Protein Kinase B beta /Akt2 Plays a Specific Role in Muscle Differentiation*

Marie VandrommeDagger §, Anne RochatDagger §, Roger Meier, Gilles CarnacDagger , Daniel Besser||, Brian A. Hemmings, Anne FernandezDagger , and Ned J. C. LambDagger **

From the Dagger  Institut de Genetique Humaine, CNRS, UPR 1142, 141 Rue de la Cardonille, 34396 Montpellier Cedex 4, France, the  Friedrich Miescher Institut, Maubeerstrasse 66, CH-4056 Basel, Switzerland, and the || Rockefeller University, Laboratory of Molecular Cell Biology, New York, New York 10021

Received for publication, June 26, 2000, and in revised form, November 20, 2001



    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Insulin-like growth factors positively regulate muscle differentiation through activation of the phosphatidylinositol 3-kinase/protein kinase B (PKB/Akt) signaling pathway. Here, we compare the role of the two closely related alpha  (Akt1) and beta  (Akt2) isoforms of PKB in muscle differentiation. During differentiation of C2.7 or L6D2 myoblasts, PKBbeta was up-regulated whereas expression of PKBalpha was unaltered. Although the two isoforms were found active in both myoblasts and myotubes, cell fractionation experiments indicated that they displayed distinct subcellular localizations in differentiated cells with only PKBbeta localized in the nuclei. In a transactivation assay, PKBbeta (either wild-type or constitutively active) was more efficient than PKBalpha in activating muscle-specific gene expression. Moreover, microinjection of specific antibodies to PKBbeta inhibited differentiation of muscle cells, whereas control or anti-PKBalpha antibodies did not. On the other hand, microinjection of the anti-PKBalpha antibodies caused a block in cell cycle progression in both non muscle and muscle cells, whereas anti-PKBbeta antibodies had no effect. Taken together, these results show that PKBbeta plays a crucial role in the commitment of myoblasts to differentiation that cannot be substituted by PKBalpha .



    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Skeletal muscle differentiation is a tightly regulated process during which proliferative myoblasts escape from the cell cycle, express a battery of muscle-specific genes, and fuse into multinucleated myotubes. This developmental process is regulated at the transcriptional level by the basic helix-loop-helix myogenic proteins including MyoD, Myf-5, myogenin, and MRF4 (1).

Insulin-like growth factors (IGFI1 and IGFII) are potent activators of muscle differentiation. An autocrine production of IGFII by myoblasts has been linked to spontaneous differentiation triggered by lowering serum concentration in the culture medium (2, 3). Indeed, C2 myoblasts overexpressing an IGFII antisense construct fail to differentiate spontaneously and lose MyoD expression (4).

Recently, Coolican and collaborators (5) showed that activation of phosphatidylinositol 3 kinases (PI3Ks) is essential for IGF-stimulated differentiation. Differentiation of L6E9, C2.7, Sol8, and chicken primary myoblasts was also shown to be dependent on PI3Ks activity (6-9). Among downstream targets of PI3K, the serine/threonine protein kinase B/Akt (PKB) was proposed as an essential downstream component for IGF-induced differentiation (9). Three different isoforms of PKB have been identified: PKBalpha (Akt1), PKBbeta (Akt2), and PKBgamma (Akt3). All of them are activated by growth factors in a PI3K-dependent manner. Phosphorylation of Thr308 (Thr309 in PKBbeta ) in the activation loop and Ser473 (Ser474 in PKBbeta ) in the C-terminal activation domain is required for full activation of PKBalpha and PKBbeta (reviewed in Refs. 10 and 11). The most studied isoform is PKBalpha /Akt1, generally referred to as PKB or Akt. PKBalpha has been implicated in several processes stimulated by IGFs such as cell survival, glucose uptake in myocytes and adipocytes, cell growth, and differentiation (reviewed in Refs. 12 and 13). Based on a dominant positive overexpression approach, a role for PKBalpha has been assigned in the PI3K-mediated differentiation of avian primary myoblasts (9), as well as in promoting cell survival when myoblasts escape from the cell cycle to differentiate (14). Several recent reports suggest that the alpha  and beta  isoforms of PKB may have distinct functions in cellular regulation. In adipocytes, PKBbeta is preferentially involved in insulin-stimulated glucose transporter 4 (GLUT4) translocation (15, 16) and is found associated with GLUT4-containing vesicles (17). PKBbeta appears to be functionally different from PKBalpha since stress activates PKBalpha and PKBgamma , but not PKBbeta (18). Little is known about PKBbeta and its potential role in myogenesis. Interestingly, PKBbeta /Akt2 mRNA is up-regulated during myogenic differentiation and has a tissue-specific distribution in mouse embryos being preferentially expressed in brown fat and muscle tissues, suggesting a preferential role for this isoform in differentiation processes (19).

