Activation of Mitochondrial Raf-1 Is Involved in the Antiapoptotic Effects of Akt¹

The Akt serine/threonine kinase (protein kinase B) is a phosphatidylinositol-3-kinase downstream effector (1, 2) that mediates the survival-promoting effects of several growth factors (3, 4). Expression of constitutively active Akt in different cell types suppresses apoptotic death induced by a variety of stimuli including growth factor withdrawal, aberrant cell cycle activity, loss of cell adhesion, and DNA damage (5). Akt is also involved in IL³-2-dependent cell cycle progression (6) and is required for leukemogenesis induced by the BCR/ABL oncogenic tyrosine kinase (7). Thus, growth factor receptor activation or expression of an oncogenic tyrosine kinase leads to phosphatidylinositol-3-kinase-dependent Akt activation, which then promotes cell survival and, in certain cell types, enhances cell cycle activity. Akt has been shown to exert its antiapoptotic effects via serine phosphorylation of the apoptosis agonist BAD (8, 9) and caspase 9 (10). Upon serine phosphorylation, BAD is sequestered in the cytoplasm by interacting with 14-3-3, a process that neutralizes its apoptotic effects dependent on the association with Bcl-2 and Bcl-X_L (11). Serine phosphorylation of caspase 9 inhibited its protease activity and promotes survival (10). BAD and caspase 9 phosphorylation might not be the only mechanisms whereby Akt promotes cell survival, because enhanced expression of Bcl-2 was also detected in hematopoietic cells expressing constitutively active Akt (6, 7). Bcl-2 has been implicated in the transport of the Raf-1 serine/threonine kinase on the mitochondrial membranes, where Raf-1 can be also involved in phosphorylation and inactivation of BAD (12).

To investigate mechanisms whereby Akt promotes survival of hematopoietic cells, constitutively active forms of Akt were introduced in the growth factor-dependent myeloid precursor 32Dcl3 cells. As expected, Akt-expressing cells survived longer than parental cells in growth factor-deprived medium. The reduced susceptibility to apoptosis of Akt-expressing cells correlated with enhanced bcl-2 expression, activation of mitochondrial and plasma membrane Raf-1, and phosphorylation of BAD. Interestingly, the apoptosis-resistant phenotype of Akt-expressing cells was not impaired by inhibition of the Raf-1-dependent MAP kinase cascade but was abrogated by expression of mitochondria-targeted, dominant-negative Raf-1. Moreover, expression of mitochondria-targeted, dominant-negative Raf-1 prevented the inactivation of the death-agonist BAD, as indicated by its detection in the nonphosphorylated form.

Together, these data support the importance of Raf-1 activation in the antiapoptosis effects of Akt.

The retroviral constructs carrying HA-tagged c-Akt, E40KAkt, and MyrAkt were described previously (7). Plasmids containing mitochondrial membrane-targeted dominant-negative or dominant-active Raf-1 mutants (MM-Raf-1 DNM or MM-Raf-1 DAM, respectively) were prepared as described (13). Dominant-negative N17 Ras mutant (N17Ras; Ref. 14) was cloned into pZIPNeoSV(x)1.

The murine IL-3 dependent 32Dcl3 myeloid precursor cell line (15) was maintained in IMDM-CM [IMDM supplemented with 10% heat-inactivated FBS, 2 mm l-glutamine, penicillin/streptomycin (100 μg/ml each), and 15% WEHI-conditioned medium as a source of IL-3]. 32Dcl3 cell clones expressing Akt constructs (wild-type c-Akt or DAMs E40KAkt or MyrAkt) were established by electroporation and subsequent selection in G418-containing medium as described (7). MM-Raf-1 mutants were introduced by electroporation, and clones were selected in puromycin-containing medium. Expression of Akt was confirmed by Western blotting with anti-HA antibody. Clones expressing MM-Raf-1 mutants were identified by detection of a M_r ∼43,000 truncated Raf-1 in the HMs but not in the LMs or Cyt fractions. Clones expressing BCR/ABL were described (7).

Subcellular fractionation was performed as described (13).

32Dlc3 parental cells or clones expressing BCR/ABL, HA-c-Akt, HA-E40KAkt, or HA-MyrAkt were starved of growth factor and serum for 5 h. Akt activation was examined as described (7) by measuring the phosphorylation of histone 2B (H2B). Raf-1 kinase assay was performed as described (16), using H1 as a substrate. Ras activation was determined as described (17) by measuring GTP-bound RAS. PKC enzymatic activity was assessed in specific anti-PKC immunoprecipitates, by in vitro kinase assay according to the manufacturer’s protocol (United Biomedical, Inc., Lake Placid, NY).

