Mechanisms controlling membrane recruitment and activation of the autoinhibited SHIP1 inositol 5-phosphatase

Abstract

Signal transduction downstream of growth factor and immune receptor activation relies on the production of phosphatidylinositol-(3,4,5)-trisphosphate (PI(3,4,5)P₃) lipids by PI3K. Regulating the strength and duration of PI3K signaling in immune cells, Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1) controls the dephosphorylation of PI(3,4,5)P₃ to generate phosphatidylinositol-(3,4)-bisphosphate. Although SHIP1 has been shown to regulate neutrophil chemotaxis, B-cell signaling, and cortical oscillations in mast cells, the role that lipid and protein interactions serve in controlling SHIP1 membrane recruitment and activity remains unclear. Using single-molecule total internal reflection fluorescence microscopy, we directly visualized membrane recruitment and activation of SHIP1 on supported lipid bilayers and the cellular plasma membrane. We find that localization of the central catalytic domain of SHIP1 is insensitive to dynamic changes in PI(3,4,5)P₃ and phosphatidylinositol-(3,4)-bisphosphate both in vitro and in vivo. Very transient SHIP1 membrane interactions were detected only when membranes contained a combination of phosphatidylserine and PI(3,4,5)P₃ lipids. Molecular dissection reveals that SHIP1 is autoinhibited with the N-terminal Src homology 2 domain playing a critical role in suppressing phosphatase activity. Robust SHIP1 membrane localization and relief of autoinhibition can be achieved through interactions with immunoreceptor-derived phosphopeptides presented either in solution or conjugated to a membrane. Overall, this work provides new mechanistic details concerning the dynamic interplay between lipid-binding specificity, protein-protein interactions, and the activation of autoinhibited SHIP1.

Keywords: SHIP1; Src homology 2 domain; phosphatase; phosphatidylinositol; phosphatidylinositol phosphatase; phosphatidylinositol signaling; phosphatidylinositol-3-kinase.

Figure 1

SHIP1(PH-PP-C2) does not strongly interact with individual PIP lipids in vitro.A, cartoon diagram showing the reported lipid-binding domains of SHIP1, which include the phosphatase domain and flanking PH-R and C2 domains. B, experimental setup for directly visualizing protein–lipid interactions on SLBs using smTIRF-M. C, representative image showing single particle detection (purple circle) and trajectories (yellow line) of three Grp1-AF647 membrane-bound molecules. Image collected in the presence of 1 pM Grp1-AF647. Membrane composition: 2% PI(3,4,5)P₃ and 98% DOPC. D, representative TIRF-M images showing bulk membrane recruitment in the presence of either 20 nM (“high density”) or (“low density”) AF488-PLCδ (250 pM), TAPP1-SNAP-AF647 (1 pM), Grp1-AF647 (1 pM), or LactC2-Dy647 (2 pM) on SLBs containing DOPC lipids plus either 2% PI(4,5)P₂, 2% PI(3,4)P₂, 2% PI(3,4,5)P₃, or 20% DOPS lipids, respectively. E, single-molecule dwell time distributions of various PIP lipid–binding domains plotted as dwell time versus log₁₀(1-cumulative distribution frequency). Curves are fit with a single or double exponential decay curve yielding the following dwell times: AF488-PLCδ (τ₁ = 24 ± 2 ms), TAPP1-SNAP-AF647 (τ₁ = 1.02 ± 0.053 s), Grp1-AF647 (τ₁ = 0.544 ± 0.007 s), or LactC2-Dy647 (τ₁ = 0.765 ± 0.191 s, τ₂ = 6.58 ± 0.539 s, α = 0.5 ± 0.04). See Table S1 for statistics. F, mNG-SHIP1(PH-PP-C2) does not robustly associated with SLBs containing PIP or PS lipids. Representative images showing bulk membrane recruitment in the presence of 20 nM mNG-SHIP1(PH-PP-C2) on SLBs containing DOPC lipids plus either 2% PI(4,5)P₂, 2% PI(3,4)P₂, 2% PI(3,4,5)P₃, or 20% DOPS, respectively. Note that image intensities were scaled in a similar manner to the top row of images shown in (C). DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; PIP, phosphatidylinositol phosphate; PI(3,4)P_2, phosphatidylinositol-(3,4)-bisphosphate; PI(3,4,5)P₃, phosphatidylinositol-(3,4,5)-trisphosphate; PS, phosphatidylserine; SHIP1, Src homology 2 domain–containing inositol 5-phosphatase 1; SLB, supported lipid bilayer; smTIRF, single-molecule total internal reflection fluorescence.

