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The Journal of Immunology, 2006, 176: 2407-2413.
Copyright © 2006 by The American Association of Immunologists

Involvement of Protein Kinase C{epsilon} in the Negative Regulation of Akt Activation Stimulated by Granulocyte Colony-Stimulating Factor1

Hong Liu2,*,{dagger}, Yaling Qiu*, Lei Xiao{ddagger} and Fan Dong3,*

* Department of Biological Sciences, University of Toledo, Toledo, OH 43606; {dagger} Department of Hematology, Hospital of Nantong University, Nantong, Jiangsu, China; and {ddagger} University Florida Shands Cancer Center Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL 32610


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Stimulation of cells with G-CSF activates multiple signaling cascades, including the serine/threonine kinase Akt pathway. We show in this study that G-CSF-induced activation of Akt in myeloid 32D was specifically inhibited by treatment with PMA, a protein kinase C (PKC) activator. PMA treatment also rapidly attenuated sustained Akt activation mediated by a carboxy truncated G-CSF receptor, expressed in patients with acute myeloid leukemia evolving from severe congenital neutropenia. The inhibitory effect of PMA was abolished by pretreatment of cells with specific PKC inhibitor GF109203X, suggesting that the PKC pathway negatively regulates Akt activation. Ro31-8820, a PKC{epsilon} inhibitor, also abrogated PMA-mediated inhibition of Akt activation, whereas rottlerin and Go6976, inhibitors of PKC{delta} and PKC{alpha}betaI, respectively, exhibited no significant effects. Furthermore, overexpression of the wild-type and a constitutively active, but not a kinase-dead, forms of PKC{epsilon} markedly attenuated Akt activation, and inhibited the proliferation and survival of cells in response to G-CSF. The expression of PKC{epsilon} was down-regulated with G-CSF-induced terminal granulocytic differentiation. Together, these results implicate PKC{epsilon} as a negative regulator of Akt activation stimulated by G-CSF and indicate that PKC{epsilon} plays a negative role in cell proliferation and survival in response to G-CSF.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The serine/threonine kinase Akt, also termed protein kinase B, plays an important role in cell survival and proliferation. Akt is activated in cells in response to diverse stimuli such as growth factors, cytokines, and hormones through the PI3K pathway (1, 2, 3, 4, 5). PI3K generates phosphoinositides PI(3,4,5)P3 and PI(3,4)P2 that target Akt via its pleckstrin homology domain to the plasma membrane, where it is phosphorylated at Thr308 and Ser473 by phosphoinositide-dependent kinase (PDK)4 1 and a yet to be identified kinase, respectively. Membrane targeting and subsequent phosphorylation result in Akt activation. Akt phosphorylates and regulates the functions of many cellular proteins implicated in apoptosis and proliferation. The activity of Akt is tightly controlled in normal cells, but frequently constitutively activated in many types of human cancer (1, 2, 3, 4, 5).

G-CSF plays a major role in granulopoiesis. Treatment of cells with G-CSF has been shown to activate Akt in a PI3K- and Src family kinase-dependent manner (6, 7). Akt is involved in the regulation of the survival, proliferation, and differentiation of myeloid cells in response to G-CSF (6, 7, 8). Notably, truncation of the C-terminal region of the G-CSF receptor, as seen in patients with acute myelogenous leukemia (AML) evolving from severe congenital neutropenia (SCN), results in markedly prolonged Akt activation (6). Apart from its role in normal granulopoiesis, Akt is constitutively activated in leukemic cells and plays a critical role in cellular transformation by leukemogenic proteins Bcr-Abl, Tel-Jak2, and Flt3/ITD (9, 10, 11, 12, 13).

PKC isozymes comprise a family of related serine-threonine kinases that can be grouped into three categories on the basis of their structural and biochemical properties (14, 15). The classical PKCs are composed of isoforms {alpha}, betaI, betaII, and {gamma}, which are calcium dependent and activated by diacylglycerol (DAG) or phorbol ester. The novel PKCs include isoforms {delta}, {epsilon}, {eta}, {theta}, and µ, which are calcium unresponsive and activated by DAG/phorbol ester. Atypical PKCs consist of isoforms {iota}{lambda} and {zeta}, which are unresponsive to both calcium and DAG/phorbol ester. Specific PKC isoforms play pivotal roles in the regulation of myeloid, erythroid, and megakaryocytic development (16, 17, 18, 19). Expressions of various PKC isoforms are strictly regulated during hemopoietic development (20, 21). Although PKC pathway has been implicated in G-CSF receptor signaling (22), its biological significance remains to be determined.