In this report we have investigated a role for PKBbeta in skeletal muscle differentiation. In contrast to the constant expression levels of PKBalpha , PKBbeta protein levels were specifically increased during differentiation of mouse C2.7 or rat L6D2 myoblasts. The two isoforms were found active in both myoblasts and myotubes with a differentiation-dependent increase in PKBbeta activity mirroring protein levels. Cell fractionation experiments indicated that the two isoforms differed in their subcellular distributions in differentiated cells with only PKBbeta localized in the nuclei. When overexpressed in C2.7 cells, PKBbeta (either wild-type or constitutively active forms) was more efficient than PKBalpha in enhancing transactivation of a muscle-specific gene. Finally, inactivation of PKBbeta through microinjection of specific antibodies impaired differentiation of C2.7, L6D2, and human myoblasts, whereas anti-PKBalpha antibodies had no effect. By contrast, injection of the anti-PKBalpha antibodies caused a block in cell cycle progression in both nonmuscle and muscle cells, whereas anti-PKBbeta antibodies had no effect. Taken together, these results show that the alpha  and beta  isoforms of PKB play different roles in myogenesis.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- C2.7 (4) or L6D2 myoblasts (a subclone of L6 cells; Ref. 20) were grown in proliferation medium (50% Dulbecco's modified Eagle's medium (Biowhittaker, France), 50% Ham's F-12 supplemented with 10% fetal calf serum (Life Technologies, Inc.). To induce muscle differentiation, myoblasts were placed in differentiation medium (Dulbecco's modified Eagle's medium, 2% fetal calf serum).

Clonal human myoblasts (CHM) were obtained from needle biopsy sample taken from the paravertebral muscle of a healthy subject, using an experimental protocol previously described for rodent (21). To obtain an homogenous population of myoblasts, cells were grown as a clonal population and individually tested for their capacity to differentiate in a low serum containing medium. Selected clones were mixed together. The population obtained called CHM (for cloned human myoblasts) can be grown for 6-10 passages. Following transfer into a differentiation medium, the majority of cells fuse into multinucleated myotubes after 36-48 h, expressing muscle-specific markers such as myogenin and troponin T.

Western Blot-- 50 µg of cellular protein extracts prepared from proliferating or differentiated C2.7 or L6D2 cells were analyzed by Western blot as previously described (21). Antibodies used were rabbit anti-PKBalpha (22), sheep anti-PKBbeta (Upstate Biotechnology), rabbit anti-myogenin (Santa-Cruz Biotechnology), and mouse anti-tubulin antibodies (Sigma). Secondary antibodies were horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies (Amersham Pharmacia Biotech). Sheep antibodies were detected using horseradish peroxidase-conjugated protein A/G (Interchim, France). Blots were developed by using an Amersham enhanced chemiluminescence reagent.

Immunoprecipitation and in Vitro Kinase Assay-- Cells (myoblasts and myotubes) were resuspended in a lysis buffer (50 mM Tris-HCl, pH 7.5, 120 mM NaCl, 1% w/v Nonidet P-40, 40 mM beta -glycerophosphate, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 4 µM leupeptin, 2 µg/ml aprotinin, 0.1 mM sodium orthovanadate) and homogenized by passages through a 21-gauge needle. Following a 10-min centrifugation at 12,000 × g, soluble proteins were quantified using a Bradford method (Bio-Rad). 200-400 µg of cell free extracts were pre-cleared by incubation with protein G-Sepharose beads (Amersham Pharmacia Biotech) and incubated overnight at 4 °C with either rabbit anti-PKBalpha or sheep anti-PKBbeta antibodies. 20 µl of protein G-Sepharose beads were added for 30 min at 4 °C, and beads were washed three times with lysis buffer containing 0.5 M NaCl, once with lysis buffer, and once with kinase buffer (50 mM Tris-HCl, pH 7.4, 1 mM phenylmethylsulfonyl fluoride, 10 mM beta -glycerophosphate). In vitro kinase assay were performed for 30 min at 30 °C by adding 20 µl of kinase buffer supplemented with 10 mM MgCl2, 50 µM [gamma -32P]ATP (Amersham Pharmacia Biotech, 1,000-2,000 cpm/pmol), 1 µM protein kinase A inhibitor peptide, and 2 µg of the Crosstide sequence (23, 24) fused to GST as substrate (see below). Proteins were subsequently separated on a 12.5% SDS-polyacrylamide gel electrophoresis. The radioactivity was analyzed by autoradiography.

