science pharmaceutical expo biotech jobs
Originally published online as doi:10.1189/jlb.0106041 on August 29, 2006

Published online before print August 29, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0106041v1
80/6/1473    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Barragán, M.
Right arrow Articles by Gil, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Barragán, M.
Right arrow Articles by Gil, J.
(Journal of Leukocyte Biology. 2006;80:1473-1479.)
© 2006 by Society for Leukocyte Biology

Regulation of Akt/PKB by phosphatidylinositol 3-kinase-dependent and -independent pathways in B-cell chronic lymphocytic leukemia cells: role of protein kinase Cβ

Montserrat Barragán*,1, Mercè de Frias*,1, Daniel Iglesias-Serret*, Clara Campàs*, Esther Castaño*, Antonio F. Santidrián*, Llorenç Coll-Mulet*, Ana M. Cosialls*, Alicia Domingo{dagger}, Gabriel Pons* and Joan Gil*,2

* Unitat de Bioquímica, Departament de Ciències Fisiològiques II, IDIBELL-Universitat de Barcelona, Campus de Bellvitge, L’Hospitalet de Llobregat, Barcelona, Spain; and
{dagger} Servei d’Hematologia, IDIBELL-Hospital Universitari de Bellvitge, Campus de Bellvitge, L’Hospitalet de Llobregat, Barcelona, Spain

2 Correspondence: Departament de Ciències Fisiològiques II, IDIBELL-Universitat de Barcelona, Campus de Bellvitge, Pavelló de Govern, 4ª planta, L’Hospitalet de Llobregat, Barcelona E-08907, Spain. E-mail: jgil{at}ub.edu


arrow
ABSTRACT
 
Apoptosis of B cell chronic lymphocytic leukemia (B-CLL) cells is regulated by the PI-3K-Akt pathway. In the present work, we have analyzed the mechanisms of Akt phosphorylation in B-CLL cells. Freshly isolated cells present basal Akt phosphorylation, which is PI-3K-dependent, as incubation with the PI-3K inhibitor LY294002 decreased Ser-473 and Thr-308 phosphorylation in most samples analyzed (seven out of 10). In three out of 10 cases, inhibition of protein kinase C (PKC) inhibited basal Akt phosphorylation. Stromal cell-derived factor-1{alpha}, IL-4, and B cell receptor activation induced PI-3K-dependent Akt phosphorylation. PMA induced the phosphorylation of Akt at Ser-473 and Thr-308 and the phosphorylation of Akt substrates, independently of PI-3K in B-CLL cells. In contrast, PKC-mediated phosphorylation of Akt was PI-3K-dependent in normal B cells. Finally, a specific inhibitor of PKCβ blocked the phosphorylation and activation of Akt by PMA in B-CLL cells. Taken together, these results suggest a model in which Akt could be activated by two different pathways (PI-3K and PKCβ) in B-CLL cells.

Key Words: apoptosis • signal transduction


arrow
INTRODUCTION
 
Apoptosis of B cell chronic lymphocytic leukemia (B-CLL) cells is regulated by multiple signal transduction pathways (reviewed in ref. [1 ]). Several reports have described the role of PI-3K in B-CLL cell survival [2 3 4 5 6 7 8 9 10 ]. Thus, it has recently been reported that inhibition of constitutively activated PI-3K induces apoptosis in B-CLL cells [4 , 5 ]. Moreover, PI-3K inhibition increases the sensitivity of B-CLL cells to fludarabine, dexamethasone, and chlorambucil ex vivo [4 , 6 ] and blocks the survival effect of engagement of the antigen receptor, phorbol esters, IL-4, albumin, and lysophosphatidic acid in B-CLL cells [2 , 4 , 8 9 10 ].

The most characterized target of PI-3K involved in cell survival is Akt, also known as protein kinase B (PKB), which is a point of convergence of several signaling pathways involved in cell proliferation, survival, differentiation, and metabolism in most cell types including B lymphocytes [11 12 13 ]. It is important that deregulation of Akt has been implicated in the development of human cancer, and this kinase could be a target for cancer therapy [14 15 16 ]. Akt resides in the cytosol in a low-activity conformation, and it is activated through recruitment to cell membranes by PI-3K lipid products and phosphorylation. Akt phosphorylation occurs at the catalytic domain (Thr-308) by 3'-phosphoinositide-dependent kinase-1 (PDK-1) [17 ]. The mechanism leading to phosphorylation of Ser-473 is unclear [18 , 19 ], and candidate Ser-473 kinases include MAPK-activated protein kinase 2 (MK2) [20 , 21 ], DNA-dependent protein kinase [22 ], PKCβ [23 ], and Rictor-mammalian target of rapamycin (mTOR) complex [24 ]. Active Akt phosphorylates and inactivates multiple proteins involved in the control of apoptosis, including Bad, caspase-9, glycogen synthase kinase-3 (GSK-3), and the Forkhead family of transcription factors [11 , 13 , 14 ].