Despite significant progress in our understanding of the molecular mechanisms by which Akt is activated, relatively less is known about the signaling events that negatively regulate Akt activation. In this work, we show that treatment with PMA, a well-known PKC activator, resulted in complete inhibition of Akt activation by G-CSF in myeloid 32D. The negative effect of PMA was abrogated by preincubation of cells with specific PKC inhibitors. We further show that PKC{epsilon}, whose expression was down-regulated during G-CSF-induced granulocytic differentiation, was involved in mediating the inhibitory effect of PMA. Our data provide the first evidence that PKC{epsilon} negatively regulates Akt activation, and add to the understanding of the complex regulatory mechanism that controls Akt activation in myeloid cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells

The 32D cells stably transfected with the wild-type and truncated (D715) forms of the G-CSF receptor have been described (23). The 32D cells used in this study did not express the endogenous G-CSF receptor. L-G cells (24) were provided by T. Honjo (Kyoto University, Kyoto, Japan). The 32D and L-G cells were grown in RPMI 1640 medium supplemented with 10% FBS and 10% WEHI-3B cell-conditioned medium as a crude source of IL-3, 100 µg/ml penicillin, and 100 µg/ml streptomycin.

Reagents

Anti-Akt Ab, phospho-specific Abs against Akt, PDK1, STAT3, STAT5 Erk1/2, JNK, and p38; and Akt kinase assay kit were purchased from Cell Signaling Technology. Anti-PKC{epsilon} Ab and anti-G-CSF receptor Ab were obtained from Santa Cruz Biotechnology and BD Biosciences, respectively. Anti-FLAG (M2) and anti-beta-actin Abs were purchased from Sigma-Aldrich. ECL kit and GelCode blue stain reagent were purchased from Pierce Biotechnology.

Expression constructs and transfection

The expression constructs for the wild-type, constitutively active (A159E), and kinase-dead (K437R) forms of PKC{epsilon} have been described (25). The 32D and L-G cells were transfected by electroporation and selected in medium containing G418 (0.8 mg/ml) 24 h after transfection. Individual clones were expanded and examined for expression of transfected proteins by Western blotting. Two independent clones for each PKC{epsilon} form were used in subsequent experiments.

Preparation of cell extracts

Cells were washed with ice-cold PBS and resuspended in lysis buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 10 mM NaF, 0.5 mM DTT, 1% Triton X-100, 1 mM PMSF, and 1 mM vanadate). After incubation on ice for 20 min, lysates were cleared by centrifugation at 12,000 rpm for 30 min at 4°C. The preparation of membrane and cytosolic extracts was essentially as described (26). Cells were resuspended in hypotonic buffer (20 mM Tris (pH 7.5), 10 mM EGTA, and 1 mM PMSF) and lysed by 50 strokes in a glass Dounce homogenizer. After centrifugation, the supernatants were collected as cytoplasmic fraction. The membrane pellets were solubilized in hypotonic buffer containing 1% Triton X-100 and centrifuged at 12,000 rpm for 30 min. The resulting supernatants were collected as membrane fractions.

Western blot analysis

Cell extracts were boiled in SDS sample buffer and resolved by SDS-PAGE before transfer to Immobilon membranes. The membranes were incubated with the appropriate Abs, and the reactive proteins were visualized by ECL.