The PKB substrate GST-GRPRTSS*FAEG (the asterisk denotes the phosphorylated serine) and the mutant substrate GST-GRPKTSSFAEG were purified from extracts of bacteria (XL1-blue) as described by the manufacturer of the pGEX-6P-1 plasmid (Amersham Pharmacia Biotech). The bacteria had been transformed with GST expression plasmids pGEX-6P-1/GRPRTSSFAEG or pGEX-6P-1/GRPKTSSFAEG, respectively. These plasmids were constructed by inserting oligonucleotides with the sequence GATCCGGAAGGCCAAGAACTTCATCGTTCGCAGAGGGTC (pGEX-6P-1/GRPRTSSFAEG) or GATCCGGAAGGCCAAAAACTTCATCGTTC GCAGAGGGTC (pGEX-6P-1/GRPKTSSF- AEG) into the BamHI and XhoI site of pGEX-6P-1.

Cell Fractionation-- Cells were collected in an ice-cold hypotonic buffer containing 10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 4 µM leupeptin, 2 µg/ml aprotinin and lysed by 10 strokes in a Dounce homogenizer. Nuclei were collected by a 10-min centrifugation at 2000 × g and rinsed once with hypotonic buffer. Protein concentrations in the cytoplasmic fraction was determined using the Bradford detection reagent (Bio-Rad). Isolated nuclei were lysed directly in a volume of 1× Laemmli buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 8% glycerol, 0.001% bromphenol blue, and 10 mM dithiothreitol) equal to that of the cytoplasmic fraction.

Transfection and Luciferase Assay-- Plasmids used for transfection were: The pECE vector either empty or encoding wild-type PKB tagged with HA (pECE-HA-PKBalpha wt, pECE-HA-PKBbeta wt), inactive PKB (pECE-HA-PKBalpha kd, pECE-HA-PKBbeta kd), or constitutively active membrane-targeted PKB (pECE-HA-PKBalpha m and -beta m; 22, 25), pRL-TK (Promega, Charbonnieres, France), and muscle creatine kinase enhancer and promoter upstream of firefly luciferase gene (MCK-luc; gift from S. Leibovitch, Institut Gustave Roussy, Villejuif, France).

Transfections were carried out using 4 µl of FuGENE and 1 µg of DNA in 35-mm dishes as described by the manufacturer (Roche Molecular Biochemicals). Transfected cells were kept in proliferation medium for 24 h and transferred to differentiation medium for indicated times before processing for Western blot or immunofluorescence as described elsewhere (21). Antibodies to HA and myogenin were used to visualize transfected and differentiated cells, respectively.

For luciferase assay, cells were transfected with 1 µg of total plasmid DNA composed of pECE/pECE-HA-PKB/MLCK-luc/RL-TK plasmids (ratio 1:1:2:1). Following incubation for 24 h in proliferation medium and 36 h in differentiation medium, transfected cells were harvested for luciferase assay. Activities of firefly and Renilla luciferases were measured sequentially using the luciferase assay kit reagent as described by the manufacturer (Promega, Charbonnieres, France).

Microinjection and Immunofluorescence Experiments-- Microinjections were carried out in growing myoblasts before induction of differentiation or in G0-synchronized REF-52 fibroblasts before serum refeeding, as previously described (26, 27). Injected antibodies were affinity-purified rabbit anti-PKBalpha antibodies (directed against amino acids 453-466 of human PKBalpha protein; Ref. 22), affinity-purified sheep anti-PKBbeta antibodies (directed against amino acids 455-469 of rat PKBbeta ; Euromedex, France), or 1 mg/ml anti-hPKBbeta IgGs (directed against amino acid 454-470 of human PKBbeta , Ref. 22). Prior to injection, purified antibodies (0.3-0.5 mg/ml) or sera were dialyzed against 100 mM Hepes, pH 7.4, and mixed when necessary with rabbit marker antibodies (1 mg/ml). Immunofluorescence analysis was carried out as previously described (21).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expression of PKBbeta , but Not PKBalpha , Is Up-regulated during C2.7 and L6D2 Cell Differentiation-- We have initially determined the expression profiles of PKBalpha and PKBbeta during differentiation of mouse C2.7 cells. To first ensure that the anti-PKB antibodies were isoform-specific, C2.7 cells transiently transfected with HA-tagged membrane-targeted forms of either PKBalpha or PKBbeta were examined by immunoblotting. The rabbit anti-PKBalpha antibody only immunoblotted the overexpressed HA-PKBalpha protein, whereas the sheep anti-PKBbeta antibody detected only the overexpressed HA-PKBbeta (Fig. 1A). In addition, at the exposure shown here, PKBbeta antibodies only detected HA overexpressed protein whereas anti-PKBalpha detected both overexpressed (upper band) and endogenous (lower faint band) proteins. As comparable amounts of overexpressed proteins were analyzed (see Fig. 1A, lane HA), it appears that the anti-PKBbeta antibodies have a lower affinity (~5-fold) than PKBalpha antibodies against their respective isoforms.



