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A Medical Research Council of Canada B. C. Lung Association Scientist. To whom correspondence should be addressed: Jack Bell Research Center, Vancouver Hospital, 2660 Oak St., Vancouver, British Columbia V6H 3Z6, Canada. Tel.: 604-875-4707; Fax: 604-875-4497
* This work was supported by the Medical Research Council of Canada (now the Canadian Institutes for Health Research), by the National Cancer Institute of Canada with core support from the British Columbia Cancer Foundation and the British Columbia Cancer Agency, and by National Institutes of Health Grants NS29632 and GM57705.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Both authors contributed equally to this work. ¶ Held a Cancer Research Society studentship. Present address: Ontario Cancer Institute, 610 University Ave., Toronto, Ontario M5E 2C3, Canada. ** Supported by the Deutsche Forschungsgemeinschaft. ¶¶ A Terry Fox Cancer Research Scientist of the National Cancer Institute of Canada, supported by funds from the Canadian Cancer Society and the Terry Fox Run.
Using bone marrow derived mast cells from SH2-containing inositol-5-phosphatase (SHIP) +/+ and −/− mice, we found that the loss of SHIP leads to a dramatic increase in Steel Factor (SF)-stimulated phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3), a substantial reduction in PI(3,4)P2, and no change in PI(4,5)P2 levels. We also found that SF-induced activation of protein kinase B (PKB) is increased and prolonged in SHIP−/− cells, due in large part to more PKB associating with the plasma membrane in these cells. Pretreatment of SHIP−/− cells with 25 μm LY294002 resulted in complete inhibition of SF-induced PI(3,4)P2, while still yielding PI(3,4,5)P3 levels similar to those achieved in SHIP+/+ cells. This offered a unique opportunity to study the regulation of PKB by PI(3,4,5)P3, in the absence of PI(3,4)P2. Under these conditions, PKB activity was markedly reduced compared with that in SF-stimulated SHIP+/+ cells, even though more PKB localized to the plasma membrane. Although phosphoinositide-dependent kinase 1 mediated phosphorylation of PKB at Thr-308 was unaffected by LY294002, phosphorylation at Ser-473 was dramatically reduced. Moreover, intracellular delivery of PI(3,4)P2 to LY294002-pretreated, SF-stimulated SHIP−/− cells increased phosphorylation of PKB at Ser-473 and increased PKB activity. These results are consistent with a model in which SHIP serves as a regulator of both activity and subcellular localization of PKB.
src homology 2
bone marrow-derived mast cells
phosphoinositide-dependent kinase 1
protein kinase B
Steel factor or stem cell factor
4)P2, phosphatidylinositol 3,4-bisphosphate
5)P2, phosphatidylinositol 4,5-bisphosphate
4,5)P3, phosphatidylinositol 3,4,5-trisphosphate
fetal calf serum
high performance liquid chromatography
Iscove's modified Dulbecco's medium
phosphatase and tensin homolog on chromosome 10
The src homology 2 (SH21)-containing inositol phosphatase (SHIP) is a 145-kDa hemopoietic-specific signaling protein (
). SHIP has been shown to inhibit immune receptor activation in both mast cells and B cells by binding to the tyrosine-phosphorylated immunoreceptor tyrosine-based inhibition motif of the inhibitory co-receptor FcγRIIB and inhibiting FcεR1- and B cell receptor-induced calcium influx, respectively (
). In addition, SHIP has been shown, even in the absence of FcγRIIB co-clustering, to play a “gatekeeper” role in IgE-mediated mast cell degranulation by setting the threshold for and limiting the degranulation process (
), we demonstrated its ability, in vitro, to hydrolyze the 5′-phosphate from phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) but not from PI(4,5)P2. More recently, however, by modifying the in vitro assay conditions, SHIP was found capable of readily hydrolyzing PI(4,5)P2 to PI(4)P (
). To resolve its phospholipid substrate specificity and to gain some insight into the normal role that SHIP plays in vivo, we generated a SHIP knockout mouse by homologous recombination in embryonic stem cells (
). These mice have allowed us to ask whether one of SHIP's normal functions is to hydrolyze PI(3,4,5)P3 and/or PI(4,5)P2in vivo. Specifically, in the present study we have utilized bone marrow-derived mast cells (BMMCs) from SHIP−/− and +/+ littermates to determine if SHIP affects PI(4,5)P2 levels and if it plays a significant role in hydrolyzing the Steel Factor (SF)-stimulated increase in PI(3,4,5)P3in vivo. We have also used these two cell types to examine the role of SHIP in the activation of the proto-oncogene, protein kinase B (PKB) (also referred to as Akt or RAC) (
), and more specifically, the role of PI(3,4,5)P3 and PI(3,4)P2 in mediating the activation of PKB. Our data reveal that SHIP is the primary enzyme responsible for hydrolyzing PI(3,4,5)P3 in SF-stimulated normal BMMCs and that PI(3,4,5)P3 is the major source for PI(3,4)P2 in these cells. However, the presence or absence of SHIP does not have any significant effect on PI(4,5)P2levels. Interestingly, although the loss of SHIP increases the levels of PI(3,4,5)P3 and enhances PKB activity, we show that the generation of PI(3,4)P2 is also essential to fully activate PKB, because this lipid is important for mediating phosphorylation of PKB at Ser-473.