Akt kinase assay

The kinase activity of Akt was analyzed using the Akt kinase assay kit, according to the manufacturer’s protocol. Cells were starved in serum-free medium for 4 h and stimulated with G-CSF for 10 min with or without PMA pretreatment. Akt was immunoprecipitated with an immobilized Akt Ab from equal amounts of whole cell extracts and incubated with a glycogen synthase kinase-3 (GSK-3) fusion protein in the presence of ATP. The reaction mixtures were separated by SDS-PAGE and transferred to Immobilon membranes. The membranes were incubated with a phospho GSK-3{alpha}beta (Ser21/9) Ab supplied in the kit. GSK3 phosphorylation was visualized by chemiluminescence.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
PMA specifically inhibited Akt activation by G-CSF

To investigate the intracellular events that negatively regulate Akt activation, we examined the effect of PMA on G-CSF-stimulated activation of Akt in myeloid 32D cells stably transfected with the wild-type G-CSF receptor (32D/WT) (23). Cells were incubated with PMA for different times before stimulation with G-CSF for 15 min. Whole cell extracts were prepared and examined for Akt phosphorylation by Western blot analysis using Abs that recognize Akt only when phosphorylated on Ser476 or Thr308 (see below). As shown in Fig. 1A, PMA treatment of cells for only 5 min effectively blocked G-CSF-stimulated activation of Akt. PMA significantly attenuated Akt activation even when added with G-CSF simultaneously or 5 min later. In contrast to Akt, G-CSF-stimulated activation of other signaling pathways, including STAT3, STAT5, p38, JNK, and Erk1/2, was not significantly affected by PMA treatment. Thus, it appeared that PMA treatment specifically inhibited Akt activation by G-CSF.


Figure 1
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  		FIGURE 1. PMA inhibited G-CSF-stimulated activation of Akt, but not other signaling pathways. A, 32D/WT cells were either untreated or treated with G-CSF for 15 min following starvation in serum-free medium for 4 h. PMA (100 nM) was added from 60 to 0 min before, or 5 min after addition of G-CSF. Phosphorylation of Akt, STAT3, STAT5, Erk1/2, JNK, and p38 was examined by Western blotting using phospho-specific Abs. The membrane was probed with Akt Ab to show sample loadings. B, 32D/WT cells were treated with PMA for 5 min, washed extensively, and incubated in serum-free medium for the indicated times before stimulation with G-CSF for 15 min. Akt phosphorylation was examined by Western blotting.

 
We then determined the duration of PMA-mediated inhibitory effect. Cells were pretreated with PMA for 15 min, washed extensively to remove PMA, and incubated in serum-free medium for different times before G-CSF stimulation. As shown in Fig. 1B, Akt was not activated by G-CSF during the first 60 min post-PMA treatment, but reactivated by G-CSF after 120 min.

The sustained Akt activation mediated by the truncated G-CSF receptor was also inhibited by PMA

It has been shown that truncation of the C-terminal region of the G-CSF receptor, as seen in patients with AML/SCN, results in prolonged activation of Akt upon G-CSF stimulation (6, 7). We were interested to know whether PKC shortened the duration of Akt activation mediated by the truncated G-CSF receptor. Cells expressing a carboxy truncated G-CSF receptor, which was expressed in AML/SCN patients (27, 28, 29), were incubated with G-CSF for 15 min before addition of PMA. PMA reduced Akt phosphorylation to nearly basal levels within 15 min after it was added to the culture (Fig. 2). In the absence of PMA, Akt remained strongly phosphorylated for at least 90 min. Thus, PMA suppressed sustained Akt activation mediated by the carboxy truncated G-CSF receptor.


Figure 2
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  		FIGURE 2. PMA down-regulated sustained Akt activation mediated by the C-terminally truncated G-CSF receptor. Cells expressing the receptor mutant lacking the C-terminal region of 98 aa were starved in serum-free medium for 4 h and subsequently stimulated with G-CSF for the indicated times. PMA or vehicle control DMSO (Ctr) was added at 15 min of G-CSF stimulation. Akt phosphorylation was examined by Western blotting.

 
The negative effect of PMA on Akt activation was blocked by PKC inhibitors

To evaluate the role of PKC pathway in G-CSF-stimulated Akt activation, we investigated whether PKC inhibitors abrogated the negative effect of PMA. The 32D/WT cells were preincubated with different PKC inhibitors before addition of PMA. PMA-mediated inhibition of Akt activation was completely reversed by GF109203X, an inhibitor of PKC{alpha}, betaI, betaII, {gamma}, {delta}, and {epsilon} isoforms, and PKC{epsilon} inhibitor Ro318220 (30), whereas Go6976, an inhibitor of PKC{alpha} and betaI (31), and PKC{delta} inhibitor rottlerin (32) had no significant effect (Fig. 3A). GF109203X and Ro318220 also blocked the inhibitory effect of PMA on the kinase activity of Akt that was stimulated by G-CSF (Fig. 3B).