Although several recent reports propose a role for Akt in B-CLL cell survival [4 , 6 7 8 ], the regulation of Akt and its role in the survival of B-CLL cells are poorly characterized. First, it is controversial whether B-CLL cells present basal Akt activity [4 5 6 7 8 ]. Second, it is not clear whether the phosphorylation of Akt is dependent on PI-3K. Thus, inhibition of PI-3K decreases Akt phosphorylation induced by plasma [3 ], anti-IgM [2 ], IL-4 [4 ], or albumin [6 ]; however, it does not affect PKC-induced Akt phosphorylation [4 ]. Third, although phorbol esters and survival signals induce phosphorylation of Akt in Ser-473 [4 ], the effect on the phosphorylation of Thr-308, necessary for full activation of Akt, has not been analyzed. Here, we examine the signal transduction pathways involved in the phosphorylation and activation of Akt in B-CLL cells.


arrow
MATERIALS AND METHODS
 
Cytokines and chemical reagents
PMA, staurosporine (Ssp), and PMSF were purchased from Sigma Chemical Co. (St. Louis, MO). Recombinant human IL-4 and stromal cell-derived factor-1{alpha} (SDF-1{alpha}) were purchased from Immunotools (Friesoythe, Germany). Bisindolylmaleimide I (Bis I), SB203580, PD98059 and LY294002 were purchased from Calbiochem-Novabiochem (San Diego, CA). LY333531 and SH-5 were purchased from Alexis Biochemicals (Lausen, Switzerland). Goat F(ab')2 anti-human IgM was purchased from Southern Biotechnology Associates (Birmingham, AL), and Triciribine was purchased from Biaffin GmbH and Co KG (Kassel, Germany).

B-CLL patients and cell isolation
B-CLL was diagnosed according to standard clinical and laboratory criteria. Cells were obtained from the Hospital de Bellvitge, L’Hospitalet de Llobregat (Spain). Written informed consent was obtained from all patients. Mononuclear cells from peripheral blood samples were isolated by centrifugation on a Ficoll/Hypaque (Seromed, Berlin, Germany) gradient and cryopreserved in liquid nitrogen in the presence of 10% DMSO.

Normal B and T lymphocyte isolation
Four whole blood samples from healthy donors were obtained from the Hospital de Bellvitge, L’Hospitalet de Llobregat. B lymphocytes or T lymphocytes were isolated using RosetteSepTM human B cell or RosetteSepTM human T cell enrichment cocktails (StemCell Technologies, Vancouver, Canada). Briefly, 1 ml RosetteSepTM was added to 20 ml whole blood. The mixed solution was incubated at room temperature for 20 min. Then, the sample was diluted with an equal volume of PBS plus 2% heat-inactivated FBS (Gibco-Invitrogen, Frederick, MD), layered on the top of Ficoll/Hypaque medium, and centrifuged at 1200 g for 20 min. B or T cells were removed from the density medium/plasma interface and washed twice with PBS plus 2% FBS before culture.

Cell culture
Cells were cultured at a concentration of 5 x 106 cells/ml in RPMI-1640 culture medium supplemented with 2 mM glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Biological Industries, Kibbutz, Beit Haemek, Israel) and 10% heat-inactivated FBS at 37°C in a humidified atmosphere containing 5% carbon dioxide.

Flow cytometry analysis of cell viability
Cell viability was determined simultaneously by double-staining with FITC-conjugated annexin V (Bender MedSystem, Vienna, Austria) and propidium iodide (PI), as described previously [4 ]. Briefly, 2 x 105 B-CLL cells were washed in PBS and resuspended in 100 µL annexin V binding buffer (10 mM HEPES, pH 7.4, 2.5 mM CaCl2, 140 mM NaCl) containing 1 µl FITC annexin V. After 15 min of incubation in the dark at room temperature, cells were diluted with 100 µl annexin V binding buffer containing 1 µg/ml PI and analyzed with a FACSCalibur (Becton Dickinson, San Jose, CA). Data analysis was performed with CellQuest software (Becton Dickinson). Cell viability was measured as the percentage of annexin V- and PI-negative cells.

Western blot analysis of protein phosphorylation
Lymphocytes from B-CLL patients were incubated at a density of 5 x 106 cells/ml for the indicated period of time, washed in ice-cold PBS, resuspended in lysis buffer (Tris-buffered saline containing 1% Triton X-100, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 5 mM EDTA, 40 mM β-glycerophosphate, 100 mM NaCl, 1 µg/ml pepstatin, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 mM benzamidine, and 1 mM PMSF), and incubated on ice for 15 min. After 1 min of vortexing, lysates were spun at 16,000 g for 15 min at 4°C, supernatants were collected, and protein concentration was determined using the Micro bicinchoninic acid protein assay reagent kit (Pierce, Rockford, IL). For detection of phosphorylated forms of Akt, 50 µg each protein lysate was separated on 12% SDS-polyacrylamide gels, transferred to Immobilon-P membranes (Millipore, Bedford, MA), and analyzed by Western blotting. After binding with HRP-conjugated secondary antibodies, blots were visualized with the ECL detection system (Amersham, Little Chalfont, Buckinghamshire, UK). Antibodies against phospho-Akt (Ser-473), phospho-ERK (Thr-202/Tyr-204), phospho-FoxO3 (member of the Forkhead box O family), and phospho-substrates of Akt were purchased from Cell Signaling Technologies (Beverly, MA). The antibody against phospho-Akt (Thr-308) was purchased from Affinity (Golden, CO), and the antibody against phospho-GSK3{alpha}/β was from Upstate Biotechnology (Lake Placid, NY). As a confirmation of equal loading and transfer proteins, blots were stripped and reprobed with antibodies against total Akt (Santa Cruz Biotechnology, Inc., CA), ERK (Upstate Biotechnology), or {alpha}-tubulin (Sigma Chemical Co.).