The current model for the activation of PKB suggests that inactive, cytosolic PKB translocates to the plasma membrane to bind to PI(3,4,5)P3 and/or PI(3,4)P2 that is generated in response to PI3K activation (
). Binding via its pleckstrin homology (PH) domain to one or both of these phosphoinositides alters PKB's conformation such that it becomes accessible to phosphorylation by two upstream kinases, PDK1 and an unidentified kinase that has been referred to as PDK2. The phosphatidylinositol-dependent kinase PDK1 (
) phosphorylates PKB in the activation loop of its kinase domain at Thr-308 while a separate kinase phosphorylates PKB in its carboxyl-terminal tail at Ser-473. A recent report has described this as Hm kinase due to its phosphorylation at the hydrophobic region of PKB (
). Thus, PKB autophosphorylation is not likely to be the mechanism by which Ser-473 is regulated. Once phosphorylated at both sites, PKB becomes locked into the fully active conformation, detaches from the plasma membrane, phosphorylates target substrates such as glycogen synthase kinase-3 (
One major controversy surrounding this model of PKB activation is whether PI(3,4,5)P3 or PI(3,4)P2 is the critical second messenger that attracts PKB and its kinases to the plasma membrane in vivo. There is substantial in vitro data suggesting that PI(3,4)P2 has a higher affinity than PI(3,4,5)P3 for PKB (
However, in support of PI(3,4,5)P3 being the critical second messenger, it also binds PKB and in one study examining the binding of di-C8-PI(3,4,5)P3 and PI(3,4)P2 to purified PKB, PI(3,4,5)P3 bound with slightly higher affinity (
) have proposed that PKB activation by PI(3,4)P2in vitro might be due to contamination of the PKB preparations with PDK1. Last, phosphorylation of PKB at Ser-473 also appears to be under the control of PI3K (
), but because the kinase has not as yet been identified, there is no data to suggest whether it may have a higher affinity for PI(3,4,5)P3 or PI(3,4)P2.
The availability of SHIP−/− BMMCs has given us the unique opportunity to modulate these two phosphoinositides, such that in the presence of 25 μm LY294002 an amount of PI(3,4,5)P3equivalent to that observed in SHIP+/+ BMMCs is produced in response to SF (Fig. 5A). Under these unique conditions, no PI(3,4)P2 is formed and we were therefore able to investigate the activation of PKB in vivo, in the presence of increased PI(3,4,5)P3 alone. Comparison of PKB levels in the membrane fraction of SF-stimulated SHIP+/+ cells, with LY294002-pretreated SHIP−/− cells, suggests that PI(3,4,5)P3 alone is sufficient in vivo to attract PKB to the plasma membrane. However, our experiments also demonstrated that membrane recruitment via PI(3,4,5)P3alone is not sufficient to drive Ser-473 phosphorylation, which was prevented by LY294002 pretreatment under these conditions. This led us to consider the possibility that the generation of PI(3,4)P2 is necessary for Ser-473 phosphorylation. To test this, we added exogenous PI(3,4)P2, which restored both Ser-473 phosphorylation and activity. These data, and our results with two structurally unrelated PI3K inhibitors, strongly support the possibility that the Ser-473 phosphorylation of PKB is dependent upon the presence of PI(3,4)P2.