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Fig. 1.   Differentiation-dependent up-regulation of PKBbeta . A, C2.7 cells were transfected with membrane-targeted HA-tagged forms of either PKBalpha or PKBbeta , and comparable amounts of overexpressed proteins (as probed with anti-HA antibodies) were analyzed by Western blot for expression of PKBalpha and PKBbeta . B, total cellular extracts were prepared from C2.7 cells before (0d) or after 1, 2, or 3 days (d) being placed in differentiation medium, and analyzed by Western blot for expression of PKBalpha , PKBbeta , myogenin (myog) as a differentiation marker, and tubulin (tub) as a loading control. C, C2.7 myotubes were separated from quiescent myoblasts by limited trypsinization after 3 (3d) or 4 days (4d) in differentiation medium. Western blot analysis of total cellular extracts from proliferative myoblasts (P), quiescent myoblasts (Q), and myotubes (M) was carried out for PKBalpha , PKBbeta , troponin T (TropT) as a differentiation marker, and tubulin (tub) as a loading control.

Extracts from proliferative or differentiating myoblasts were probed by Western blot for expression of PKB isoforms, myogenin as a differentiation marker, and tubulin as a loading control. As shown in Fig. 1B, PKBalpha protein level remains constant throughout differentiation. In contrast, PKBbeta expression is up-regulated, as early as 24 h after induction of differentiation, a time that coincides with appearance of the early differentiation marker myogenin. A specific up-regulation of PKBbeta was also seen during differentiation of rat L6D2 myoblasts (data not shown). Moreover, when compensating for the different affinities of the anti-PKBalpha and anti-PKBbeta antibodies (see Fig. 1A), PKBalpha and PKBbeta proteins may be expressed at similar levels in myoblasts, but PKBbeta becomes the major isoform expressed in myotubes.

We have previously shown that, upon induction of differentiation, a subset of myoblasts escape from the differentiation process and remain in a quiescent undifferentiated mononucleated state (21). To investigate if the observed increase in PKBbeta level was directly linked to differentiation, C2.7 myotubes were separated from quiescent cells, and expression of PKBbeta was examined by Western blot in these two subpopulations. PKBbeta is expressed at similar low levels in proliferative and nondifferentiated quiescent myoblasts (Fig. 1C, compare lanes P and Q), whereas it shows a significant increase in differentiated myotubes (Fig. 1C, lane M). Expression levels of PKBalpha are similar in all tested conditions. As expected for a marker of differentiation, troponin T was detected only in the myotube population.

Taken together, these results show that PKBalpha and PKBbeta are differentially expressed during differentiation of muscle cells, with PKBbeta being up-regulated in a differentiation-dependent manner.

Activity and Subcellular Distribution of PKBalpha and PKBbeta during Differentiation-- Activation of either PKBalpha or PKBbeta occurs through the same mechanisms. Active PI3Ks allow translocation of PKB to the plasma membrane, where its PH domain binds to the PI3K lipid products. Following phosphorylation by two upstream activating kinases, PKB translocates into the nucleus (22, 25). In our culture model, myoblasts are allowed to differentiate by lowering serum concentration in the medium. This spontaneous differentiation is possible because of an autocrine production of IGFs by differentiating myoblasts. We first estimated the relative activity of each isoform during the process of spontaneous differentiation by using a kinase assay. PKBalpha and PKBbeta were immunoprecipitated from C2.7 myoblasts or myotubes, and their activity assessed against the Crosstide peptide fused to GST (GST-C) as a substrate. As shown in Fig. 2A, PKBalpha and PKBbeta were found to phosphorylate Crosstide in both myoblasts and myotubes. PKBalpha activity was slightly increased in myotubes in comparison to myoblasts with similar amounts of protein immunoprecipitated at both stages. PKBbeta activity was significantly up-regulated in differentiated myotubes when compared with myoblasts. This increase in PKBbeta activity corresponds to an augmentation of immunoprecipitated proteins. For both isoforms, no activity was detected when a mutated non-phosphorylatable Crosstide peptide was used as substrate testifying for specificity of the assay (data not shown).



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Fig. 2.   Subcellular localization and activity of PKBalpha and PKBbeta in myoblasts and myotubes. A, PKBalpha and PKBbeta were immunoprecipitated from proliferative C2.7 myoblasts and myotubes and their activity assessed against the peptide Crosstide fused to GST (GST-C) as substrate. The amount of immunoprecipitated proteins were controlled by Western blot (WB). B, cytoplasmic and nuclear fractions were prepared from C2.7 myoblasts (P) and myotubes (M) and analyzed by Western blot for expression of PKBalpha and PKBbeta . Nuclei (N) were lysed in a volume of Laemmli buffer equivalent to cytosolic fraction (C) and whole cell extract proteins (WCE).

