INTRODUCTION

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 p21^ras/mitogen-activated protein kinase network and the other is the PI¹ 3-kinase pathway. Following ligand binding, the common  subunit of receptors for interleukin (IL)-3, IL-5, and granulocyte/macrophage colony-stimulating factor (GM-CSF) becomes tyrosine-phosphorylated (1, 2). Several other signaling proteins including JAK-2 (3), STAT-5 (4), p46, and p52 Shc (5, 6), p42/44 mitogen-activated protein kinases (7, 8), and SH-PTP2 (9) 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 (3), 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 p21^ras activator, m-SOS1, have been shown to be involved in the activation of p21^ras (5, 6, 10), and activation of PI 3-kinase in hemopoietic cells is a result of cytokine-induced association of PI 3-kinase with specific tyrosine-phosphorylated proteins (11, 12, 13, 14). A tyrosine-phosphorylated 70-kDa substrate was shown to associate with PI 3-kinase following treatment of cells with IL-3 or GM-CSF (14), although this substrate was subsequently shown to be the tyrosine phosphatase, SH-PTP2, and its association may be indirect in a complex with Grb-2 (9).

The involvement of p21^ras in mitogenesis is well understood, but the role of PI 3-kinase, which is thought to be essential as well (15), is less well characterized. Recent evidence suggests that two serine/threonine kinases, p70 S6 kinase (16, 17) and PKB (18), are thought to be activated downstream of PI 3-kinase. In hemopoietic cells, we showed previously that IL-3, IL-5, and GM-CSF as well as IL-4 and Steel factor all activate PI 3 kinase (14), and indeed, this signal is important in the ability of cytokines to inhibit apoptosis (19).

An intriguing set of observations made in the past few years has suggested that PI 3,4-P₂ and PI 3,4,5-P₃ can activate several novel and atypical PKC isoforms (PKC, -, -, and -) (20, 21). 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 (22, 23), 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 (24, 25, 26).

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, PKC, 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 PKC in response to FcRI receptor activation in another cell type does not lead to any change in association between PKC 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 PKC and is somehow modulated by specific receptor-mediated events.

EXPERIMENTAL PROCEDURES

TF-1 cells, a human erythroleukemia cell line (a kind gift from Dr. T. Kitamura, DNAX, Palo Alto, CA), were maintained in RPMI 1640 (Life Technologies, Inc.) with 10% fetal bovine serum (Intergen) plus 5 ng/ml IL-5 (purified recombinant human IL-5 prepared in a baculovirus expression system was kindly provided by Dr. D. Nicholson, Merck-Frosst, Kirkland, Quebec). In experiments in which cells were stimulated with cytokines, human recombinant cytokines were used; IL-3 and GM-CSF were from R&D Systems and IL-5 as described above. RBL-2H3 cells (a kind gift from Dr. John Rivera, NIH) were maintained in RPMI 1640 with 10% fetal bovine serum. Cells were trypsinized and passaged every 3 days.

TF-1 cells, grown in complete RPMI with IL-5, were placed in cytokine-free medium with 1% serum 18 h prior to assay. Cells (5 × 10⁶/500 µl) were washed three times in Hanks' balanced salt solution buffered with 20 mM Hepes, pH 7.4, and then preincubated in RPMI 1640 buffered with 20 mM Hepes, pH 7.4, at 37 °C for 30 min. Recombinant human GM-CSF (50 ng/ml), IL-3 (100 ng/ml), or IL-5 (500 ng/ml) were added for 15 min. Cells were solubilized in 500 µl of solubilization buffer (50 mM Tris-Cl, pH 7.7, 1% Triton X-100, 10% glycerol, 100 mM NaCl, 2.5 mM EDTA, 10 mM NaF, 0.2 mM Na₃VO₄, 1 mM Na₂MoO₄, 40 µg/ml phenylmethylsulfonyl fluoride, 1 µM pepstatin, 0.5 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor), centrifuged to remove debris, and then mixed with immunoprecipitating antibodies (anti-PKC mouse monoclonal (Transduction Laboratories; 2 µg/500 µl of solubilization buffer), anti-PKC rabbit polyclonal IgG (Life Technologies, Inc.; 4 µg/500 µl of cell lysate), or anti-p85 rabbit polyclonal antiserum (raised against the p85 SH2-glutathione S-transferase fusion protein; a kind gift from Dr. Melanie Welham; 1 µl/500 µl of cell lysate) for 1 h on ice. Following addition of protein A-Sepharose beads (Pharmacia Biotech Inc.), samples were rotated at 4 °C for 1 h. The beads were washed 5 times with cold solubilization buffer. For immunoblots, proteins were eluted with SDS sample buffer containing 2-mercaptoethanol and boiled at 90 °C for 1 min prior to loading on 8.0% polyacrylamide gels using an acrylamide:bis ratio of 118:1. Proteins were transferred onto nitrocellulose using an LKB Novablot semi-dry transfer apparatus. The nitrocellulose blots were preincubated with Tris-buffered saline (5% bovine serum albumin, 1% ovalbumin, 0.05% NaN₃) when blotted with anti-phosphotyrosine antibody 4G10 (Upstate Biotechology Inc., Lake Placid, NY), anti-p85, or anti-p110 (Santa Cruz Biotechology), or they were blocked using 5% skim milk powder for immunoblots with anti-PKC, -, -, -, -µ, or - (Santa Cruz Biotechology).

