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Recipient of a Medical Research Council of Canada/British Columbia Lung Association Fellowship. To whom correspondence should be addressed: Dept. of Medicine, Jack Bell Research Centre, University of British Columbia, 2660 Oak St., Vancouver, B.C. V6H 3Z6, Canada. Tel.: 604-875-4707; Fax: 604-875-4497;
* This work was supported by a grant from the Medical Research Council of Canada. 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. ‡ Recipient of a Heart and Stroke Foundation of Canada traineeship.
Phosphatidylinositol (PI) 3-kinase is activated as a result of cytokine-induced association of the enzyme with specific tyrosine-phosphorylated proteins. PI 3-kinase lipid products, PI 3,4-P2 and PI 3,4,5-P3, have been shown, in vitro, to directly activate novel and atypical protein kinase C (PKC) isozymes. However, the mechanism by which PI 3-kinase may be involved in regulation of PKC isoforms in vivo is presently unknown. We investigated a possible relationship by looking for associations between these enzymes. We found that in a human erythroleukemia cell line, as well as in rabbit platelets, PI 3-kinase and PKCδ associate in a specific manner that is modulated by cell activation. Granulocyte-macrophage colony-stimulating factor treatment of cells caused increased association of PKCδ and PI 3-kinase as did treatment of platelets with platelet-activating factor. Results using two PI 3-kinase inhibitors, wortmannin and LY-294002, showed that the former inhibited this association, while the latter did not, suggesting that PI 3-kinase lipid products may not be a prerequisite for the PI 3-kinase/PKCδ association. Our results also suggest that tyrosine phosphorylation of PKCδ is not involved in its association with PI 3-kinase.
Cytokine stimulation of mitogenesis of hematopoietic cells is known to occur through at least two signaling pathways, both of which depend on tyrosine phosphorylation of key regulatory proteins; one is the p21ras/mitogen-activated protein kinase network and the other is the PI
3-kinase pathway. Following ligand binding, the common β subunit of receptors for interleukin (IL)-3, IL-5, and granulocyte/macrophage colony-stimulating factor (GM-CSF) becomes tyrosine-phosphorylated (
) become tyrosine-phosphorylated as well. This cascade of tyrosine phosphorylation is thought to be initiated by the activation of the receptor-associated JAK-2 kinase and the subsequent phosphorylation of the receptor (
), although many of the details of the subsequent events have yet to be worked out. Tyrosine phosphorylation of Shc, followed by SH2-mediated association with the adaptor protein Grb-2 and the p21ras activator, m-SOS1, have been shown to be involved in the activation of p21ras (
). These results were obtained from in vitro assays, but the mechanism whereby PKC isoforms may be activated by PI 3-kinase in vivo is not known. The activation of PKC activity by cytokines has been reported in a few publications (
), but it is not clear how this may occur. In the case of cytokines such as IL-3, IL-5, and GM-CSF, there is no evidence that a classical PI-phospholipase C pathway is operating, although evidence for increased production of diacylglycerol derived from phosphatidylcholine has been reported (
In an effort to further delineate pathways operating downstream of the related receptors for IL-3, IL-5, and GM-CSF, we have examined the role of PKC isoforms. This is a complex task, as there are at least 11 distinct family members that may be regulated by several independent mechanisms. In this report, we focus on the potential interaction of one specific PKC isoform, PKCδ, with the PI 3-kinase enzyme. We find that the two enzymes can be co-immunoprecipitated as shown both by immunoblotting and enzyme activities. Furthermore, the association is modulated by cytokines, particularly by GM-CSF, in a human hemopoietic cell line. In another system, platelets activated by platelet-activating factor (PAF), we can also demonstrate an increase in the association between the two enzymes following activation of the cells. In addition, increased tyrosine phosphorylation of PKCδ in response to FcεRI receptor activation in another cell type does not lead to any change in association between PKCδ and PI 3-kinase. Therefore, we have discovered an association between PI 3-kinase and one specific PKC isoform that is probably independent of tyrosine phosphorylation of PKCδ and is somehow modulated by specific receptor-mediated events.