To characterize further the relative activity of each isoforms during spontaneous differentiation, we also looked for the subcellular distribution of PKBalpha and PKBbeta in myoblasts and myotubes, as nuclear translocation of PKB has been described to follow its activation (22, 25). Cytoplasmic and nuclear extracts of mouse C2.7 or rat L6D2 myoblasts and myotubes were prepared and immunoblotted with anti-PKBalpha or anti-PKBbeta antibodies. As shown in Fig. 2B, PKBalpha and PKBbeta were found distributed in both cytoplasm and nuclei at similar levels in proliferative C2.7 myoblasts. By contrast, only PKBbeta was still detected in C2.7 myotube nuclei whereas PKBalpha was detected only in the cytoplasm. Similar results have been observed when using L6D2 cell extracts (data not shown).

Taken together, these data show that PKBalpha and PKBbeta are both active in myoblasts and myotubes, but differ in their subcellular distribution in myotubes with only PKBbeta still detectable in the nucleus. This difference in subcellular localization strongly suggests a different function for each isoform during muscle differentiation.

PKBbeta Is More Efficient than PKBalpha in Transactivating Muscle-specific Gene in C2.7 Cells-- To examine whether the two PKB isoforms display similar efficiency in promoting muscle differentiation, we used a classical transactivation assay with a luciferase reporter plasmid (MCK-luc) containing the -1256 base pair upstream region from the muscle creatine kinase, which mediates differentiation-dependent activity in cultured skeletal muscle cells (28, 29). Thus, activation of MCK-luc should allow a quantitative measurement of the extent of differentiation. Wild-type and mutant (constitutively active or inactive) forms of PKBalpha and PKBbeta were cotransfected with the MCK-luc construct into C2.7 cells. In each case, plasmid pRL-TK was cotransfected as an internal control for transfection efficiency. After transfection, cells were placed in proliferation medium for 24 h, and incubated for an additional 36 h in differentiation medium before harvesting and measurement of the reporter gene transactivation by luciferase assay. As shown in Fig. 3, constitutively active PKBalpha (alpha m) and PKBbeta (beta m) significantly promoted MCK-luc activity (up to 3- and 7-fold, respectively) with PKBbeta m showing a 2-fold higher efficiency than PKBalpha m. Significantly, wild-type PKBbeta also increased transactivation of the reporter gene to a level similar to that obtained with the constitutively active PKBalpha , whereas wild-type PKBalpha appeared as inefficient as constitutively inactive mutants, in promoting MCK-luc activity. Expression levels of the different PKB constructs were checked by Western blot and were found to be similar in all tested conditions (data not shown). Taken together, these results show that PKBbeta (either wild-type or constitutively active) is more efficient than PKBalpha in enhancing muscle-specific gene expression.



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Fig. 3.   PKBbeta is more efficient than PKBalpha in enhancing muscle-specific gene expression. C2.7 myoblasts were transfected with the MCK-luc reporter construct and pRL-TK plasmid, together with plasmids encoding for wild-type (alpha wt and beta wt), constitutively active (alpha m and beta m), or kinase dead (alpha kd and beta kd) forms of PKBalpha and PKBbeta . After 24 h in proliferation medium followed by 36 h of incubation in differentiation medium, transfected cells were harvested and luciferase activity was measured and corrected with respect to pRL-TK activity. MCK-luc activities are expressed as -fold activation relative to that obtained in empty pECE cotransfected cells set at 1. Shown are the mean values of duplicates obtained in two independent experiments.

Microinjected anti-PKBbeta Antibodies Inhibit Muscle Differentiation-- To further investigate the differences between PKBalpha and PKBbeta in myogenic cells, we examined whether targeted inhibition of individual PKB isoforms, through microinjection of specific antibodies, could interfere with differentiation. Such an approach has been successfully used to demonstrate the involvement of key regulatory proteins such as the coactivator p300 (30) or the serum response factor (26, 31) in skeletal muscle differentiation.

Affinity-purified anti-PKBalpha , anti-PKBbeta antibodies mixed with rabbit marker antibodies, or marker antibodies alone were injected into both C2.7 or L6D2 myoblasts. After microinjection cells were switched to the differentiation medium for 48 h and processed for immunofluorescence. As shown in Fig. 4, only microinjection of anti-PKBbeta antibodies blocked differentiation of L6D2 and C2.7 cells by 50% and 48%, respectively, whereas neither anti-PKBalpha nor marker antibody (data not shown; see also Ref. 26), had a significant effect on myogenin expression and fusion of microinjected cells into myotubes.