Statistical analysis
Data were analyzed using the SPSS (Chicago, IL) 11.5 software package. Results are shown as mean ± SEM of values obtained in independent experiments. The Student’s t-test was used to compare the differences between samples.


arrow
RESULTS
 
Role of PI-3K and PKC in basal Akt phosphorylation
First, we analyzed the basal phosphorylation state of Akt in freshly isolated and cryopreserved B-CLL cells from 12 different patients. Comparison of freshly isolated with cryopreserved cells showed that phosphorylation of Akt at Ser-473 and Thr-308 was lost after isolation and one cryopreservation/thawing cycle (Fig. 1A ). This basal Akt phosphorylation present in fresh cells is PI-3K-dependent, as incubation with the PI-3K inhibitor LY294002 decreased Ser-473 and Thr-308 phosphorylation in most samples analyzed (seven out of 10; Fig. 1B , Patients 5–7). In addition, in three out of 10 samples, basal Akt phosphorylation was PKC-dependent, as incubation with the PKC inhibitor Bis I decreased Ser-473 and Thr-308 (Fig. 1B , Patient 6) or Ser-473 Akt phosphorylation (Fig. 1B , Patient 7). Finally, in some samples (three out of 10), basal Akt phosphorylation was not affected by PI-3K or PKC inhibitors (Fig. 1B , Patient 8).


Figure 1
View larger version (57K):
[in this window]
[in a new window]

  		Figure 1. B-CLL cells have constitutive Akt activity, which is blocked by PI-3K or PKC inhibition. (A) Akt is phosphorylated (P-Akt) at Ser-473 and Thr-308 in freshly isolated, primary B-CLL lymphocytes. Freshly isolated B-CLL cells were lysed 1 h after separation from peripheral blood (freshly isolated, F) or 1 h after thawing (cryopreserved, C). Representative results with fresh or cryopreserved samples derived from four patients from 12 analyzed are shown. (B) Fresh B-CLL cells were incubated for 1 h in the presence of 20 µM LY294002, 5 µM Bis I, or in medium alone (Ct). Representative results with fresh samples derived from four patients out of 10 analyzed are shown. Whole protein lysates from different samples were analyzed by immunoblotting with specific antibodies as indicated.

Two candidates to maintain basal Akt phosphorylation in B-CLL cells are SDF-1{alpha} and IL-4, which are synthesized by nurse-like cells and T lymphocytes, respectively, and induce survival of B-CLL cells [4 , 25 ]. SDF-1{alpha} induced Akt phosphorylation at Ser-473 and Thr-308 (Fig. 2A ). This phosphorylation was inhibited by LY294002 but not by Bis I (n=3), indicating that SDF-1{alpha} induced Akt phosphorylation by a PI-3K-dependent mechanism. Similar results were obtained with IL-4 and CD40 ligand (data not shown). Furthermore, activation of BCR induced Akt phosphorylation by a PKC-independent and PI-3K-dependent mechanism. In contrast, PMA-induced Akt phosphorylation was not inhibited by LY294002 (Fig. 2A) .


Figure 2
View larger version (45K):
[in this window]
[in a new window]

  		Figure 2. PI-3K-dependent and PI-3K-independent Akt phosphorylation in B-CLL and normal B cells. (A) Cryopreserved B-CLL cells were preincubated for 1 h with 20 µM LY294002 or 5 µM Bis I and incubated with 10 nM PMA, 50 ng/ml SDF-1{alpha}, or 10µg/ml goat F(ab')2 anti-human IgM for an additional 20 min (n=3). (B) Normal B and T cells were preincubated in the presence of 20 µM LY294002 or 5 µM Bis I for 1 h prior to stimulation with 10 nM PMA for an additional 20 min. Whole protein lysates from different samples were analyzed by immunoblotting with specific antibodies as indicated.

To examine whether this PMA-induced, PI-3K-independent phosphorylation of Ser-473 was specific to B-CLL cells, we analyzed the effect of PMA on Akt phosphorylation in several cell lines and in normal B and T lymphocytes. PMA did not induce Ser-473 phosphorylation in any of the cell lines analyzed, including EHEB (human B cell chronic lymphocytic leukemia), JVM-2 (human B-prolymphocytic leukemia), BAF-3 (mouse pro B cells), HeLa (human cervix carcinoma), and Swiss 3T3 (mouse Swiss albino embryo fibroblasts) (data not shown). Similarly to B-CLL cells, treatment of human B and T lymphocytes from normal donors with PMA induced the phosphorylation of Ser-473 and Thr-308, and this phosphorylation was inhibited by Bis I. In contrast to B-CLL and normal T cells, PMA-induced phosphorylation of Akt was inhibited by LY294002 in normal B cells (n=2; Fig. 2B ).

PMA-induced Akt phosphorylation activates Akt in B-CLL cells
To further characterize Akt phosphorylation by PMA, dose-response and time-course experiments were performed in B-CLL cells. PMA induced Ser-473 phosphorylation at 10 nM in all cases analyzed (n=4; Fig. 3A ). Incubation of cells with PMA induced Ser-473 phosphorylation after 1 min, with a peak at 10–30 min, which was maintained for at least 6 h in all cases analyzed (n=5; Fig. 3B ).


Figure 3
View larger version (41K):
[in this window]
[in a new window]

  		Figure 3. PMA induces Akt phosphorylation in a dose- and time-dependent manner. Cryopreserved B-CLL cells were incubated (A) with increasing concentrations of PMA for 20 min or (B) with 10 nM PMA for the indicated periods of time. At the end of each time, cells were harvested and lysed, and Akt phosphorylation in Ser-473 was analyzed by Western blot as described in Materials and Methods. Results from one representative patient sample are shown.

We next analyzed whether PMA treatment increases Akt activity in B-CLL cells using phospho-specific antibodies generated against the Akt substrate consensus sequence [26 ]. As expected, some Akt substrates were phosphorylated in basal conditions from freshly isolated cells, and LY294002 and Bis I treatment decreased this phosphorylation (Fig. 4A ). It is important that treatment with PMA increased the phosphorylation of several Akt substrates. The most prominent phosphorylated Akt substrates had an apparent molecular mass of ~30, 52, 66, 80, 95, and 120 kDa. PMA-induced phosphorylation of these proteins was not inhibited by LY294002 (only phosphorylation of 30-kDa protein was slightly decreased) and completely blocked by Bis I. The 30-kDa protein has been identified previously as S6 ribosomal protein [26 ]. Using specific antibodies against known Akt substrates, we found that the 52-kDa and the 95-kDa proteins correspond to GSK3β and FoxO3, respectively (Fig. 4B) .


