These studies further characterized the important role of the PI 3-kinase and p38 MAPK pathways in myogenesis, revealing a reciprocal cross-talk and activation that is essential for efficient myoblast differentiation. We demonstrate that Akt is differentially phosphorylated during myogenesis and that its kinase activity only increases when both sites are phosphorylated. Whereas phosphorylation of AktT308 is relatively constant during myogenesis, AktS473 phosphorylation changes dramatically and has a key role in determining Akt kinase activity. Our findings also highlight a key role for Akt2 and not Akt1 in myoblast differentiation. Overall Akt2 activity was sensitive to both p38 and PI 3-kinases but via different mechanisms. Whereas p38 regulates transcription from the Akt2 gene, PI 3-kinase appears essential for subsequent Akt2 phosphorylation. Complementary to p38-mediated transactivation of Akt2, PI 3-kinase also regulated p38 activity upstream of MKK6, demonstrating bidirectional communication and positive feedback characteristic of myogenic regulation. Further, Kaneko et al. (30
) have demonstrated that Akt2 (once activated) can feed forward and activate its own promoter, thus providing a mechanism for sustained Akt activation.
The idea of communication between PI 3-kinase and p38 MAPK in myogenesis has been excluded in previous studies (39
), which concluded that the pathways were independent and parallel and exhibited no cross-activation or cross-inhibition. In support of this, cooperative but independent signaling by PI 3-kinase and p38 has been observed in other systems (see, e.g., reference 41
). Our observations suggest that activation is strictly temporally regulated, with p38 activation preceding increased Akt kinase activity by 24 h; it is therefore possible that cross-activation only occurs at specific time points. Given this time delay in p38 and Akt activation, it is difficult to reconcile this idea with a hypothesis according to which p38 directly activates Akt. However, our observation that a key role of p38 is that of stimulating Akt transcription is compatible with these temporal observations.
The embryonic death of p38α-null mice demonstrates the importance of p38 in development and that other p38 isoforms cannot compensate (5
). Targets for p38 include the key myogenic transcription factors MyoD (46
) and MEF2 (66
) and the structural protein caveolin-3 (which is essential for myoblast fusion) (24
). The p38 kinase target MK2 and p38 itself mutually enhance each other's functionality: MK2 activity is very much reduced in p38α-null mice (5
), and MK2 stabilizes p38 and can dictate its cellular localization (31
). However, the precise role of each of the p38 isoforms is not well defined. The inhibition of myoblast differentiation by SB203580 indicates that the p38α and p38β isoforms are important, and microarray analysis has defined MyoD- and p38α/p38β-activated genes in myogenesis (11
); use of SB203580-resistant forms of p38α and p38β demonstrated that both are required for maximal induction of myogenic target genes (39
). Further, the p38γ isoform may also induce myoblast differentiation (37
). Each isoform may therefore exhibit specific temporal or spatial expression or may activate nonoverlapping downstream targets. For example, p38α and p38β are more effective than the p38γ and p38δ isoforms in MEF2A and C phosphorylation (63
) but the p38γ and p38δ isoforms have been reported to be more efficient at inducing myogenic differentation of myoblasts cotransfected with MyoD (65
). It has also been suggested that p38α and p38β have opposing roles with respect to effects on apoptosis (reviewed in reference 42
). Even though the dominant-negative p38 construct used in the study presented here was a p38α sequence, it may well have sequestered key p38 partner molecules (e.g., scaffold proteins) and inhibited activity of other isoforms. In theory, high-level expression of MKK6EE would activate all isoforms (6
). Therefore, our studies confirm the importance of p38α and p38β but have not excluded functions for p38γ and p38δ in myogenesis.
In terms of Akt activation, the identity of the kinase responsible for AktT308 phosphorylation (PDK1) is unequivocally established whereas that for AktS473, the putative PDK2, has not been determined. It is not known whether a novel kinase remains to be cloned or whether existing kinases have the role of AktS473 phosphorylation. Alessi et al. (4
) first demonstrated that MK2 was capable of phosphorylating AktS473 in cell extracts. Rane et al. (47
) confirmed p38 MAPK-mediated phosphorylation of AktS473 in stimulated neutrophils and coimmunoprecipitation of Akt with p38, MK2, and Hsp27. Even though our study strongly supports the idea of a p38-mediated induction of Akt2 activity in myogenesis via transcriptional mechanisms, we do not have evidence that the p38 or its targets are acting as a PDK2. Our data do suggest that once Akt2 is synthesized, appropriate PDK2-like mechanisms exist in differentiating myoblasts that are highly PI 3-kinase dependent and appear to be more active towards Akt2 than Akt1. As far as we are aware, the specificity of PDK1 or putative PDK2 enzymes for different Akt isoforms has not been investigated. Related observations of ovarian cancer cells (in which lysophosphatidic acid and sphingosine-1-phosphate induce coordinated AktS473 and AktT308 phosphorylation) support the idea of p38-mediated activation of Akt (9
); regulation of AktS473 phosphorylation was p38 dependent but (in marked contrast to the results seen with myoblasts) also required MEK activation, which inhibits myogenesis. Inhibition (using SB203580 blockage of p38 activation) of cardiac myocyte differentiation did not reduce PI 3-kinase activity (17
); Akt activity was not determined, but these observations are not incompatible with p38 regulation of the PI 3-kinase pathway downstream of PI 3-kinase itself.
