Journal: The Journal of Biological Chemistry

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

doi: 10.1016/j.jbc.2023.105022

Figure Lengend Snippet: 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)P 3 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)P 2 , 2% PI(3,4)P 2 , 2% PI(3,4,5)P 3 , or 20% DOPS lipids, respectively. E , single-molecule dwell time distributions of various PIP lipid–binding domains plotted as dwell time versus log 10 (1-cumulative distribution frequency). Curves are fit with a single or double exponential decay curve yielding the following dwell times: AF488-PLCδ (τ 1 = 24 ± 2 ms), TAPP1-SNAP-AF647 (τ 1 = 1.02 ± 0.053 s), Grp1-AF647 (τ 1 = 0.544 ± 0.007 s), or LactC2-Dy647 (τ 1 = 0.765 ± 0.191 s, τ 2 = 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)P 2 , 2% PI(3,4)P 2 , 2% PI(3,4,5)P 3 , 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)P 3 , 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.

Article Snippet: The following lipids were used to generate small unilamellar vesicles (SUVs): 1,2-dioleoyl- sn -glycero-3-phosphocholine (18:1 DOPC; Avanti, catalog no.: 850375C), 1,2-dioleoyl- sn -glycero-3-phospho- l -serine (18:1; Avanti, catalog no.: 840035C), 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine (Avanti, catalog no.: 850725C), 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine- N -[4-(p-maleimidomethyl)cyclohexane-carboxamide] (18:1 MCC-PE; Avanti, catalog no,: 780201C), d -myo-phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P 3 diC16; Echelon, catalog no.: P-3916-100ug).

Techniques: In Vitro, Binding Assay, Single Particle, Membrane, Fluorescence

Journal: The Journal of Biological Chemistry

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

doi: 10.1016/j.jbc.2023.105022

Figure Lengend Snippet: SHIP1 catalyzes the dephosphorylation of PI(3,4,5)P 3 with 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)P 3 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)P 2 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)P 3 . 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)P 3 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 log 10 (1-cumulative distribution frequency). Curves fit with a single or double exponential decay curve yielding the following dwell times: 150 mM NaCl buffer (τ 1 = 25 ± 1 ms) or 75 mM NaCl buffer (τ 1 = 9 ± 2 ms, τ 2 = 56 ± 7 ms, α = 0.44 ± 0.13). Membrane composition: 88% DOPC, 2% PI(3,4,5)P 3 , 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)P 3 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)P 3 , ±10% PS. See Table S1 for statistics. DOPC, 1,2-dioleoyl- sn -glycero-3-phosphocholine; PI(3,4,5)P 3 , 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.

Article Snippet: The following lipids were used to generate small unilamellar vesicles (SUVs): 1,2-dioleoyl- sn -glycero-3-phosphocholine (18:1 DOPC; Avanti, catalog no.: 850375C), 1,2-dioleoyl- sn -glycero-3-phospho- l -serine (18:1; Avanti, catalog no.: 840035C), 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine (Avanti, catalog no.: 850725C), 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine- N -[4-(p-maleimidomethyl)cyclohexane-carboxamide] (18:1 MCC-PE; Avanti, catalog no,: 780201C), d -myo-phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P 3 diC16; Echelon, catalog no.: P-3916-100ug).

Techniques: De-Phosphorylation Assay, Activity Assay, In Vitro, Membrane, Concentration Assay, Fluorescence

Journal: The Journal of Biological Chemistry

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

doi: 10.1016/j.jbc.2023.105022

Figure Lengend Snippet: 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)P 3 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)P 3 . DOPC, 1,2-dioleoyl- sn -glycero-3-phosphocholine; PI(3,4,5)P 3 , phosphatidylinositol-(3,4,5)-trisphosphate; SH2, Src homology 2; SHIP1, Src homology 2 domain–containing inositol 5-phosphatase 1; TIRF, total internal reflection fluorescence.

Article Snippet: The following lipids were used to generate small unilamellar vesicles (SUVs): 1,2-dioleoyl- sn -glycero-3-phosphocholine (18:1 DOPC; Avanti, catalog no.: 850375C), 1,2-dioleoyl- sn -glycero-3-phospho- l -serine (18:1; Avanti, catalog no.: 840035C), 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine (Avanti, catalog no.: 850725C), 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine- N -[4-(p-maleimidomethyl)cyclohexane-carboxamide] (18:1 MCC-PE; Avanti, catalog no,: 780201C), d -myo-phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P 3 diC16; Echelon, catalog no.: P-3916-100ug).

Techniques: De-Phosphorylation Assay, Concentration Assay, Activity Assay, Membrane, Fluorescence

Journal: The Journal of Biological Chemistry

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

doi: 10.1016/j.jbc.2023.105022

Figure Lengend Snippet: Phosphot yrosine peptides promote membrane recruitment and activ ation 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)P 3 , 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) (τ 1 = 54 ± 4 ms, τ 2 = 872 ± 82 ms, α = 0.62) and mNG-SHIP1(ΔCT) (τ 1 = 55 ± 5 ms, τ 2 = 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)P 2 was monitored in the presence of 20 nM Grp1-AF647. Plots were inverted to display the production of PI(3,4)P 2 , rather than the depletion of PI(3,4,5)P 3 . D – J , membrane composition: 96% DOPC, 2% PI(3,4,5)P 3 , 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)P 2 , phosphatidylinositol-(3,4)-bisphosphate; PI(3,4,5)P 3 , 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.

Article Snippet: The following lipids were used to generate small unilamellar vesicles (SUVs): 1,2-dioleoyl- sn -glycero-3-phosphocholine (18:1 DOPC; Avanti, catalog no.: 850375C), 1,2-dioleoyl- sn -glycero-3-phospho- l -serine (18:1; Avanti, catalog no.: 840035C), 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine (Avanti, catalog no.: 850725C), 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine- N -[4-(p-maleimidomethyl)cyclohexane-carboxamide] (18:1 MCC-PE; Avanti, catalog no,: 780201C), d -myo-phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P 3 diC16; Echelon, catalog no.: P-3916-100ug).

Techniques: Membrane, Activity Assay, Derivative Assay, Fluorescence