Figure 4
View larger version (48K):
[in this window]
[in a new window]

  		Figure 4. PMA induces Akt activation in B-CLL cells. (A) Cryopreserved B-CLL cells from a representative patient out of four analyzed were incubated with 20 µM LY294002 or 5 µM Bis I for 1 h before addition of 10 nM PMA to the culture for an additional 20 min. Cells were lysed, and whole extracts were analyzed by Western blot with a specific antibody against phosphorylated Akt substrates, as described in Materials and Methods. (B) Cryopreserved B-CLL cells were incubated with 10 nM PMA for 20 min. Whole protein lysates were analyzed by immunoblotting with phospho-Akt substrates, phospho-FoxO3, and phospho-GSK3{alpha}/β. Results from one representative patient sample from three analyzed are shown.

Effect of protein kinase inhibitors on PKC-induced Akt phosphorylation
To analyze the signal transduction pathways involved in PMA-induced Akt phosphorylation, we used selective protein kinase inhibitors (Fig. 5 ). As previously shown, the effect of PMA on Akt phosphorylation was blocked completely by Bis I, a selective inhibitor of classical and novel isoforms of PKC. The p38-MK2 and MEK-ERK pathways are activated downstream of PKC. However, inhibition of p38 MAPK and MEK by SB203580 and PD98059, respectively, did not affect Ser-473 phosphorylation. Furthermore, our results show that PMA-induced Ser-473 phosphorylation was blocked completely by 50 nM Ssp, indicating that a Ssp-sensitive kinase is involved in this phosphorylation in B-CLL cells. Neither SH-5, which prevents phosphatidylinositol-3-phosphate binding to pleckstrin homolog (PH) domain, nor triciribine, an inhibitor of Akt phosphorylation, inhibited PMA-induced Akt phosphorylation (Fig. 5B) . Finally, neither mTOR nor calmodulin-dependent pathways play a role in PKC-induced Ser-473 phosphorylation in B-CLL cells, as rapamycin (an inhibitor of mTOR) and W13 (an inhibitor of calmodulin-dependent proteins) did not affect PMA-induced Akt phosphorylation (data not shown).


Figure 5
View larger version (38K):
[in this window]
[in a new window]

  		Figure 5. Effect of protein kinase inhibitors on PMA-induced Akt phosphorylation. (A) Cryopreserved B-CLL cells were preincubated in the presence of 5 µM Bis I, 20 µM LY294002 (LY29), 10 µm SB203580 (SB), 20 µM PD98059 (PD), and 50 nM Ssp for 1 h prior to stimulation with 10 nM PMA. After 20 min of stimulation, the cells were lysed, and Akt and ERK phosphorylation was analyzed. Results from one representative patient sample from four analyzed are shown. (B) Cryopreserved B-CLL cells were preincubated in the presence of 5 µM Bis I, 1 µM Triciribine (TCN), 20 µM LY294002, and 10 µM SH-5 for 1 h prior to stimulation with 10 nM PMA (n=3).

PKCβ induces Akt phosphorylation independently of PI-3K in B-CLL cells
While we were performing the present study, it was reported that classic PKC isoforms, particularly, PKCβII, can regulate Akt activity by directly phosphorylating the critical residue Ser-473 in vitro and in Fc{epsilon}RI-stimulated mast cells [23 ]. Thus, we examined whether Akt phosphorylation depends on the activity of this PKC isoform. As shown in Figure 6A , Akt phosphorylation on Ser-473 and Thr-308 was inhibited by the PKCβ inhibitor LY333531 [27 ] in a dose-dependent manner. Time-course experiments showed that inhibition of PKCβ blocked Akt phosphorylation at Ser-473 and Thr-308 at all times analyzed (Fig. 6B) . In contrast, PKCβ was not required for Ser-473 phosphorylation in B-CLL cells stimulated with IgM or SDF-1{alpha} (Fig. 6C) . Basal and PMA-induced phosphorylation of Akt substrates was inhibited by LY333531 (Fig. 6D) , indicating that a PKCβ-dependent pathway activates Akt in B-CLL cells. Similarly to B-CLL cells, treatment of human B and T lymphocytes from normal donors with PMA induced the phosphorylation of Ser-473 and Thr-308, and this phosphorylation was inhibited by LY333531 (Fig. 6E) . It is surprisingly that 1 µM LY333531, a dose that inhibited PMA-induced Akt phosphorylation, did not affect viability of B-CLL cells from six different patients (control, 60%±11%; PMA, 73%±8%; LY333531, 59%±18%; PMA+LY333531, 74%±11%). Finally, we analyzed the effect of the combination of LY294002 and PKC inhibitors on the viability of B-CLL cells. Bis I and LY294002 had an additive effect, but LY333531 did not increase the apoptotic effect of LY294002 (Fig. 7 ).


