Phospholipid-binding sites of phosphatase and tensin homolog (PTEN): exploring the mechanism of phosphatidylinositol 4,5-bisphosphate activation

Abstract

The lipid phosphatase activity of the tumor suppressor phosphatase and tensin homolog (PTEN) is enhanced by the presence of its biological product, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). This enhancement is suggested to occur via the product binding to the N-terminal region of the protein. PTEN effects on short-chain phosphoinositide (31)P linewidths and on the full field dependence of the spin-lattice relaxation rate (measured by high resolution field cycling (31)P NMR using spin-labeled protein) are combined with enzyme kinetics with the same short-chain phospholipids to characterize where PI(4,5)P2 binds on the protein. The results are used to model a discrete site for a PI(4,5)P2 molecule close to, but distinct from, the active site of PTEN. This PI(4,5)P2 site uses Arg-47 and Lys-13 as phosphate ligands, explaining why PTEN R47G and K13E can no longer be activated by that phosphoinositide. Placing a PI(4,5)P2 near the substrate site allows for proper orientation of the enzyme on interfaces and should facilitate processive catalysis.

Keywords: 31P Field Cycling; Enzyme Kinetics; Micelles; Nuclear Magnetic Resonance (NMR); Phosphatase and Tensin Homolog (PTEN); Phosphatidylinositol Phosphatase; Phosphoinositide; Phospholipid-binding Site; Spin-labeled Protein.

FIGURE 1.

Ribbon diagrams of the crystal structure of PTEN in the absence and presence of substrate-containing membrane. A, PTEN crystal structure (Protein Data Bank code 1D5R) with colors from blue to red for N to C termini. Yellow spheres represent sulfur atoms of Cys residues that are spin-labeled in the NMR experiments. The tartrate molecule bound at the postulated active site is in van der Waals sphere representation. The arrow indicates a postulated movement of the two domains upon membrane binding. B, model for membrane-bound PTEN with the missing N-terminal peptide docked as an interfacial helix (pink). The sulfur of Cys-124 is depicted as a yellow sphere and a diC₆PIP₃ molecule (magenta) is docked in the active site. The postulated conformational change is based on that suggested by Kalli et al. (17).

FIGURE 2.

Purification of recombinant PTEN and variants. Top, SDS-PAGE analysis of fractions from the Ni-NTA column; mass standards are indicated on the gel. Bottom, CD spectra for recombinant PTEN (–) and R47A (- - -) are indicated.

FIGURE 3.

Effect of PI(4,5)P₂ and other amphiphiles on PTEN hydrolysis of different substrates. A, relative specific activities for PTEN toward 0.1 mm diC₈PI(3,4)P₂, DOPI(3,4)P₂/POPC (0.05 mm/0.95 mm) LUVs, DOPI(3,4)P₂/POPC (0.1 mm/0.9 mm) LUVs, and 0.5 mm diC₈PI(3)P with PI(4,5)P₂ are shown, with substrate/PI(4,5)P₂ = 0.5 (■) or 1.0 (\\\\). B, effect of varying the chain length of substrate and activator. Substrates include 0.1 mm diC₆PI(3,4)P₂ or 0.1 mm diC₈PI(3,4)P₂ with dihexanoyl- or dioctanoyl-PI(4,5)P₂ added, and substrate/PI(4,5)P₂ = 0.5 (■) or 1.0 (\\\\). C, effect of PIP₂ and Triton X-100 on PTEN hydrolysis of different substrates. Substrates include diC₈PI(3,4)P₂/Triton X-100 (0.1 to 0.4 mm), DOPI(3,4)P₂/Triton X-100 (0.1 to 0.4 mm), and diC₈PI(3)P/Triton X-100 (0.5 to 2 mm). The concentration of PI(4,5)P₂ (either the dioctanoyl compound or DOPI(4,5)P₂ depending on the substrate) was half (■) or the same (\\\\) as that of the substrate. The dashed lines in A–C would represent an activity with the PI(4,5)P₂ equivalent to that in its absence. D, diC₇PC inhibition of PTEN activity toward 0.5 mm diC₈PI(3)P (■, □) or 0.1 mm diC₈PI(3,4)P₂ (●, ○) in the absence (filled symbols) or presence (open symbols) of Triton X-100 equivalent to four times the substrate concentration. Error bars here (and in other plots of enzyme activities) represent standard deviations from the repeats for each experimental condition.

FIGURE 4.