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Fig. 4.   Microinjection of anti-PKBbeta but not anti-PKBalpha antibodies prevents differentiation of C2.7 and L6D2 cells. A, proliferative myoblasts were microinjected with affinity purified anti-PKBalpha or anti-PKBbeta antibodies. Cells were placed in differentiation medium and 48 h later were fixed and stained for the injected antibodies and myogenin. Shown are fluorescence micrographs of cells injected with anti-PKBalpha (panels A-C) or anti-PKBbeta (panels D-F), and stained for the injected antibodies (panels A and D), myogenin (panels B and E), and the superimposition of the two (panels C and F). The histogram summarizes the data obtained from two different sets of microinjection experiments done in both C2.7 and L6D2 cells. Percent of myogenin expression refers to the percentage of microinjected cells expressing myogenin. The number (n) of cells injected with anti-PKBalpha (alpha ) and anti-PKBbeta (beta ) antibodies is indicated.

To confirm the requirement of PKBbeta for muscle differentiation, we performed a second set of microinjection experiments. We used the rabbit anti-hPKBbeta antibodies directed against amino acids 454-470 of the human protein (22). Because these antibodies recognized with high affinity the human protein, injection was carried out in CHM (see "Materials and Methods") as described above. As shown in Fig. 5, microinjection of anti-hPKBbeta IgGs into CHM efficiently blocked differentiation. The majority of injected cells remained mononucleated with ~20% expressing myogenin. Inversely, in control experiments in which myoblasts were microinjected with pre-immune serum, 60% of injected cells were either found in multinucleated myotubes or in mononucleated myocytes expressing myogenin. This percentage of differentiation was similar to that of surrounding noninjected cells.



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Fig. 5.   Microinjection of anti-human PKBbeta antibodies blocks differentiation of human myoblasts. CHM cells were injected with anti-hPKBbeta IgGs or pre-immune serum as control and incubated in differentiation medium for 36-48 h. Cells were analyzed by immunofluorescence as described in Fig. 4. Shown are fluorescence micrographs of cells injected with pre-immune serum (panels A-C) or anti-hPKBbeta antibodies (panels D-F), and stained for the injected antibodies (panels A and D), myogenin (panels B and E) and the superimposition of the two (panels C and F). The histogram summarizes the data obtained in two independent experiments with the percentage of myogenin expression in surrounding noninjected cells (indicated as 0), cells injected with the pre-immune serum (PI), or cells injected with anti-hPKBbeta IgGs. n = number of injected cells.

To confirm that the function of PKBalpha is not necessary for the transition from myoblast to myotube, we further performed microinjection experiments to prove that anti-PKBalpha antibodies were inhibitory to PKBalpha . For that purpose, we monitored the effect of microinjected anti-PKB antibodies on cell proliferation of REF-52 fibroblasts, which express both PKBalpha and PKBbeta as probed by immunoblotting (data not shown). Indeed, a recent report described that PKBalpha is part of a Ras-PI3K-glycogen synthase kinase-3beta transduction pathway that positively regulates cyclin D1, a key component of the cell cycle involved in progression of cells through G1 phase (32). Serum-starved REF-52 fibroblasts were injected with anti-PKBalpha or anti-PKBbeta antibodies, refed with serum to enter the cell cycle and incubated in a medium containing BrdUrd for 20-22 h to follow passage through S phase via DNA synthesis. As shown in Fig. 6, BrdUrd incorporation was inhibited in 90% of anti-PKBalpha injected cells, whereas anti-PKBbeta had no significant effect on S phase entry with 73% of injected cells having passed through S phase. These data indicate that anti-PKBalpha antibodies are active in inhibiting PKBalpha . Similarly, injection of anti-PKBalpha antibodies blocked proliferation of C2.7 cells, whereas injected anti-PKBbeta antibodies had no effect (data not shown).



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Fig. 6.   Microinjection of anti-PKBalpha but not anti-PKBbeta antibodies impedes proliferation. G0-synchronized REF-52 fibroblasts were microinjected with anti-PKBalpha (A) or anti-PKBbeta (B) antibodies and following serum refeeding, incubated with BrdUrd for 20-22 h to follow S phase completion. Shown are fluorescence micrographs of cells double-stained for the injected antibodies (red) and for incorporation of BrdUrd (green). The histogram summarizes the data obtained from two different microinjection experiments. Percent of DNA synthesis refers to the percentage of microinjected cells that have incorporated BrdUrd. The number of cells injected with anti-PKBalpha (alpha ) or anti-PKBbeta (beta ) is indicated.