Figure 6
View larger version (21K):
[in this window]
[in a new window]

  		Figure 6. Inhibition of PKCβ blocks PMA-induced Akt phosphorylation and activation. (A) Cryopreserved B-CLL cells were preincubated in the presence of 5 µM Bis I, 20 µM LY294002, and increasing concentrations of LY333531 for 1 h prior to stimulation with 10 nM PMA for an additional 20 min. (B) Cryopreserved B-CLL cells were preincubated in the presence or absence of 1 µM LY333531 for 1 h prior to stimulation with 10 nM PMA for the indicated periods of time. (C) Cryopreserved B-CLL cells were preincubated for 1 h with 20 µM LY294002 or 1 µM LY333531 and incubated with 50 ng/ml SDF-1{alpha} or 10 µg/ml goat F(ab')2 anti-human IgM for an additional 20 min. (D) Inhibition of PKCβ blocks PMA-induced phosphorylation of Akt substrates. B-CLL cells were preincubated in the presence of 1 µM LY333531 for 1 h prior to stimulation with 10 nM PMA for an additional 20 min. Results from one representative patient sample from three analyzed are shown. (E) Normal B and T cells were preincubated in the presence of 1 µM LY333531 for 1 h prior to stimulation with 10 nM PMA for an additional 20 min. Results from one representative sample from two analyzed are shown.


Figure 7
View larger version (10K):
[in this window]
[in a new window]

  		Figure 7. Effect of the combination of LY294002 with PKC inhibitors. B-CLL cells were incubated with 20 µM LY294002, 5 µM Bis I, and 1 µM LY333531, alone or combinations thereof, for 48 h (n=11). Cell viability was measured by analysis of phosphatidylserine exposure and PI uptake as described in Materials and Methods. Data are shown as the mean value ± SEM. *,P < 0.01, LY294002 + Bis I versus LY294002; #, P < 0.001, LY294002 + Bis I versus Bis I.


arrow
DISCUSSION
 
Our results show that PKCβ induces the phosphorylation and activation of Akt independently of PI-3K in B-CLL cells. Activation of PKC is sufficient to induce Akt phosphorylation in some cell types [4 , 23 , 28 29 30 31 32 33 34 35 ]. Thus, we reported that phorbol esters induce Akt phosphorylation at Ser-473, independently of PI-3K [4 ]. In addition, it has been reported that incubation of human platelets with phorbol esters induces Akt phosphorylation [32 , 33 ] and that phorbol esters induce Akt phosphorylation in murine T lymphocytes and the myeloid cell line FDC-P1 [34 , 35 ].

PKC-mediated phosphorylation of Akt is independent of PI-3K in B-CLL cells and in normal T cells, but it is surprising that this phosphorylation depends on PI-3K in normal B lymphocytes. The reason for this difference is unknown. One possibility is that B-CLL cells have lost this dependency during transformation. A second possibility is that this is a characteristic of the normal B cell subpopulation from which B-CLL cells develop. In fact, B-CLL cells express CD5 and ZAP-70, two proteins that are expressed in normal T cells but not in normal B cells [36 ]. The understanding of this difference between B-CLL cells and normal B lymphocytes could be exploited in the future for a more targeted and selective therapy.

Recently, it has been reported that classic PKC isoforms, particularly PKCβII, regulate Akt activity by directly phosphorylating the critical residue Ser-473 in vitro and in Fc{epsilon}RI-stimulated mast cells [23 ]. Thus, PKCβ is a candidate to directly phosphorylate Akt at Ser-473 in B-CLL cells. Whether or not PKCβ is involved in the phosphorylation of Akt in platelets and myeloid cells is unknown. Our results indicate that PMA-induced Akt phosphorylation in normal human B and T lymphocytes is dependent on PKCβ. B-CLL cells express high levels of PKCβ protein [37 ], and this raises the possibility that PKCβ could be deregulated in these leukemia cells. Furthermore, it is possible that PKCβ plays a role in the activation of Akt in other leukemias.

In vitro studies demonstrate that PKCβ directly phosphorylates Akt at Ser-473 without affecting the phosphorylation status of the Thr-308 residue [23 ]. However, in B-CLL cells, inhibition of PKCβ decreases Ser-473 and Thr-308 phosphorylation. As Thr-308 phosphorylation is not inhibited by LY294002 in response to PMA, these results suggest that a protein kinase acting downstream of PKCβ mediates the phosphorylation of Thr-308 independently of PI-3K in B-CLL cells. Alternatively, PKCβ could induce the activation of PDK-1 independently of PI-3K, as reported for the tyrosine kinase rearranged in transformation/papillary thyroid carcinomas [38 ]. Finally, the decrease in Thr-308 phosphorylation induced by LY333531 is also consistent with the model, in which Ser-473 phosphorylation precedes the phosphorylation of Thr-308 by constitutively active PDK-1 [18 , 39 ].

It is interesting that inhibition of PKCβ-induced Akt activity did not affect viability of B-CLL cells. These results indicate that Akt activity is not essential for the survival effect of PKC activation and that other pathways induced by PKC are sufficient to maintain cell viability. Whether or not Akt activation is essential for the survival effect of SDF-1{alpha} and IL-4 is presently unknown. Although the inhibition of PI-3K blocks the survival effect of IL-4, LY294002 induces apoptosis in cryopreserved cells with dephosphorylated Akt [4 ], suggesting the existence of other critical survival pathways downstream of PI-3K. Finally, Bis I but not PKCβ inhibitor has additive effects on apoptosis induction when combined with the PI-3K inhibitor. This suggests that when PKCβ is inhibited, other PKC-dependent survival pathways remain activated in B-CLL cells.

In conclusion, these results and those reported previously [2 3 4 ] indicate that B-CLL cells have two convergent signal transduction pathways to phosphorylate and activate Akt (Fig. 8 ). The first pathway is PI-3K-dependent and corresponds to the classical PI-3K-Akt pathway described in most models. This pathway is activated by BCR engagement [2 ], albumin [3 ], IL-4 [4 ], lysophosphatidic acid [9 ], and SDF-1{alpha} (this report; ref. [40 ]) in B-CLL cells and involves PDK-1 and probably the recently identified Ser-473 kinase Rictor-mTOR complex [24 ]. The second pathway depends on PKCβ, is independent of PI-3K, and contributes to the basal-constitutive Akt activity present in B-CLL cells. Finally, these results suggest that multiple redundant pathways are involved in the survival of B-CLL cells, and that likely, it is necessary to analyze the complex network of survival pathways to select the best therapeutic targets.


