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. 2007 Oct 15;407(2):255-66.
doi: 10.1042/BJ20070558.

The control of phosphatidylinositol 3,4-bisphosphate concentrations by activation of the Src homology 2 domain containing inositol polyphosphate 5-phosphatase 2, SHIP2

Ian H Batty  1 , Jeroen van der Kaay, Alex Gray, Joan F Telfer, Miles J Dixon, C Peter Downes
Affiliations

The control of phosphatidylinositol 3,4-bisphosphate concentrations by activation of the Src homology 2 domain containing inositol polyphosphate 5-phosphatase 2, SHIP2

Ian H Batty et al. Biochem J. .

Abstract

Activation of class Ia PI3K (phosphoinositide 3-kinase) produces PtdInsP3, a vital intracellular mediator whose degradation generates additional lipid signals. In the present study vanadate analogues that inhibit PTPs (protein tyrosine phosphatases) were used to probe the mechanisms which regulate the concentrations of these molecules allowing their independent or integrated function. In 1321N1 cells, which lack PtdInsP3 3-phosphatase activity, sodium vanadate or a cell permeable derivative, bpV(phen) [potassium bisperoxo(1,10-phenanthroline)oxovanadate (V)], increased the recruitment into anti-phosphotyrosine immunoprecipitates of PI3K activity and of the p85 and p110a subunits of class Ia PI3K and enhanced the recruitment of PI3K activity stimulated by PDGF (platelet-derived growth factor). However, neither inhibitor much increased cellular PtdInsP3 concentrations, but both diminished dramatically the accumulation of PtdInsP3 stimulated by PDGF or insulin and markedly increased the control and stimulated concentrations of PtdIns(3,4)P2. These actions were accounted for by the ability of PTP inhibitors to stimulate the activity of endogenous PtdInsP3 5-phosphatase(s), particularly SHIP2 (Src homology 2 domain containing inositol polyphosphate 5-phosphatase 2) and to inhibit types I and II PtdIns(3,4)P2 4-phosphatases. Thus bpV(phen) promoted the translocation of SHIP2 from the cytosol to a Triton X-100-insoluble fraction and induced a marked (5-10-fold) increase in SHIP2 specific activity mediated by enhanced tyrosine phosphorylation. The net effect of these inhibitors was, therefore, to switch the signal output of class I PI3K from PtdInsP3 to PtdIns(3,4)P2. A key component controlling this shift in the balance of lipid signals is the activation of SHIP2 by increased tyrosine phosphorylation, an effect observed in HeLa cells in response to both PTP inhibitors and epidermal growth factor.