Figure 2

SHIP1(PH-PP-C2) plasma membrane localization is insensitive to dynamic changes in PI(3,4,5)P₃lipid composition.A, cartoon diagramming UV-dependent photoconversion and plasma membrane binding of a mEos-tagged cytoplasmic protein in cells. B, representative images showing localization of mEos-LactC2 before and after photoconversion with 405 nm UV light (“low” and “high” laser power). Localization of mEos-LactC2 was visualized using smTIRF-M in PLB-985 cells. C, molecular brightness distribution of single plasma membrane–localized mEos-LactC2 molecules. D, stepwise photobleaching of a single plasma membrane–localized mEos-LactC2 molecule compared with the background fluorescence of the cell. E, single-molecule dwell distributions of mEos-LactC2 and mEos3.2-Grp1. Curves were fit to a single exponential decay curve to calculate the following dwell times: mEos-LactC2 (τ₁ = 371 ± 7 ms, N = 12 cells) and mEos-Grp1 (τ₁ = 392 ± 5 ms, N = 10 cells). F, plot showing the translocation of Akt1-mScarlet to the plasma membrane following stimulation of PLB-985 cells with 10 nM fMLF. G, bulk membrane localization of nonphotoconverted mEos-SHIP1(PH-PP-C2) does not change in response to stimulating PLB-985 cells with 10 nM fMLF (arrow). Traces represent the single cell plasma membrane intensity of mEos-SHIP1(PH-PP-C2) measured by TIRF-M. H, representative image showing single particle detection of mNG-SHIP1 in live PLB-985 cells. I, single-molecule dwell time distributions of photoconverted mEos-SHIP1(PH-PP-C2) in PLB-985 cells ± 10 nM fMLF plotted as dwell time versus log₁₀(1-cumulative distribution frequency). Curves are fit with a single exponential decay curves, and both curves yield the following dwell times for mEos-SHIP1(PH-PP-C2): τ₁ = 38 ± 3 ms (pre-fMLF; N = 4 cells) and τ₁ = 37 ± 5 ms (post-fMLF; N = 5 cells). A single dwell time was measured for each cell with n = 421 to 4267 molecules tracked per cell. Dwell times are reported as mean ± SD. See Table S1 for statistics. fMLF, N-formyl-methionine-leucine-phenylalanine; PI(3,4,5)P₃, phosphatidylinositol-(3,4,5)-trisphosphate; SHIP1, Src homology 2 domain–containing inositol 5-phosphatase 1; TIRF, total internal reflection fluorescence.

Figure 3

SHIP1 catalyzes the dephosphorylation of PI(3,4,5)P₃with first-order kinetics.A, experimental design for measuring SHIP1–lipid phosphatase activity in vitro on supported lipid bilayers using TIRF-M. B, kinetics of 20 nM SHIP1(PH-PP-C2). PI(3,4,5)P₃ dephosphorylation measured using 20 nM Grp1-AF647. C, bulk membrane localization of 20 nM mNG-SHIP1 during catalysis shown in (B). D, PS lipids enhance SHIP1 phosphatase activity. Representative kinetics traces of 4 nM mNG-SHIP1 (PH-PPtase-C2 domain) in the absence and presence of PS lipids. Production of PI(3,4)P₂ was monitored by the presence of 20 nM Grp1-AF647. Initial membrane composition: 88 to 98% DOPC, 0 to 10% PS lipids, and 2% PI(3,4,5)P₃. E, quantification of reaction half time in (D). Bars equal mean values (N = 2 reactions per concentration, error = SD). F, quantification of bulk localization measured in the presence of 20 nM mNG-SHIP1(PH-PPtase-C2) on SLBs containing 2% PI(3,4,5)P₃ and 0, 5, 10% PS lipids (N = 10 fluorescent intensity measurements per membrane, error = SD). G, single-molecule dwell time measured in the presence of 200 pM mNG-SHIP1(PH-PPtase-C2). Data plotted as dwell time versus log₁₀(1-cumulative distribution frequency). Curves fit with a single or double exponential decay curve yielding the following dwell times: 150 mM NaCl buffer (τ₁ = 25 ± 1 ms) or 75 mM NaCl buffer (τ₁ = 9 ± 2 ms, τ₂ = 56 ± 7 ms, α = 0.44 ± 0.13). Membrane composition: 88% DOPC, 2% PI(3,4,5)P₃, 20% PS. H, quantification of bulk localization measured in the presence of 20 nM mNG-SHIP1(PH-PPtase-C2) on SLBs containing 2% PI(3,4,5)P₃ and 0-20% PS lipids. I, full-length SHIP1 is stimulated by PS lipids but exhibits lower activity compared with mNG-SHIP1(PH-PPtase-C2). Reaction half times comparing phosphatase activity in the presence of 4 nM full-length mNG-SHIP1 or 4 nM mNG-SHIP1(PH-PPtase-C2). Membrane composition: 88 to 98% DOPC, 2% PI(3,4,5)P₃, ±10% PS. See Table S1 for statistics. DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; PI(3,4,5)P₃, phosphatidylinositol-(3,4,5)-trisphosphate; PS, phosphatidylserine; SHIP1, Src homology 2 domain–containing inositol 5-phosphatase 1; SLB, supported lipid bilayer; TIRF, total internal reflection fluorescence.