It is also important to note that PDK1-mediated phosphorylation of PKB at Thr-308 in SHIP−/− BMMCs is unaffected by 25 μmLY294002 (Fig. 6B), suggesting that PI(3,4,5)P3alone is sufficient to impart the conformational change within PKB to allow its phosphorylation by PDK1. In addition, our results demonstrate that little or no phosphorylation of Ser-473 is required to dock PDK-1 and mediate phosphorylation of Thr-308. This could be in contrast to other AGC kinases, such as RSK2, in which phosphorylation of the Ser-473-equivalent residue is thought to provide a docking site for PDK-1 to allow efficient activation loop phosphorylation (
We thus propose a model of SF-stimulated PKB activation in BMMCs in which PKB and PDK1 are attracted via PI(3,4,5)P3 whereas a Ser-473 kinase is attracted via PI(3,4)P2 to the plasma membrane (Fig. 9). Another possibility could be that PI(3,4)P2 inactivates a phosphatase responsible for the turnover of Ser-473 phosphorylation, and at this point we cannot rule out this possibility. Hemmings and co-workers (
) have used a membrane-inducible PKB allele to suggest that Ser-473 phosphorylation could be under the control of a PI3K-dependent phosphatase activity, but the mechanism for this is currently unknown. The same group found that hyperosmotic stress caused dephosphorylation of PKB at Ser-473 while also causing elevation of PI(3,4,5)P3 levels and depression of PI(3,4)P2, thus likely affecting 5-phosphatase activity (
). PI(3,4)P2 inhibition of a Ser-473 phosphatase could also allow for the possibility that this residue is a target of autophosphorylation by PKB. In either case, our data show that generation of PI(3,4)P2 leads to accumulation of Ser-473 phosphorylation. Of note, it has been proposed that, because PDK1 has such a high affinity for PI(3,4,5)P3 (
) have reported that ILK may act as the putative PDK2. This protein has a PH-like domain that appears to be capable, at least in vitro, of binding to both PI(3,4,5)P3 and, to a lesser extent, PI(3,4)P2. More recent data further supports the role of ILK as a direct regulator of PKB activity (
). Until some insight into the in vivo affinities of ILK for PI(3,4)P2 is obtained, our present findings cannot help in determining whether ILK is serving as the kinase that phosphorylates Ser-473 in BMMCs.
Interestingly, with regards to SHIP, our studies to date as well as others have shown that this inositol-5-phosphatase plays a negative role in proliferation (
). However, the data presented herein suggest that SHIP may also play a positive role by generating PI(3,4)P2 and thus activating PKB. This raises several interesting points about the regulation of PKB. It seems that nature has set in place the requirement of both PI(3,4,5)P3 and PI(3,4)P2for its activation. Loss of SHIP may not super-potentiate PKB activity, because PI(3,4)P2 levels are reduced, and this may explain why we have not observed any hemopoietic malignancies in mice lacking this hemopoietically expressed gene (
) and perhaps greater PKB activity than can be obtained in SHIP-depleted cells, and this may facilitate tumor formation. Studies are currently underway to directly compare PKB activity in response to cytokine stimulation of SHIP−/− and PTEN−/− ES cell-derived mast cells to assess this possibility.
In summary, we have shown that SHIP is the primary enzyme responsible for hydrolyzing PI(3,4,5)P3 and generating PI(3,4)P2 in SF-stimulated BMMCs. We have also shown that SHIP plays a critical role in regulating PKB activity in these cells and that the overall effect of the large increase in PI(3,4,5)P3 (and a decrease in PI(3,4)P2levels) is increased activation of PKB. This is consistent with reports by Liu et al. (
). However, our data reveal that, while the loss of SHIP enhances PKB activity, the generation of PI(3,4)P2in SHIP−/− BMMCs, although substantially lower than in SHIP+/+ BMMCs in response to SF, is essential to fully activate PKB by enhancing its phosphorylation at serine 473. Our results also suggest that SF-stimulated SHIP−/− and +/+ BMMCs, with their dramatically different PI(3,4,5)P3/PI(3,4)P2 ratios, could prove very useful for future studies comparing the potential role of these two phosphoinositides both in recruitment of target proteins and in regulation of signaling cascades.
We thank Vivian Lam for excellent technical support and Christine Kelly for help in typing the manuscript. We thank S. Ozaki (University of Utah) for providing the shuttles, Echelon Research Laboratories (Salt Lake City, UT) for providing cargo phosphoinositides, and J. Shope and D. DeWald (Utah State University) for sharing phosphoinositide shuttling protocols prior to publication. We thank Dr. Bayard Clarkson, Memorial Sloan-Kettering Cancer Center, New York, NY for his generous gift of anti-SHIP2 antibody.
Proc. Natl. Acad. Sci. U. S. A.1996; 93: 1689-1693