Effect of PI(4,5)P₂ on the activity of recombinant PTEN and variants K13E, R47K, and R47G in monomer and vesicle assay systems. The ratio of the specific activity with PI(4,5)P₂ added to the activity in its absence is shown for WT and the three mutant proteins. In the monomer substrate mixture diC₈PI(3,4)P₂ is 0.1 mm with either 0.05 mm (■) or 0.1 mm (///) diC₈PI(4,5)P₂. Vesicles (▩) were composed of DOPI(3,4)P₂/POPC (0.05 to 0.95 mm) with 0.05 mm DOPI(4,5)P₂. The specific activities of wild type PTEN toward each substrate in the absence of PIP₂ are 45 ± 4 nmol/mg min for diC₈PI(3,4)P₂ and 0.59 ± 0.02 nmol/mg min for DOPI(3,4)P₂/POPC LUVs.

FIGURE 5.

Dependence of R_P-e(0) on the distance of a ³¹P nucleus from an unpaired electron and the τ_P-e correlation time predicted for two different ratios of protein to the ³¹P-containing amphiphile. Different τ_P-e values are 25 (– – –), 50 (—), 100 (- - -), 150 (···), and 200 (— — —) ns.

FIGURE 6.

Field dependence of the PR₁E for diC₆PI and diC₆PI(4,5)P₂. A, R₁ for the ³¹P of diC₆PI (3 mm) caused by 4.9 μm spin-labeled PTEN; the inset shows the ΔR₁ of this phosphodiester from 0.004 to 4 T after subtraction of the profile obtained using nonlabeled PTEN. B, R₁ for the ³¹P resonances of diC₆PI(4,5)P₂ (3 mm) in the presence of 5.1 μm spin-labeled PTEN: P-1 (●), P-4 (▵), and P-5 (▴). The inset in B shows the R₁ profile for 3 mm I(1,4,5)₃ in the presence of 4.9 μm spin-labeled PTEN: P-4 (●), P-1 phosphate (○), and P-5 phosphate (□). Note that none of these ³¹P resonances exhibit the low field rise in R₁ indicative of a small molecule-protein complex. Error bars for R₁ values (here and in subsequent plots) were obtained from a least squares fit of the ln(intensity) versus time determining R₁ at that particular field.

FIGURE 7.

Field dependence of the PR₁E for diC₈PI and diC₈PI(4,5)P₂. A, R₁ for the ³¹P of diC₈PI (3.0 mm) in the presence of 2.0 μm unlabeled (○) or spin-labeled (●) PTEN is shown as a function of magnetic field. B, ΔR₁ representing the difference due to the spin-labeled PTEN is shown; the line represents a fit with two dispersions with τ_P-e values of 5 and 200 ns. C, ΔR₁ for diC₈PI(4,5)P₂ (3.0 mm) in the presence of 4.9 μm spin-labeled PTEN (solid symbols): P-1 (●), P-4 (▴), and P-5 (■).

FIGURE 8.

Field dependence of the ³¹P PR₁E for diC₈PI and diC₈PI(4,5)P₂ in the presence of spin-labeled PTEN C124S. A, effect of 4.3 μm spin-labeled C124S on 3 mm diC₈PI(4,5)P₂: P-1 (●); P-4 (□); (▴) P-5. B, comparison of PRE for diC₈PI (3 mm) produced by spin-labeled 2.0 μm native PTEN (○) or 2.2 μm spin-labeled variant C124S (●); the field dependence was fit with τ_P-e = 200 ns, and the arrow approximates the amount of R₁ contributed by a spin label on Cys-124.

FIGURE 9.

Ribbon diagrams of the model of diC₆PI(4,5)P₂ binding to PTEN at a discrete site near the active site are compatible with the ³¹P electron distances to the Cys-124 spin label. A, view of the phosphatase domain as seen from the membrane. The sulfur of Cys-124 is the yellow sphere to which the 2,2,5,5-tetramethyl-1-oxyl-3-methyl methanethiosulfonate is attached. The PI(4,5)P₂ molecule, placed according to the surface features and to the relative distances determined by NMR, forms hydrogen-bonded contacts with Arg-47 and Lys-13, both critical residues for the PIP₂ activation to be observed. B, surface representation of the phosphatase domain, in a similar view to that in A, colored by the electrostatic potential (red, negative, and blue, positive) with both substrate and activator docked to their sites. The PI(3,4,5)P₃ molecule (magenta) is docked at the active site marked by the dark loop and the yellow sphere depicting Cys-124. The PI(4,5)P₂ molecule (in cyan) occupies the same site as in A. The N-terminal helix is docked into the phosphatase domain, and its positive electrostatic potential complements the charges of the domain to create a positive membrane binding profile.

FIGURE 10.

Effect of spin-labeled K13E PTEN (4.1 μm) on the field dependence of R₁ for phospholipid mixed micelles of 3 mm diC₈PI (○) and 3 mm diC₈PI(4,5)P₂ (P-1 (●), P-4, (Δ), P-5 (■)).