative to that of wild-type PHLPP2. The graph shows means ± SEM of three separate experiments. (b) Position of the mutated residues in the homology model of PHLPP2. (c) Sequence alignment of subdomains 1, 2, 8, and 11 of the PP2C family showing conserved RXXXD, DGXXG, DG, and GXXDN (colored orange, green, yellow, and cyan, respectively). Mn²⁺-coordinating acidic residues are colored red. Below is a schematic of PP2Cα (left) showing four asparates that coordinate the two bound Mn²⁺ ions (magenta spheres) and a homology model of PHLPP2 showing equivalent acidic residues and structurally important Phe residues.