Additional candidates besides p38 MAPK exist for the role of PDK2. For example, stress activation of Akt is independent of p38 (50
). Insulin also induces Akt phosphorylation that is not dependent on p38 activity, and MK2 activity is minimally stimulated by insulin (4
). ILK has been proposed as a possible PDK2 in prostate cancer cells (45
) and in immortalized macrophages (55
), and insulin indeed stimulates ILK-mediated phosphorylation of AktS473 (20
). Transfection of myoblasts with dominant-negative ILK partially inhibited AktS473 phosphorylation (I. Gonzalez and J. M. Pell, unpublished observations), indicating that this might be a mechanism for Akt phosphorylation during myogenesis. One proposal is therefore that several kinases exist that can assume a PDK2-like function and whose specificity is stimulus or cell dependent or is dictated by a distinct affinity for a particular Akt isoform. Alternatively, it is possible that a single PDK2-type enzyme exists but that several phosphatases differentially regulate the AktS473 site.
Of the three Akt isoforms (Akt1, Akt2, and Akt3), Akt2 is highly expressed in insulin-responsive tissues such as skeletal muscle, heart, liver, and adipose tissue (7
). Akt2 protein and mRNA levels specifically increase during myoblast and adipocyte differentiation (7
), suggesting that Akt2 rather than Akt1 is important during myoblast differentiation in physiological conditions. Studies in vivo and in vitro have demonstrated that Akt1 and Akt2 have different functions and do not compensate for each other. Akt1-null mice exhibit modest growth retardation and increased susceptibility to apoptotic stimuli (14
), whereas Akt2-null mice grow normally but develop type II diabetes (16
). Akt1 and Akt2 double-null mice die at birth and exhibit severe muscle hypoplasia (43
). In consistency with these observations, microinjection of myoblasts with antibodies specific to either Akt2 or Akt1 demonstrated that Akt1 is important for cell cycle progression whereas Akt2 stimulated myoblast differentiation (57
). Akt2, but not Akt1, localizes to the nuclei of myoblasts (40
), and IGFs can stimulate nuclear localization of wild-type Akt (8
). This finding casts doubt on the validity of studies conducted using constitutively active myristoylated Akt (which may remain membrane bound) (8
). p21 phosphorylation status (48
) and localization (67
) may be determined by Akt localization, with cytoplasmic p21 stimulating cell survival and nuclear p21 inducing cell cycle withdrawal. Finally, the Akt2 promoter (but not the Akt1 promoter) contains multiple E boxes that are transactivated by MyoD, leading to downstream activation of MEF2 proteins and the formation of highly transcriptionally active MyoD/MEF2 complexes (30
). These recent findings suggest a role for Akt2 rather than Akt1 in myogenesis. Our data support and extend this hypothesis, suggesting differential roles for Akt isoforms and demonstrating a novel functional link between the PI 3-kinase and p38 pathways, Akt2 transcription, and Akt activity in myoblast differentiation.
Our findings suggest not only p38 transactivation of Akt2 but also reciprocal activation of p38 MAPK pathways upstream of MKK6 by PI 3-kinase. Thus, constitutively active PI 3-kinase stimulated p38 phosphorylation and kinase activity. Communication between PI 3-kinase and p38 in myogenesis is controversial; increases in p38 activation independent of PI 3-kinase activation have been reported for skeletal myoblasts (52
), but PI 3-kinase-regulated p38 activity has been observed in cardiac myoblasts (17
). It has been suggested that PI 3-kinase can activate p38 via Rac/Cdc42 (54
); further, PI 3-kinase is activated via E-cadherin-mediated cell-cell contact in enterocytes, activating p38 and stimulating differentiation (34
In conclusion, we have revealed novel links between the p38 MAPK and the PI 3-kinase/Akt pathways that occur at several levels. PI 3-kinase can regulate activity of the p38 MAPK pathway (which then induces transactivation of the Akt2 promoter). Subsequent Akt2 kinase activity is mainly determined by phosphorylation of S473 and is PI 3-kinase dependent. Intriguingly, both Akt transcription and activation mechanisms appear to be specific for Akt2 but not for Akt1. We thus demonstrate reciprocal feedforward activation mechanisms that are characteristic of myogenic regulation.