RESULTS AND DISCUSSION
The association of PKC isoforms δ and ε with PI 3-kinase was examined in the erythroleukemia cell line TF-1. Cell lysates from TF-1 cells, immunoprecipitated with antibodies to either PKCδ or PKCε and analyzed by immunoblotting with antibodies to the p85 or the p110 subunits of PI 3-kinase, showed that PI 3-kinase was being co-immunoprecipitated (Fig. 1, A and B, and Fig. 2). In reciprocal experiments, immunoprecipitation with antibody to p85 co-immunoprecipitated PKCδ and -ε, as detected by immunoblotting. Other PKC isoforms found in TF-1 cells include α, β, µ, ζ, and θ.
S. L. Ettinger and V. Duronio, unpublished observations.
None of the latter PKC isoforms were found to co-immunoprecipitate with PI 3-kinase, as determined by immunoblotting following immunoprecipitation with anti-p85 antibody, either in untreated cells or following stimulation with GM-CSF, IL-3, or IL-5 (data not shown).
We next determined if this association was inducible by cytokine stimulation. After 15 min of stimulation with GM-CSF, there was an increased association of PI 3-kinase in anti-PKCδ immunoprecipitates. This increased association was marginal in IL-5-treated cells but was not evident in IL-3-treated cells (Fig. 1, A and B). There was no detectable increase in PI 3-kinase protein in any of the PKCε immunoprecipitates (Fig. 2). The difference in the effect using GM-CSF compared with IL-3 was very reproducible at this time point, but further studies should be done to test additional times.
To investigate whether the PI 3-kinase co-immunoprecipitated with PKCδ or PKCε remains active, anti-PKCδ and anti-PKCε immunoprecipitates were analyzed for PI 3-kinase activity in vitro. GM-CSF stimulation of the cells led to increased PI 3-kinase activity associated with PKCδ compared with control samples or IL-5- or IL-3-stimulated cells (Fig. 3A), thereby correlating with the detection of PI 3-kinase in immunoblots. Anti-PKCε immunoprecipitates also contained active PI 3-kinase; however, the PI 3-kinase activity was not affected by cytokine treatment of cells.
We next studied the role of PI 3-kinase activity in mediating the association with PKCδ, taking advantage of two potent inhibitors of PI 3-kinase, wortmannin (WM) and LY-294002. Cells were pretreated with 100 nM WM or 50 µM LY-294002 prior to cytokine stimulation, conditions that are known to inhibit the majority of PI 3-kinase activity (
M. P. Scheid and V. Duronio, submitted for publication.
WM decreased the association of PKCδ with PI 3-kinase in all cases (Fig. 3, A and B), but LY-294002 had no effect (data not shown). The relative decreases were consistently more pronounced in GM-CSF- and IL-5-treated cells than in IL-3-treated cells. WM is known to bind covalently to the active site of the p110 subunit, while LY-294002 is a competitive inhibitor. The contrasting results between WM and LY-294002 suggest that the PI 3-kinase/PKCδ association is not modulated by the lipid products of PI 3-kinase activity but may be affected by alterations of the p110 subunit caused by WM. Alternatively, we cannot rule out the possibility that WM is having its effect on the PI 3-kinase/PKCδ association independently of its effect on PI 3-kinase activity. Other inhibitory effects of WM have been described (
), and in our laboratory we have recently shown that WM has inhibitory effects on other kinases that are not seen with concentrations of LY-294002 that cause an equivalent inhibition of PI 3-kinase activity.3
Recent reports have described tyrosine phosphorylation of PKCδ following stimulation with 12-O-tetradecanoylphorbol-13-acetate, carbachol, substance P, or FcεRI cross-linking (
). In TF-1 cells, a faint band is detected in anti-phosphotyrosine blots of anti-PKCδ immunoprecipitates, and this band comigrates with PKCδ (data not shown). However, we have been unable to detect any differences in the levels of tyrosine phosphorylation following cytokine stimulation. Phosphoamino acid analysis of PKCδ will be necessary to confirm whether PKCδ is being tyrosine-phosphorylated to any significant extent. In another independent system, we have confirmed the results of Haleem-Smith et al. (
), showing that cross-linking of the FcεRI receptor in RBL-2H3 cells leads to a large increase in tyrosine phosphorylation of PKCδ (Fig. 4A), but interestingly, no PI 3-kinase was present in PKCδ immunoprecipitates, either before or after receptor activation (Fig. 4B). Together, these results strongly suggest that the association between PI 3-kinase and PKCδ that we have observed is independent of PKCδ tyrosine phosphorylation and is therefore not likely to be mediated by the SH2 domains of the PI 3-kinase p85 subunit.