Taken together, these results show that PKBbeta plays a specific function in triggering differentiation of myoblasts, a role for which PKBalpha appears unable to substitute.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The PI3K/PKB (Akt) pathway has been recently implicated in IGF-activated muscle differentiation and hypertrophy as well as IGF-induced muscle cell survival (9, 14, 33, 34). Except for the work by Fujio et al. (Ref. 9, as discussed below), none of these studies investigated a functional difference between the PKB isoforms. In this report, we provide evidence that PKBalpha and PKBbeta play distinct roles in myogenesis and demonstrate that PKBbeta is necessary to trigger muscle cell differentiation.

PKBalpha and -beta Differ in Their Expression and Subcellular Localizations during Differentiation-- The first difference between PKBalpha and PKBbeta in muscle cells can be seen at the expression level, with only PKBbeta protein being up-regulated when C2.7 and L6D2 myoblasts are induced to differentiate and maintained at high level in differentiated cells. Inversely, PKBalpha protein levels remain constant during differentiation. An increase in PKBbeta protein and/or mRNA level was previously reported in differentiating Sol8 (6), C2C12 myoblasts (19), and MyoD-converted 10T1/2 fibroblasts (35), showing that up-regulation of PKBbeta is not restricted to our cell models, and most likely occurs at the transcriptional level. In this context, it is quite surprising that Fujio et al. (9) described an induction of PKBalpha protein during differentiation of C2C12 cells. This may be explained by insufficient specificity of the anti-PKBalpha antibody used in that study. We found both PKBalpha and PKBbeta active in phosphorylating the Crosstide peptide in both myoblasts and myotubes. Whereas PKBalpha was slightly activated during differentiation, PKBbeta activity was up-regulated in a manner corresponding to the increase in protein levels. This is consistent with the fact that both isoforms are activated in a PI3K-dependent manner (22, 25). In fact, a major difference between the two isoforms lies in their subcellular distribution in differentiated cells, with only PKBbeta maintained in the nuclear compartment. Both PKBalpha and PKBbeta were found in myoblast nuclei and cytoplasm, but in contrast to PKBbeta , PKBalpha was no more detectable in myotube nuclei. The mechanisms by which PKB kinases translocate to the nucleus following membrane activation have been poorly studied. The oncogene for Tcl1, which is involved in the development of mature T-cell leukemia, was recently shown to be a cofactor of PKB. Tcl1, by interacting with PKB, enhances its activity by promoting its nuclear translocation (36). Because Tcl1 is restricted to certain lymphoid cells, other molecules, perhaps related to Tcl1, responsible for PKB nuclear translocation may exist in differentiated cells and remain to be identified. The unique nuclear localization of PKBbeta in myotubes suggests that it may stimulate differentiation by phosphorylating nuclear substrates. In this context, it may appear puzzling that membrane-targeted forms, albeit incapable to translocate to the nucleus, display activation of muscle-specific genes. Constitutively active mutants may be able to activate endogenous PKB. Indeed, activation of PKB appears to involve oligomerization of PKB molecules (37, 38). In addition, PKB may be responsible for its auto-activation by phosphorylating Ser473 (Ser474 in PKBbeta ) referred to as hypothetical PDK2 site (39). Constitutive membrane-targeted mutants may be able to oligomerize with and phosphorylate endogenous PKB, thus explaining an activation effect that would involve nuclear localization of endogenous PKB. Interestingly, PI3K was recently shown to positively regulate the transcriptional activity of myocyte enhancer factor 2 family members, which cooperate with myogenic factors in the regulation of muscle-specific genes including myogenin (40). As such, it will be interesting to determine whether myocyte enhancer factor 2 proteins constitute a direct target of PKBbeta .