Figure 3
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  		FIGURE 3. PKC mediated the inhibitory action of PMA. A, Effects of different PKC inhibitors on PMA-mediated inhibition of Akt activation by G-CSF. The 32D/WT cells were preincubated with Go6976 (Gö; 3 µM), GF109203X (GFX; 5 µM), rottlerin (Rot; 20 µM), and Ro318820 (Ro; 1 µM) before treatment with PMA for 5 min. Cells were subsequently stimulated with G-CSF for 15 min, and Akt phosphorylation was examined. B, Assay of Akt kinase activity. Cells were treated as above. Akt was immunoprecipitated from equal amounts of whole cell extracts and incubated with the GSK-3 fusion protein. The phosphorylation of the GSK-3 fusion protein was determined by immunoblotting with the phospho GSK-3{alpha}beta (Ser21/9) Ab (upper panel). The membrane was stained with GelCode blue stain reagent to show the GSK-3 fusion protein (lower panel). C, Membrane translocation of PKC{epsilon} upon PMA stimulation. The 32D/WT cells and 32D/WT cells overexpressing PKC{epsilon} were treated with PMA for 15 min. Membrane and cytosolic extracts were prepared and examined for PKC{epsilon} by Western blot analysis. The levels of beta-actin in cytosolic extracts and the G-CSF receptor in membrane extracts were also determined to show sample loadings.

 
The results obtained with the different PKC inhibitor suggested that the classical and/or novel PKC isoforms, including PKC{epsilon}, might be involved in the negative regulation of Akt activation. We further investigated whether PKC{epsilon} was activated by PMA treatment by examining its membrane translocation. As shown in Fig. 3C, PKC{epsilon} was translocated to plasma membrane upon PMA treatment in 32D/WT cells as well as in 32D/WT cells overexpressing PKC{epsilon}.

PMA treatment did not affect PDK1 phosphorylation and membrane localization

PDK1 is involved in Akt activation by phosphorylating Akt at Thr308. PDK1 is activated by phosphorylation of Ser241 located in the activation loop of its kinase domain (33), which correlates with Akt activation (34). To assess whether PMA treatment influenced PDK1 activation, we examined PDK1 phosphorylation at Ser241 by Western blot analysis. PDK1 phosphorylation was not altered by PMA treatment (Fig. 4A). Because PDK1 activation may also involve its translocation to the plasma membrane (35, 36), we investigated whether PMA treatment altered PDK1 membrane localization. As shown in Fig. 4B, treatment of 32D cells with PMA did not significantly affect the membrane localization of PDK1.


Figure 4
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  		FIGURE 4. PDK1 and PP2A were not the targets of regulation by the PKC pathway. A, Effect of PMA on PDK1 phosphorylation. The 32D/WT cells were treated with PMA for 5 min with or without preincubation with the different PKC inhibitors, as indicated for 30 min. Cells were subsequently stimulated with G-CSF for 15 min and examined for PDK1 phosphorylation by Western blot analysis. B, Effect of PMA on PDK1 membrane localization. The 32D/WT cells were treated as above. Membrane and cytosolic extracts were prepared, and equal amounts of extracts were examined for the presence of different proteins, as indicated. C, Effect of okadaic acid on PMA-mediated attenuation of Akt activation. The 32D/WT cells were incubated with okadaic acid for 30 min at concentrations, as indicated, before PMA treatment. Cells were then stimulated with G-CSF for 15 min. Akt phosphorylation was examined.

 
The negative effect of PMA was not abolished by inhibition of protein phosphatase 2A (PP2A) activity

PP2A has been shown to dephosphorylate Akt and negatively regulate its activity (37, 38, 39, 40). To assess the potential involvement of PP2A in PMA-mediated inhibition of Akt activation, we examined whether the inhibitory effect of PMA was abolished by pretreatment of cells with okadaic acid, a PP2A inhibitor. Preincubation of 32D/WT cells with okadaic acid at concentrations up to 50 µM had no significant effect on PMA-mediated inhibition of Akt activation (Fig. 4C and data not shown). Okadaic acid alone did not affect Akt phosphorylation induced by G-CSF. Together, the results suggested that PP2A might not play a major role in the regulation of Akt activation by G-CSF in 32D cells.