To determine if PI 3-kinase associates with PKC isoforms in any other signaling systems we next investigated rabbit platelets treated with the potent agonist, PAF. PAF-treated platelets also showed an increase in PI 3-kinase protein co-immunoprecipitated with PKCδ, compared with controls, as determined on immunoblots using anti-p85 antibodies (Fig. 5A). This association was again inhibited by WM. The PI 3-kinase was active in the PKCδ immunoprecipitates as determined by an in vitro PI 3-kinase assay (Fig. 5B).
We also characterized the serine/threonine kinase activity in anti-p85 immunoprecipitates and to date have found Ca2+-independent protein serine/threonine kinase activity as evidenced by phosphorylation of a Ser-containing peptide substrate corresponding to the PKCε pseudo-substrate site. There were no increases in kinase activity in response to calcium, and the activity was at least partially lipid-dependent. These features are consistent with the characteristics of PKCδ and PKCε activities. We are investigating this further to determine the role PI 3-kinase may play in regulation of the PKC activity, particularly that of PKCδ.
The importance of PI 3-kinase in signal transduction has been pointed out in numerous publications over the past few years. There have been many proteins shown to associate with PI 3-kinase following stimulation of cells, generally as a result of tyrosine-phosphorylated proteins associating via the PI 3-kinase p85 SH2 domains. This type of association is known to result in PI 3-kinase activation, independent of the tyrosine phosphorylation of the p85 subunit itself (
). We demonstrate here for the first time that at least two members of the PKC family of enzymes, PKCδ and PKCε, also associate with PI 3-kinase, and in the case of PKCδ, the association is increased upon stimulation of hemopoietic cells with GM-CSF or upon activation of platelets. In our system, PKCδ appears to be constitutively tyrosine-phosphorylated to a small extent, as seen on immunoblots. However, we cannot discern differences in levels of tyrosine phosphorylation with cytokine stimulation. Several PKC isoforms contain the consensus sequence recognized by PI 3-kinase SH2 domains in the p85 subunit including PKCδ, which has YNYM (
). However, the lack of increased tyrosine phosphorylation of PKCδ following cytokine stimulation, along with the lack of PKCδ/PI 3-kinase association in a system in which PKCδ is heavily tyrosine-phosphorylated suggests that we are observing an association that is unlike previously demonstrated interactions with PI 3-kinase. However, we do not know whether we are observing a direct interaction or one that also involves other proteins.
In the TF-1 cells, IL-3, IL-5, and GM-CSF are each able to provide a complete mitogenic stimulus, while only GM-CSF and, to a lesser extent, IL-5 are able to cause increased PI 3-kinase/PKCδ association. Coupled with the finding that PAF activation of platelets is able to increase the association, the results suggest that the functional role of this interaction may be unrelated to the mitogenic effect of cytokines. There have been conflicting reports regarding the role of PKCδ in mitogenesis (
), the association of PI 3-kinase with various proteins may be unrelated to a role for that enzyme in mitogenesis. One might also speculate that the PI 3-kinase/PKCδ association could be related to the effects of GM-CSF and perhaps IL-5 on inducing differentiation, effects that are distinct from those of IL-3. Further studies will be required to delineate the exact function of this novel association as well as other systems in which this type of regulation may be occurring.
We thank David Fong for technical assistance and Dr. Michael Gold for helpful discussions.