PKBalpha and PKBbeta Have Distinct Functions in Myogenesis-- The high specificity of the antibodies directed against PKBalpha and PKBbeta (see Fig. 1) prompted us to use a microinjection assay to discriminate between the role played by each isoform in muscle differentiation. The antibodies used are directed against the C-terminal regulatory domain of PKBalpha and PKBbeta , and, as such, upon binding they may interfere with the kinase activation. Microinjection of two different anti-PKBbeta antibodies efficiently blocked differentiation of C2.7, L6D2, and human myoblasts, respectively, an effect that was not observed after injection of anti-PKBalpha antibodies. The efficiency of injected PKBalpha antibodies in inhibiting endogenous PKBalpha was attested by its inhibitory effect on the G1 to S transition of REF-52 fibroblasts and C2.7 myoblasts. Indeed, PKBalpha was recently shown to control expression of cyclin D1, a key component of G1 progression (32, 41). Moreover, the tumor suppressor MMAC/PTEN, a lipid phosphatase that dephosphorylates PI3K-generated 3'-phosphatidylinositides, was shown to modulate G1 to S phase progression by regulating PKB signaling pathway (42, 43). Inversely, anti-PKBbeta antibodies had no effect on cell proliferation, indicating that inhibition of differentiation by these antibodies is not dependent on cell-cycle events. In accordance with our results, inhibition of PKBbeta protein expression was shown to suppress the tumorigenicity of human pancreatic cancer cells without affecting proliferation as estimated by the population doubling time in culture (44). In conclusion, our results strongly suggest that PKBalpha and PKBbeta isoforms play specific and distinct roles in myogenesis. First, we show that PKBbeta is necessary for differentiation of either, mouse, rat, or human myoblasts, implying that the role for PKBbeta /Akt2 in myogenesis is conserved in mammalian cells. Second, our results tend to indicate that PKBalpha cannot directly regulate the commitment to differentiate but is more likely involved in proliferation. Interestingly, recent reports have implicated PKBalpha in myocyte viability (9, 34). We have observed a loss in anti-PKBalpha injected cells when placed into differentiation medium, in agreement with the fact that PKBalpha may be the isoform involved in anti-apoptotic signaling. The isoform-specific function of PKBbeta in inducing muscle differentiation may appear contradictory to previous reports showing a role for PKBalpha in the myogenic-inducing activity of IGF. This discrepancy highlights inherent problems of overexpression assays. In these reports, a role for PKBalpha in IGF activated differentiation (9) or hypertrophy (33) has been demonstrated by overexpressing constitutively active forms of the kinase. Accordingly, our results show that constitutively active forms of PKBalpha and PKBbeta were both able to enhance muscle-specific gene transactivation, although PKBbeta appeared 2-fold more effective. However, when assessing wild-type isoforms, only PKBbeta displayed a myogenic activity (see Fig. 3). The constitutive active forms may escape from accurate regulation such as association with regulatory partners. This notion is exemplified by the two related isoforms, p110alpha and p110beta catalytic subunits of class IA PI3Ks. Studies based on overexpression assay of an activated mutant of the p110 subunits or dominant negative p85 regulatory subunit (which binds both p110alpha and p110beta ) could not discriminate between the two isoforms that were assumed to play similar roles in cellular regulation. However, by using an antibody microinjection approach, recent reports clearly show that p110alpha and p110beta have different functions in either platelet-derived growth factor- and insulin-activated actin reorganization in aortic endothelial cells (45), insulin-responsive glucose transport activity in adipocytes (46), or epidermal growth factor-stimulated actin nucleation in breast cancer cells (47). Interestingly, p110beta but not p110alpha was shown to associate with the GLUT4 glucose transporter compartment (48) and play a crucial role in transmitting the signals to translocate GLUT4 (46), a biological process in which PKBbeta is specifically involved (15, 16). Although in this case there is no direct evidence that PI3Kbeta activates PKBbeta , this example illustrates how cellular functions may be influenced by subcellular localization.

In summary, we have demonstrated a specific role of PKBbeta in triggering muscle differentiation. The characterization of substrates that are specifically phosphorylated by PKBbeta during differentiation is critical to understand how the kinase is involved in this process. A good candidate is glycogen synthase kinase-3beta , which, in addition to being a target of PKB, belongs to the Wnt signaling pathway known to positively regulate differentiation (reviewed in Ref. 49). Other potential in vivo downstream targets of PKBbeta include p70S6 kinase and transcription factors such as the forkhead family member FHRK1 (50). We are currently investigating which of these signaling molecules may be modulated by PKBbeta and as such involved in the control of myogenesis.


    ACKNOWLEDGEMENTS

We thank Patric Turowski for technical advice and helpful discussions and Olivier Zugasti for help in PKB kinase assay. We are grateful to Yegor Vassetzky for comments on the manuscript.


    FOOTNOTES

* This work was supported by grants from Association Francaise contre les Myopathies, by Association pour la Recherche contre le Cancer Grant 9484, and by Human Frontiers Science Program Organisation Collaborative Grant 533/1996-M.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.

§ These authors contributed equally to this work.

** To whom correspondence should be addressed. Tel.: 33-499-61-99-66; Fax: 33-499-61-99-69; E-mail: ned.lamb@igh.cnrs.fr.

Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.M005587200


    ABBREVIATIONS

The abbreviations used are: IGF, insulin-like growth factor; PKB, protein kinase B; PI3K, phosphatidylinositol 3-kinase; HA, hemagglutinin; GLUT4, glucose transporter 4; GST, glutathione S-transferase; BrdU, bromodeoxyuridine; CHM, clonal human myoblasts.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


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