Overexpression of PKC{epsilon} inhibited Akt activation by G-CSF

To directly demonstrate the involvement of PKC{epsilon} in the negative regulation of Akt activation, we examined whether overexpression of PKC{epsilon} and the constitutively active PKC{epsilon} AE (25, 41) affected Akt activation. When overexpressed, a portion of PKC{epsilon} was constitutively translocated to the membrane in 32D/WT cells (Fig. 3C). G-CSF-stimulated activation of Akt was markedly attenuated upon overexpression of PKC{epsilon} or PKC{epsilon} AE, but not the kinase-dead PKC{epsilon} KR (Fig. 5A). Notably, treatment of cells with PKC inhibitors GF109203X and Ro318220, but not with Go6976 and rottlerin, restored Akt activation (Fig. 5B). GF109203X and Ro318220 appeared less effective in abrogating the inhibitory effect of PKC{epsilon} AE, suggesting that the two inhibitors may be less efficient in suppressing the activity of PKC{epsilon} AE.


Figure 5
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  		FIGURE 5. Effects of overexpression of the different forms of PKC{epsilon} on Akt activation by G-CSF. A, 32D/WT cells were stably transfected with the empty vector (Ctr) or expression constructs for the wild-type (WT), constitutively active (AE), and the kinase-dead (KR) forms of PKC{epsilon}, as indicated. Cells were starved in serum-free medium for 4 h before stimulation with G-CSF for 15 min. Akt phosphorylation was examined by Western blotting. B, 32D/WT cells expressing the WT or AE form of PKC{epsilon} were pretreated with Go6976 (Gö), GF109203X (GFX), rottlerin (Rot), or Ro318820 (Ro) for 30 min before G-CSF stimulation. C, 32D/WT cells transfected with the empty vector (Ctr), PKC{epsilon}, or PKC{epsilon} AE were left untreated or treated with PMA for 5 min before stimulation with IL-3 for 15 min. Akt phosphorylation was examined by Western blot analysis.

 
In addition to G-CSF, IL-3 has also been shown to stimulate Akt activation. We investigated whether IL-3-stimulated Akt activation was affected by PKC{epsilon} overexpression. As shown in Fig. 5C, Akt was activated by IL-3 in 32D/WT cells transfected with the empty vector, but not in cells overexpressing PKC{epsilon} and PKC{epsilon} AE. Thus, PKC{epsilon} inhibited Akt activation that was stimulated by G-CSF and IL-3.

Overexpression of PKC{epsilon} inhibited cell proliferation and survival stimulated by G-CSF

We further examined G-CSF- and IL-3-dependent proliferation and survival of 32D/WT cells overexpressing PKC{epsilon}, PKC{epsilon} AE, or PKC{epsilon} KR. Cells were cultured in IL-3- or G-CSF-containing medium. Cell numbers and viability were determined on different days. The 32D/WT cells transfected with PKC{epsilon} and PKC{epsilon} AE barely grew and lost viability rapidly when cultured in G-CSF-containing medium (Fig. 6). Few living cells could be seen after culture in G-CSF for 5 or 6 days. The 32D/WT cells transfected with the empty vector or PKC{epsilon} KR showed comparable growth and survival. Notably, although PKC{epsilon} inhibited IL-3-stimulated Akt activation, overexpression of PKC{epsilon} and PKC{epsilon} AE had no or only minimal effect on the proliferation and survival of 32D/WT cells cultured in IL-3. Thus, PKC{epsilon} specifically inhibited cell proliferation and survival stimulated by G-CSF.


Figure 6
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  		FIGURE 6. Effects of overexpression of the different forms of PKC{epsilon} on cell proliferation and survival stimulated by G-CSF or IL-3. The 32D/WT cells stably transfected with the empty vector (Ctr) or with the WT, AE, and KR forms of PKC{epsilon} were cultured in medium containing G-CSF (A and B) or IL-3 (C and D). The numbers of viable cells (A and C) and cell viabilities (B and D) were determined daily for up to 6 days using the trypan blue exclusion method. Comparable results were obtained with two independent clones for each PKC{epsilon} form.