Figure 4

Full-length (FL) SHIP1 is autoinhibited by the N-terminal SH2 domain.A, domain organization of FL SHIP1 and truncation mutants used in supported lipid bilayer TIRF-M assays. B, kinetic traces showing the dephosphorylation of PI(3,4,5)P₃ in the presence 10 nM mNG-SHIP1 (1–1188 aa), mNG-SHIP1(PH-PPtase-C2), mNG-SHIP1(ΔSH2), and mNG-SHIP1(ΔCT). C, quantification of reaction half times measured in the presence of 10 nM SHIP1, FL, and truncation mutants. Bars equal mean values (N = 4–5 technical replicates, error = SD). D, plot shows the relationship between SHIP1 concentration and phosphatase activity. Comparing mNG-SHIP1 (1–1188 aa), mNG-SHIP1(PH-PPtase-C2), mNG-SHIP1(ΔSH2), and mNG-SHIP1(ΔCT). B–D, initial membrane composition: 98% DOPC and 2% PI(3,4,5)P₃. DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; PI(3,4,5)P₃, phosphatidylinositol-(3,4,5)-trisphosphate; SH2, Src homology 2; SHIP1, Src homology 2 domain–containing inositol 5-phosphatase 1; TIRF, total internal reflection fluorescence.

Figure 5

Phosphotyrosine peptides promote membrane recruitment and activation of SHIP1.A, experimental design for measuring SHIP1 phosphatase activity in the presence of phosphotyrosine peptides derived from ITIM motif of the FcγRIIB receptor (pY-ITIM) in solution or membrane conjugated. B, 100 μM pY-ITIM in solution stimulates the phosphatase activity of 20 nM full-length (FL) SHIP. Initial membrane composition: 96% DOPC, 2% PI(3,4,5)P₃, and 2% MCC-PE (quenched/nonreactive). C, quantification of reaction half times in (B). D, representative TIRF-M images showing localization of 5 nM mNG-SHIP1(FL, 1–1188 aa), mNG-SHIP1(ΔCT), mNG-SHIP1(ΔSH2), and mNG-SHIP1(R30A) in the presence of pY-ITIM conjugated to an SLB. E, representative TIRF-M images showing the detection and tracking of mNG-SHIP1 (1–1188 aa) bound to a pY membrane. F, single-molecule dwell time distribution measured in the presence of 20 pM mNG-SHIP1(1–1188 aa) or 20 pM mNG-SHIP1(ΔCT) on SLBs containing membrane-conjugated pY-ITIM peptide. Curves fit with a double exponential decay curve (black dashed line) yielding the following dwell times: mNG-SHIP1 (1–1188 aa) (τ₁ = 54 ± 4 ms, τ₂ = 872 ± 82 ms, α = 0.62) and mNG-SHIP1(ΔCT) (τ₁ = 55 ± 5 ms, τ₂ = 941 ± 17 ms, α = 0.61). Dwell times are reported as mean ± SD. See Table S1 for statistics. G–J, SHIP1 phosphatase activity measured in the absence and presence of the membrane-conjugated pY-ITIM peptide. Phosphatase activity was measured using the following SHIP1 solution concentrations: (G) 0.1 nM mNG-SHIP1(FL, 1–1188 aa), (H) 0.1 nM mNG-SHIP1 (ΔCT), (I) 5 nM mNG-SHIP1(ΔSH2), (J) 5 nM mNG-SHIP1(R30A). Production of PI(3,4)P₂ was monitored in the presence of 20 nM Grp1-AF647. Plots were inverted to display the production of PI(3,4)P₂, rather than the depletion of PI(3,4,5)P₃. D–J, membrane composition: 96% DOPC, 2% PI(3,4,5)P₃, 2% MCC-PE-(pY conjugated). DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; ITIM, immunoreceptor tyrosine inhibitory motif; MCC-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide; PI(3,4)P₂, phosphatidylinositol-(3,4)-bisphosphate; PI(3,4,5)P₃, phosphatidylinositol-(3,4,5)-trisphosphate; pY, tyrosine-phosphorylated motif; SHIP1, Src homology 2 domain–containing inositol 5-phosphatase 1; SLB, supported lipid bilayer; TIRF, total internal reflection fluorescence.