Following immunoprecipitation as described above, beads were used to assay for PI 3-kinase activity as described (14, 27). Briefly, the beads were washed twice with solubilization buffer and three times with 10 mM Tris-Cl buffer, pH 7.4. Phosphatidylinositol (10 µg/sample) in 30 mM Hepes, pH 7.4, and 40 µl of kinase buffer (30 mM Hepes, pH 7.4, 30 mM MgCl₂, 50 µM ATP, 200 µM adenosine, [³²P]ATP 10 µCi/µl) were added to the beads. The reaction was stopped after 15 min with 1 N HCl. Chloroform:methanol (1:1) was used to extract the lipid, and the labeled PI-3-P was separated by thin layer chromatography using oxalate-treated aluminum-backed silica gels (EM Scientific) and solvent comprised of NH₄OH:water:methanol:chloroform. Rabbit platelets were prepared as described (28).

FcRI Cross-linking in RBL-2H3 Cells

RBL-2H3 cells were trypsinized and washed in RPMI with serum. Cells were passively sensitized by incubation in the same medium with supernatant from a monoclonal antibody line producing IgE specific for dinitrophenol (a kind gift from Dr. John Rivera, NIH) at a dilution of 1:25. After 1 h at 37 °C, cells were washed twice in Tyrode's buffer, pH 7.4, with Ca²⁺ and Mg²⁺ added, and resuspended in the same buffer at 5 × 10⁶ cells/ml. Cross-linking of the FcRI receptor was achieved by addition of dinitrophenol-human serum albumin (Sigma) at a final concentration of 400 ng/ml. At various times, cells were centrifuged, then solubilized, and PKC-immunoprecipitated as described above.

RESULTS AND DISCUSSION

The association of PKC isoforms  and  with PI 3-kinase was examined in the erythroleukemia cell line TF-1. Cell lysates from TF-1 cells, immunoprecipitated with antibodies to either PKC or PKC 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 PKC and -, as detected by immunoblotting. Other PKC isoforms found in TF-1 cells include , , µ, , and .² 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).

Anti-PKC immunoprecipitates from TF-1 cell lysates contain PI 3-kinase.

TF-1 cell lysates immunoprecipitated with anti-PKC antibody and analyzed by immunoblotting contain PI 3-kinase.

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-PKC 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 PKC 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 PKC or PKC remains active, anti-PKC and anti-PKC 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 PKC 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-PKC immunoprecipitates also contained active PI 3-kinase; however, the PI 3-kinase activity was not affected by cytokine treatment of cells.

Cell lysates immunoprecipitated with anti-PKC contain active PI 3-kinase.

We next studied the role of PI 3-kinase activity in mediating the association with PKC, 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 (29, 30).³ WM decreased the association of PKC 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/PKC 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/PKC association independently of its effect on PI 3-kinase activity. Other inhibitory effects of WM have been described (31), 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 PKC following stimulation with 12-O-tetradecanoylphorbol-13-acetate, carbachol, substance P, or FcRI cross-linking (32, 33, 34). In TF-1 cells, a faint band is detected in anti-phosphotyrosine blots of anti-PKC immunoprecipitates, and this band comigrates with PKC (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 PKC will be necessary to confirm whether PKC is being tyrosine-phosphorylated to any significant extent. In another independent system, we have confirmed the results of Haleem-Smith et al. (34), showing that cross-linking of the FcRI receptor in RBL-2H3 cells leads to a large increase in tyrosine phosphorylation of PKC (Fig. 4A), but interestingly, no PI 3-kinase was present in PKC immunoprecipitates, either before or after receptor activation (Fig. 4B). Together, these results strongly suggest that the association between PI 3-kinase and PKC that we have observed is independent of PKC tyrosine phosphorylation and is therefore not likely to be mediated by the SH2 domains of the PI 3-kinase p85 subunit.

Tyrosine phosphorylation of PKC does not result in its association with PI 3-kinase.

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 PKC, 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 PKC immunoprecipitates as determined by an in vitro PI 3-kinase assay (Fig. 5B).

Platelet lysates immunoprecipitated with anti-PKC antibody also contain PI 3-kinase.

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 PKC 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 PKC and PKC activities. We are investigating this further to determine the role PI 3-kinase may play in regulation of the PKC activity, particularly that of PKC.

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 (35). We demonstrate here for the first time that at least two members of the PKC family of enzymes, PKC and PKC, also associate with PI 3-kinase, and in the case of PKC, the association is increased upon stimulation of hemopoietic cells with GM-CSF or upon activation of platelets. In our system, PKC 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 PKC, which has YNYM (36), and PKC, which has YEMM (37). However, the lack of increased tyrosine phosphorylation of PKC following cytokine stimulation, along with the lack of PKC/PI 3-kinase association in a system in which PKC 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/PKC 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 PKC in mitogenesis (38, 39). Furthermore, as suggested by Myers et al. (40), 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/PKC 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.