Figure 8
View larger version (21K):
[in this window]
[in a new window]

  		Figure 8. Signal transduction pathways involved in the activation of Akt in B-CLL cells. Proposed mechanisms for basal and induced Akt activation in response to different survival factors. B-CLL cells present a basal Akt phosphorylation dependent on PI-3K and PKCβ basal activities (marked in gray). Different survival factors induce Akt activation in B-CLL cells by two different pathways: the classical pathway (PI-3K-dependent), which is induced by IL-4, SDF-1{alpha}, BCR, and lysophosphatidic acid (LPA), and the novel pathway proposed in this manuscript, which is induced by PKCβ activation. Dotted line represents an unknown pathway to explain the increase in Thr-308 phosphorylation induced by PKCβ (see Discussion).


arrow
ACKNOWLEDGEMENTS
 
This study was supported by a grant from the Ministerio de Educación y Ciencia and FEDER (SAF2004-00265) to J. G. and by fellowships from the "José Carreras International Foundation Against Leukemia" to M. B., "AGAUR-Generalitat de Catalunya" to M. d. F., and "Ministerio de Educación y Ciencia" to D. I-S., A. F. S., L. C-M., and A. M. C. We thank the Unitat de Biologia (Serveis Cientificotècnics, Universitat de Barcelona) for technical support on flow cytometry and R. Rycroft for language assistance.


arrow
FOOTNOTES
 
1 These authors contributed equally to this work. Back

Received January 20, 2006; revised May 12, 2006; accepted July 3, 2006.