 
In addition to 32D cells, Akt activation in myeloid L-G cells was also inhibited by PMA treatment (data not shown). L-G cells expressed the endogenous G-CSF receptor and underwent terminal granulocytic differentiation in response to G-CSF (24). To examine whether PKC{epsilon} overexpression in L-G cells also inhibited G-CSF-dependent proliferation and survival, we stably transfected L-G cells with the expression constructs for PKC{epsilon} and PKC{epsilon} AE. The expression of PKC{epsilon} and PKC{epsilon} AE was confirmed by Western blot analysis (Fig. 7A). L-G cells showed transient and modest growth in G-CSF-containing medium (Fig. 7B). Overexpression of PKC{epsilon} and PKC{epsilon} AE in L-G cells resulted in rapid loss of viability, and essentially no living cells were seen after culture in G-CSF for 3 days.


Figure 7
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  		FIGURE 7. Effects of overexpression of PKC{epsilon} and PKC{epsilon} AE on the proliferation and survival of L-G cells. A, L-G cells were stably transfected with empty vector or PKC{epsilon} and PKC{epsilon} AE, and examined for expression of PKC{epsilon} proteins by Western blotting. B, Cells as indicated were washed and cultured in G-CSF-containing medium. The numbers of vial cells were determined on different days.

 
Expression of PKC{epsilon} was down-regulated when 32D/WT and L-G cells were induced to differentiate

G-CSF induced the terminal granulocytic differentiation of 32D/WT and L-G cells (24, 27). We investigated whether the expression of PKC{epsilon} altered during G-CSF-induced granulocytic differentiation. Expression of PKC{epsilon} was examined by Western blot analysis. As shown in Fig. 8, PKC{epsilon} protein decreased gradually when 32D/WT and L-G cells were induced to differentiate, and was barely detectable after culture of the cells in G-CSF for 5 or 6 days.


Figure 8
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  		FIGURE 8. Expression of PKC{epsilon} in myeloid cells at different stages of granulocytic differentiation. The 32D/WT (A) and L-G (B) cells were induced to differentiate with G-CSF for days, as indicated. Whole cell extracts were prepared and examined for PKC{epsilon} by Western blotting. The membrane was reprobed with anti-beta-actin Ab to show sample loadings.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Akt plays an important role in the survival, proliferation, and differentiation of myeloid cells in response to G-CSF (6, 7, 8). In this work, we have shown that PKC activator PMA specifically inhibits G-CSF-stimulated activation of Akt, but not the other signaling pathways in myeloid 32D. PMA also inhibits Akt activation in myeloid L-G cells and pro-B Ba/F3 cells stably transfected with the G-CSF receptor (data not shown). The inhibitory effect of PMA is abrogated by preincubation of cells with specific PKC inhibitors, indicating that the PKC pathway negatively regulates Akt activation by G-CSF. We have further demonstrated that the inhibitory effect of PMA is mediated, at least in part, by PKC{epsilon}. Notably, expression of PKC{epsilon} is down-regulated during G-CSF-induced granulocytic differentiation, and overexpression of PKC{epsilon} suppresses the survival and proliferation of 32D and L-G cells stimulated by G-CSF.

PMA has previously been shown to inhibit Akt activation in different cell types (42, 43, 44, 45). Interestingly, the PKC isoforms involved in mediating PMA effect were different in these studies. For instance, activation of PKC{alpha} by PMA was shown to inhibit insulin-induced Akt phosphorylation in vascular smooth muscle cells, whereas PKC{delta} was suggested to be involved in PMA-induced inhibition of Akt phosphorylation in a prostate cancer cell line (44, 45). In 32D cells, however, the negative effect of PMA was not abolished by preincubation of cells with PKC{alpha}betaI inhibitor Go6976 (31) and PKC{delta} inhibitor rottlerin (32), suggesting that PKC{alpha}, betaI, and {delta} are unlikely the major players in PMA-induced inhibition of Akt activity. Our data implicate PKC{epsilon} as an important mediator of the inhibitory effect of PMA. However, it is of note that G-CSF-stimulated Akt activation was not always completely blocked by overexpression of PKC{epsilon} and PKC{epsilon} AE in 32D cells (Fig. 5A). Further studies are needed to investigate whether other PKC isoforms such as PKCbetaII and {gamma} are also involved in PMA-induced inhibition of Akt activation.