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Figures

Figure 1
Figure 1. Sodium vanadate attenuates receptor-mediated accumulation of PtdInsP3 but potentiates that of PtdIns(3,4)P2
Cells pre-labelled with [3H]inositol were treated for 30 min in the absence (circles) or presence (triangles) of 1 mM sodium vanadate then further incubated as indicated in the absence (open symbols) or presence (closed symbols) of PDGF (50 ng/ml) and the [3H]PIs then determined. The results are expressed as a percentage of the maximum accumulation of either PtdInsP3 (A) or PtdIns(3,4)P2 (B) and represent the means±S.E.M. of three experiments performed in duplicate.
Figure 2
Figure 2. bpV(phen) enhances PDGF-stimulated PI3K activity but switches its cellular product from PtdInsP3 to PtdIns(3,4)P2
(A–C) Cellular PI concentrations. Cells labelled with [3H]inositol were incubated as indicated with 50 ng/ml PDGF (●), 0.1 mM bpV(phen) (△) or both reagents (▲) before the extraction and measurement of the [3H]PIs shown. The concentration of each lipid is expressed as a percentage of the total cellular PI and the results reflect the mean values of duplicate measurements in a single experiment representative of two that gave similar results. (D) PI3K activity. Cells were incubated as indicated with 50 ng/ml PDGF (●), 0.1 mM bpV(phen) (△) or both reagents (▲) and the PI3K activity present in anti-phosphotyrosine immunoprecipitates was then measured. The results represent the means±S.E.M. of three experiments performed in duplicate.
Figure 3
Figure 3. bpV(phen) stimulates a time- and concentration-dependent recruitment of class Ia PI3K that accounts for the cellular accumulation of PtdIns(3,4)P2
(A) The recruitment of class Ia PI3K. Cells were incubated for the times and with the concentrations of bpV(phen) indicated and the recruitment of the p85 and p110α subunits of class Ia PI3K into anti-phosphotyrosine immunoprecipitates was then determined. The results show duplicate determinations from one experiment representative of three which gave similar data. (B) Time-course of PI3K activity. Cells were incubated with bpV(phen) at 0.01mM (●) or 0.1 mM (▲) for the times indicated and the PI3K activity associated with anti-phosphotyrosine immunoprecipitates was then measured. The results are the means±S.E.M. of three experiments performed in duplicate and are expressed relative to the maximum activity achieved. (C) Concentration-dependence of PI3K activity. Cells were incubated for 10 min (▲) or 30 min (●) with the concentrations of bpV(phen) indicated before measurement of the PI3K activity as described above. The results are expressed as a percentage of the maximum activity achieved at the separate times and represent the means±S.E.M. of five experiments performed in duplicate (▲) or the mean and range of two experiments performed in duplicate (●). (D) The cellular accumulation of PtdIns(3,4)P2. Cells labelled with [3H]inositol were treated for 30 min without (circles and triangles) or with (squares) wortmannin (100 nM), then further incubated with 0.01mM (●) or 0.1 mM (▲ and ■) bpV(phen) for the times indicated before the measurement of PtdIns(3,4)P2. Values are the means of duplicate determinations in a single experiment representative of two which gave similar results and show PtdIns(3,4)P2 concentrations as a percentage of total cellular PI.
Figure 4
Figure 4. Sodium vanadate increases the rate of cellular PtdInsP3 5-phosphatase activity
Cells labelled with [3H]inositol were treated for 30 min in the absence (●) or presence (▲) of 1 mM sodium vanadate then incubated for 10 min in the presence of PDGF (50 ng/ml) before the addition of wortmannin (10 μM) and the [3H]PtdInsP3 remaining at the intervals shown was then measured. The results are expressed as percentages of the PtdInsP3 concentrations achieved in the absence and presence of sodium vanadate respectively, immediately prior to addition of wortmannin and represent the means±S.E.M. of five experiments performed in duplicate.
Figure 5
Figure 5. bpV(phen) stimulates the tyrosine phosphorylation and translocation of SHIP2
(A) Tyrosine phosphorylation of endogenous SHIP2. Cells were incubated for the times and with the concentrations of bpV(phen) indicated, then lysed and the SHIP2 protein immunoprecipitated with an anti-SHIP2 antibody and its tyrosine phosphorylation determined by SDS/PAGE and immunoblotting. The results show duplicate determinations from a single experiment representative of three that gave similar results. The lane labelled M contained markers only. (B) Translocation of GFP–SHIP2. Cells expressing GFP–SHIP2 were treated for 30 min in the absence [(i) and (ii)] or presence (iii) of wortmannin (100 nM) then further incubated for 20 min without (i) or with [(ii) and (iii)] bpV(phen) (20 μM) before the cells were fixed and examined by fluorescence microscopy. The images shown are representative of two experiments. (C) Translocation of endogenous SHIP2. Cells were incubated for 30 min with and without bpV(phen) (20 μM) and the cellular fractions indicated then prepared and analysed for SHIP2 by SDS/PAGE and immunoblotting. The results are from one experiment of three which gave similar results. The lane labelled M contained markers only.
Figure 6
Figure 6. The specific activity of SHIP2 is increased by bpV(phen)- or EGF-stimulated tyrosine phosphorylation
(A) and (B) 1321N1 cells were incubated for 30 min in the absence or presence of bpV(phen) (20 μM) as indicated and then lysed and SHIP2 immunoprecipitates analysed either for (A) SHIP2 protein and SHIP2 tyrosine phosphorylation or for (B) the corresponding SHIP2 5-phosphatase activity from control (○) and stimulated (●) cells. The results show (A) triplicate determinations and (B) the means±S.D. of the corresponding 5-phosphatase activity from the same samples in a single experiment representative of three which gave similar results. (C) and (D) Immunoprecipitates of SHIP2 from 1321N1 cells incubated as controls or treated for 30 min with bpV(phen) (20 μM) were analysed either for (C) 5-phosphatase activity or (D) SHIP2 tyrosine phosphorylation either directly (untreated) or after incubation for 30 min at 30 °C with GST–SHP1 (0.4 mg/ml) (GST–SHP1) or with buffer alone (Mock). The results in (C) show the mean and range of duplicate determinations from a single experiment and in (D) the corresponding duplicate anti-phosphotyrosine immunoblots and are representative of three experiments which gave similar results. (E) and (F) HeLa cells were incubated in the absence or presence of EGF (50 ng/ml) or bpV(phen) (100 μM) for the times indicated then lysed and SHIP2 immunoprecipitates analysed either for (E) SHIP2 5-phosphatase activity or for (F) SHIP2 protein and SHIP2 tyrosine phosphorylation. The results show the means±S.D. activity measured in a 30 min assay or the corresponding triplicate immunoblots for the same samples and are representative of five experiments which gave similar results. The vertical line dividing the upper panel in (F) indicates where images from the same immunoblot have been merged to eliminate a molecular mass marker lane and allow alignment with the lower panel.
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