arrow
REFERENCES
 
    1
  1. Barragán, M., Campàs, C., Bellosillo, B., Gil, J. (2003) Protein kinases in the regulation of apoptosis in B-cell chronic lymphocytic leukemia Leuk. Lymphoma 44,1865-1870[CrossRef][Medline]
    2. 2
  2. Bernal, A., Pastore, R. D., Asgary, Z., Keller, S. A., Cesarman, E., Liou, H. C., Schattner, E. J. (2001) Survival of leukemic B cells promoted by engagement of the antigen receptor Blood 98,3050-3057[Abstract/Free Full Text]
    3. 3
  3. Wickremasinghe, R. G., Ganeshaguru, K., Jones, D. T., Lindsay, C., Spanswick, V. J., Hartley, J. A., Wadhwa, M., Thorpe, R., Hoffbrand, A. V., Prentice, H. G., Mehta, A. B. (2001) Autologous plasma activates Akt/protein kinase B and enhances basal survival and resistance to DNA damage-induced apoptosis in B-chronic lymphocytic leukaemia cells Br. J. Haematol. 114,608-615[CrossRef][Medline]
    4. 4
  4. Barragán, M., Bellosillo, B., Campàs, C., Colomer, D., Pons, G., Gil, J. (2002) Involvement of protein kinase C and phosphatidylinositol 3-kinase pathways in the survival of B-cell chronic lymphocytic leukemia cells Blood 99,2969-2976[Abstract/Free Full Text]
    5. 5
  5. Ringshausen, I., Schneller, F., Bogner, C., Hipp, S., Duyster, J., Peschel, C., Decker, T. (2002) Constitutively activated phosphatidylinositol-3 kinase (PI-3K) is involved in the defect of apoptosis in B-CLL: association with protein kinase C{delta} Blood 100,3741-3748[Abstract/Free Full Text]
    6. 6
  6. Jones, D. T., Ganeshaguru, K., Anderson, R. J., Jackson, T. R., Bruckdorfer, K. R., Low, S. Y., Palmqvist, L., Prentice, H. G., Hoffbrand, A. V., Mehta, A. B., Wickremasinghe, R. G. (2003) Albumin activates the AKT signaling pathway and protects B-chronic lymphocytic leukemia cells from chlorambucil- and radiation-induced apoptosis Blood 101,3174-3180[Abstract/Free Full Text]
    7. 7
  7. Cuní, S., Pérez-Aciego, P., Pérez-Chacón, G., Vargas, J. A., Sanchez, A., Martin-Saavedra, F. M., Ballester, S., Garcia-Marco, J., Jorda, J., Durantez, A. (2004) A sustained activation of PI3K/NF-{kappa}B pathway is critical for the survival of chronic lymphocytic leukemia B cells Leukemia 18,1391-1400[CrossRef][Medline]
    8. 8
  8. Plate, J. M. (2004) PI3-kinase regulates survival of chronic lymphocytic leukemia B-cells by preventing caspase 8 activation Leuk. Lymphoma 45,1519-1529[CrossRef][Medline]
    9. 9
  9. Hu, X., Haney, N., Kropp, D., Kabore, A. F., Johnston, J. B., Gibson, S. B. (2005) Lysophosphatidic acid (LPA) protects primary chronic lymphocytic leukemia cells from apoptosis through LPA receptor activation of the anti-apoptotic protein AKT/PKB J. Biol. Chem. 280,9498-9508[Abstract/Free Full Text]
    10. 10
  10. Nedellec, S., Renaudineau, Y., Bordron, A., Berthou, C., Porakishvili, N., Lydyard, P. M., Pers, J. O., Youinou, P. (2005) B cell response to surface IgM cross-linking identifies different prognostic groups of B-chronic lymphocytic leukemia patients J. Immunol. 174,3749-3756[Abstract/Free Full Text]
    11. 11
  11. Franke, T. F., Hornik, C. P., Segev, L., Shostak, G. A., Sugimoto, C. (2003) PI3K/Akt and apoptosis: size matters Oncogene 22,8983-8998[CrossRef][Medline]
    12. 12
  12. Brazil, D. P., Yang, Z. Z., Hemmings, B. A. (2004) Advances in protein kinase B signaling: AKTion on multiple fronts Trends Biochem. Sci. 29,233-242[CrossRef][Medline]
    13. 13
  13. Fruman, D. A. (2004) Phosphoinositide 3-kinase and its targets in B-cell and T-cell signaling Curr. Opin. Immunol. 16,314-320[CrossRef][Medline]
    14. 14
  14. Luo, J., Manning, B. D., Cantley, L. C. (2003) Targeting the PI3K-Akt pathway in human cancer: rationale and promise Cancer Cell 4,257-262[CrossRef][Medline]
    15. 15
  15. Altomare, D. A., Testa, J. R. (2005) Perturbations of the AKT signaling pathway in human cancer Oncogene 24,7455-7464[CrossRef][Medline]
    16. 16
  16. Cheng, J. Q., Lindsley, C. W., Cheng, G. Z., Yang, H., Nicosia, S. V. (2005) The Akt/PKB pathway: molecular target for cancer drug discovery Oncogene 24,7482-7492[CrossRef][Medline]
    17. 17
  17. Alessi, D. R., Cohen, P. (1998) Mechanism of activation and function of protein kinase B Curr. Opin. Genet. Dev. 8,55-62[CrossRef][Medline]
    18. 18
  18. Woodgett, J. R. (2005) Recent advances in the protein kinase B signaling pathway Curr. Opin. Cell Biol. 17,150-157[CrossRef][Medline]
    19. 19
  19. Dong, L. Q., Liu, F. (2005) PDK2: the missing piece in the receptor tyrosine kinase signaling pathway puzzle Am. J. Physiol. Endocrinol. Metab. 289,E187-E196[Abstract/Free Full Text]
    20. 20
  20. Alessi, D. R., Andjelkovic, M., Caudwell, B., Cron, P., Morrice, N., Cohen, P., Hemmings, B. A. (1996) Mechanism of activation of protein kinase B by insulin and IGF-1 EMBO J. 15,6541-6551[Medline]
    21. 21
  21. Rane, M. J., Coxon, P. Y., Powell, D. W., Webster, R., Klein, J. B., Pierce, W., Ping, P., McLeish, K. R. (2001) p38 Kinase-dependent MAPKAPK-2 activation functions as 3-phosphoinositide-dependent kinase-2 for Akt in human neutrophils J. Biol. Chem. 276,3517-3523[Abstract/Free Full Text]
    22. 22
  22. Feng, J., Park, J., Cron, P., Hess, D., Hemmings, B. A. (2004) Identification of a PKB/Akt hydrophobic motif Ser-473 kinase as DNA-dependent protein kinase J. Biol. Chem. 279,41189-41196[Abstract/Free Full Text]
    23. 23
  23. Kawakami, Y., Nishimoto, H., Kitaura, J., Maeda-Yamamoto, M., Kato, R. M., Littman, D. R., Leitges, M., Rawlings, D. J., Kawakami, T. (2004) Protein kinase C βII regulates Akt phosphorylation on Ser-473 in a cell type- and stimulus-specific fashion J. Biol. Chem. 279,47720-47725[Abstract/Free Full Text]
    24. 24
  24. Sarbassov, D. D., Guertin, D. A., Ali, S. M., Sabatini, D. M. (2005) Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex Science 307,1098-1101[Abstract/Free Full Text]
    25. 25
  25. Burger, J. A., Tsukada, N., Burger, M., Zvaifler, N. J., Dell’Aquila, M., Kipps, T. J. (2000) Blood-derived nurse-like cells protect chronic lymphocytic leukemia B cells from spontaneous apoptosis through stromal cell-derived factor-1 Blood 96,2655-2663[Abstract/Free Full Text]
    26. 26
  26. Zhang, H., Zha, X., Tan, Y., Hornbeck, P. V., Mastrangelo, A. J., Alessi, D. R., Polakiewicz, R. D., Comb, M. J. (2002) Phosphoprotein analysis using antibodies broadly reactive against phosphorylated motifs J. Biol. Chem. 277,39379-39387[Abstract/Free Full Text]
    27. 27
  27. Jirousek, M. R., Gillig, J. R., Gonzalez, C. M., Heath, W. F., McDonald, J. H., III, Neel, D. A., Rito, C. J., Singh, U., Stramm, L. E., Melikian-Badalian, A., Baevsky, M., Ballas, L. M., Hall, S. E., Winneroski, L. L., Faul, M. M. (1996) (S)-13-[(Dimethylamino)methyl]-10,11,14,15-tetrahydro-4,9:16, 21-dimetheno-1H, 13H-dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecene-1,3(2H)-d ione (LY333531) and related analogues: isozyme selective inhibitors of protein kinase C β J. Med. Chem. 39,2664-2671[CrossRef][Medline]
    28. 28
  28. Li, W., Zhang, J., Flechner, L., Hyun, T., Yam, A., Franke, T. F., Pierce, J. H. (1999) Protein kinase C-{alpha} overexpression stimulates Akt activity and suppresses apoptosis induced by interleukin 3 withdrawal Oncogene 18,6564-6572[CrossRef][Medline]
    29. 29
  29. Tenzer, A., Zingg, D., Rocha, S., Hemmings, B., Fabbro, D., Glanzmann, C., Schubiger, P. A., Bodis, S., Pruschy, M. (2001) The phosphatidylinositide 3'-kinase/Akt survival pathway is a target for the anticancer and radiosensitizing agent PKC412, an inhibitor of protein kinase C Cancer Res. 61,8203-8210[Abstract/Free Full Text]
    30. 30
  30. Gliki, G., Wheeler-Jones, C., Zachary, I. (2002) Vascular endothelial growth factor induces protein kinase C (PKC)-dependent Akt/PKB activation and phosphatidylinositol 3'-kinase-mediates PKC {delta} phosphorylation: role of PKC in angiogenesis Cell Biol. Int. 26,751-759[CrossRef][Medline]
    31. 31
  31. Bae, Y. S., Kim, Y., Park, J. C., Suh, P. G., Ryu, S. H. (2002) The synthetic chemoattractant peptide, Trp-Lys-Tyr-Met-Val-D-Met, enhances monocyte survival via PKC-dependent Akt activation J. Leukoc. Biol. 71,329-338[Abstract/Free Full Text]
    32. 32
  32. Kroner, C., Eybrechts, K., Akkerman, J. W. (2000) Dual regulation of platelet protein kinase B J. Biol. Chem. 275,27790-27798[Abstract/Free Full Text]
    33. 33
  33. Kim, S., Jin, J., Kunapuli, S. P. (2004) Akt activation in platelets depends on Gi signaling pathways J. Biol. Chem. 279,4186-4195[Abstract/Free Full Text]
    34. 34
  34. Bauer, B., Jenny, M., Fresser, F., Uberall, F., Baier, G. (2003) AKT1/PKB{alpha} is recruited to lipid rafts and activated downstream of PKC isotypes in CD3-induced T cell signaling FEBS Lett. 541,155-162[CrossRef][Medline]
    35. 35
  35. Blalock, W. L., Navolanic, P. M., Steelman, L. S., Shelton, J. G., Moye, P. W., Lee, J. T., Franklin, R. A., Mirza, A., McMahon, M., White, M. K., McCubrey, J. A. (2003) Requirement for the PI3K/Akt pathway in MEK1-mediated growth and prevention of apoptosis: identification of an Achilles heel in leukemia Leukemia 17,1058-1067[CrossRef][Medline]
    36. 36
  36. Chiorazzi, N., Rai, K. R., Ferrarini, M. (2005) Chronic lymphocytic leukemia N. Engl. J. Med. 352,804-815[Free Full Text]
    37. 37
  37. Alkan, S., Huang, Q., Ergin, M., Denning, M. F., Nand, S., Maududi, T., Paner, G. P., Ozpuyan, F., Izban, K. F. (2005) Survival role of protein kinase C (PKC) in chronic lymphocytic leukemia and determination of isoform expression pattern and genes altered by PKC inhibition Am. J. Hematol. 79,97-106[CrossRef][Medline]
    38. 38
  38. Kim, D. W., Hwang, J. H., Suh, J. M., Kim, H., Song, J. H., Hwang, E. S., Hwang, I. Y., Park, K. C., Chung, H. K., Kim, J. M., Park, J., Hemmings, B. A., Shong, M. (2003) RET/PTC (rearranged in transformation/papillary thyroid carcinomas) tyrosine kinase phosphorylates and activates phosphoinositide-dependent kinase 1 (PDK1): an alternative phosphatidylinositol 3-kinase-independent pathway to activate PDK1 Mol. Endocrinol. 17,1382-1394[Abstract/Free Full Text]
    39. 39
  39. Scheid, M. P., Marignani, P. A., Woodgett, J. R. (2002) Multiple phosphoinositide 3-kinase-dependent steps in activation of protein kinase B Mol. Cell. Biol. 22,6247-6260[Abstract/Free Full Text]
    40. 40
  40. Nishio, M., Endo, T., Tsukada, N., Ohata, J., Kitada, S., Reed, J. C., Zvaifler, N. J., Kipps, T. J. (2005) Nurselike cells express BAFF and APRIL, which can promote survival of chronic lymphocytic leukemia cells via a paracrine pathway distinct from that of SDF-1{{alpha}} Blood 106,1012-1020[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 BloodHome page
C. Holler, J. D. Pinon, U. Denk, C. Heyder, S. Hofbauer, R. Greil, and A. Egle
PKC{beta} is essential for the development of chronic lymphocytic leukemia in the TCL1 transgenic mouse model: validation of PKC{beta} as a therapeutic target in chronic lymphocytic leukemia
Blood, March 19, 2009; 113(12): 2791 - 2794.
[Abstract] [Full Text] [PDF]