The mechanism by which PKC{epsilon} negatively regulates Akt activation is still speculative. PMA treatment had no significant effect on PDK1 phosphorylation and membrane localization. The inhibitory effect of PMA was not abolished by PP2A inhibitor okadaic acid. These results suggest that PDK1 and PP2A may not be the targets of regulation by PMA. Akt activation by G-CSF is PI3K and Src family kinase dependent (6, 7). The Src family kinases Lyn and Hck are activated by G-CSF and are required for G-CSF-stimulated cell proliferation and survival (46, 47). Notably, PMA has been shown to inhibit the activation of PI3K and Lyn stimulated by GM-CSF in human neutrophils (48). Thus, it is possible that PMA-mediated inhibition of Akt activation may result from the negative effect of PMA on PI3K and Lyn activation. However, the possibility cannot be excluded that PKC{epsilon} might target other components of the G-CSF receptor/Akt-signaling pathway.

Our results are in contrast to several recent studies that implicate PKC{epsilon} as a positive regulator of Akt activation in nonhematopoietic cells (49, 50, 51). Akt appeared to act downstream of PI3K and PDK1, as suggested by the fact that a kinase-dead PKC{epsilon} mutant inhibited insulin-induced activation of Akt, but not PI3K and PDK1, in Chinese hamster ovary cells (49). Furthermore, PKC{epsilon} was shown to interact with Akt in prostate cancer cells and mouse heart, and to directly phosphorylate and activate Akt in vitro (50, 51). Thus, it is plausible that the role of PKC{epsilon} in Akt activation may depend on the types of cells and stimuli.

The biological significance of PKC{epsilon}-mediated inhibition of Akt activation in granulopoiesis remains to be explored. Overexpression of PKC{epsilon}, which results in its constitutive membrane translocation (see Fig. 3C), inhibits the survival and proliferation of myeloid 32D and L-G cells stimulated by G-CSF, consistent with its negative role in Akt activation. Although PKC{epsilon} also inhibits IL-3-stimulated Akt activation, overexpression of PKC{epsilon} or PKC{epsilon} AE has no significant effect on the proliferation and survival of 32D and L-G cells cultured in IL-3 (Figs. 6 and 7, and data not shown), indicating that PKC{epsilon} specifically suppresses G-CSF-dependent cell proliferation and survival. Notably, expression of PKC{epsilon} was down-regulated in 32D and L-G cells that were induced with G-CSF to undergo terminal granulocytic differentiation. Myeloid progenitor cells gradually lose the abilities for proliferation and survival with terminal granulocytic differentiation. It is possible that PKC{epsilon} down-regulation may be necessary for the survival of myeloid progenitor cells in order for them to complete the differentiation process. It is also of note that the sustained Akt activation mediated by the carboxy truncated G-CSF receptor, which is associated with AML development in patients with SCN, is rapidly attenuated by PMA treatment in 32D cells. Akt is frequently constitutively activated in leukemia and may contribute to leukemogenesis (9, 10, 11, 12, 13). Given its negative role in Akt activation and in the survival and proliferation of myeloid cells, it would be interesting to examine PKC{epsilon} expression in leukemic cells and to investigate the effect of stimulating PKC{epsilon} activity on leukemogenic transformation.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by Grants RO1CA92172 (to F.D.) and RO1 CA88815 (to L.X.) from the National Institutes of Health. Back

2 Current address: Department of Hematology, Hospital of Nantong University, No. 20 Xi Si Road, Nantong, Jiangsu, China. Back

3 Address correspondence and reprint requests to Dr. Fan Dong, Department of Biological Sciences, University of Toledo, 2801 West Bancroft Street, Toledo, OH 43606. E-mail address: fdong{at}utnet.utoledo.edu Back

4 Abbreviations used in this paper: PDK, phosphoinositide-dependent kinase; AML, acute myelogenous leukemia; DAG, diacylglycerol; GSK, glycogen synthase kinase; PP2A, protein phosphatase 2A; SCN, severe congenital neutropenia. Back

Received for publication April 27, 2005. Accepted for publication November 22, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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