Home page
 JCOHome page
S. Hewamana, T. T. Lin, C. Rowntree, K. Karunanithi, G. Pratt, R. Hills, C. Fegan, P. Brennan, and C. Pepper
Rel A Is an Independent Biomarker of Clinical Outcome in Chronic Lymphocytic Leukemia
J. Clin. Oncol., February 10, 2009; 27(5): 763 - 769.
[Abstract] [Full Text] [PDF]

Home page
 Endocr Relat CancerHome page
S. Grozinsky-Glasberg, I. Shimon, M. Korbonits, and A. B Grossman
Somatostatin analogues in the control of neuroendocrine tumours: efficacy and mechanisms
Endocr. Relat. Cancer, September 1, 2008; 15(3): 701 - 720.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
M. Y. Follo, S. Mongiorgi, C. Bosi, A. Cappellini, C. Finelli, F. Chiarini, V. Papa, M. Libra, G. Martinelli, L. Cocco, et al.
The Akt/Mammalian Target of Rapamycin Signal Transduction Pathway Is Activated in High-Risk Myelodysplastic Syndromes and Influences Cell Survival and Proliferation
Cancer Res., May 1, 2007; 67(9): 4287 - 4294.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0106041v1
80/6/1473    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Barragán, M.
Right arrow Articles by Gil, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Barragán, M.
Right arrow Articles by Gil, J.