Raf-1 and BAD phosphorylation was measured in the specific immunoprecipitates obtained from lysates of cells incubated for 3 h with [³²P]P_i in phosphate-free medium deprived of growth factor and serum as described (13). The phosphorylated form of MAP kinase was detected by Western blot using anti-active-MAP kinase antibody (Promega Corp., Madison, WI) in total cell lysates obtained from growth factor- and serum-starved cells.

Cells (10⁵/ml) were cultured in growth factor-free medium and apoptotic cells were counted using TACS1 Klenow in situ apoptosis detection kit (Trevigen, Inc., Gaithersburg, MD).

For retroviral infection, cells were infected with the pZIPNeoSV(x)1-N17Ras retrovirus as described (7). Briefly, the packaging cell line Bosc23 was transfected with the N17Ras-containing retrovirus using the calcium phosphate precipitation method. Cells expressing dominant-active Akt were added to the monolayer of packaging cells (1:1 ratio) 48 h after transfection. Seventy-two h later, nonadherent cells were collected and used for the experiments.

To assess the biological effects of Akt, we analyzed apoptosis susceptibility and proliferation of growth factor-dependent murine hematopoietic precursor 32Dcl3 cells transfected with HA-tagged, wild-type c-Akt, Akt DAM E40KAkt, or MyrAkt. Western blotting with an anti-HA antibody confirmed ectopic expression of Akt in newly established clones (Fig. 1, upper panel). In vitro Akt kinase assay using anti-HA and anti-Akt immunoprecipitates (Fig. 1, middle and bottom panels, respectively) indicated that Akt remained active after IL-3 and serum starvation of cell clones expressing E40KAkt, MyrAkt, and BCR/ABL (positive control) but not of clones expressing c-Akt or of parental cells (negative control).

As compared with parental cells, apoptosis induced by growth factor withdrawal was delayed in E40KAkt- and MyrAkt-expressing cells (Fig. 2, Akt panel). Consistent with other reports (4, 6), these cell transfectants did not grow in the absence of IL-3 (data not shown). Similar results were obtained when cells were cultured in medium lacking both IL-3 and serum (data not shown).

Unlike c-Akt-expressing cells, IL-3- and serum-starved 32Dcl3 cells expressing constitutively active Akt showed the phosphorylated, slowly migrating form of BAD and enhanced Bcl-2 levels (data not shown), in accord with previous findings (6, 7, 8). Moreover, these cells showed an accumulation of Raf-1 in the membrane and mitochondria fractions (LM and HM, respectively; Fig. 3,A). Constitutively active Akt induced the phosphorylation of Raf-1 (Fig. 3,B, upper panel) and stimulated its enzymatic activity as detected by measuring Raf-1 kinase activity in anti-Raf-1 immunoprecipitates from total cell lysates and from HM (mitochondrial) and LM (cytoplasmic) membranes but not Cyt fractions (Fig. 3 B, lower panels). Akt-dependent activation of Raf-1 was accompanied by phosphorylation and stimulation of MAP kinase enzymatic activity (data not shown).

The requirement of Raf-1 activation in the antiapoptosis effects of Akt was assessed after inhibition of the MAP kinase cascade activated by plasma membrane Raf-1 or after suppressing the activity of mitochondrial Raf-1.

Inhibition of MAP kinase activation by the specific MEK inhibitor PD98059 (18) did not accelerate apoptosis of cells expressing dominant-active Akt (E40KAkt or MyrAkt) after serum and/or IL-3 withdrawal (data not shown).

To assess the role of mitochondrial Raf-1 activity in the reduced susceptibility to apoptosis of 32Dcl3 cells expressing constitutively active Akt, a plasmid carrying the mitochondrial membrane-targeted dominant-negative or the dominant-active Raf-1 mutant (MM-Raf-1 DNM and MM-Raf-1 DAM, respectively) was introduced by electroporation into these cells as well as into parental 32Dcl3 cells. Consistent with previous studies (12, 13) expression of MM-Raf-1 DAM in IL-3-starved parental cells induced transient protection from apoptosis, whereas expression of MM-Raf-1 DNM had no measurable effects (Fig. 2, Raf panel). Expression of MM-Raf-1 DNM resulted in the abrogation of the antiapoptotic activity of E40KAkt and MyrAkt (Fig. 2, E40KAkt + Raf and MyrAkt + Raf panels). Coexpression of constitutively active Akt and MM-Raf-1 DAM did not have synergistic antiapoptotic effects, although each ectopically expressed protein induced transient protection from apoptosis (Fig. 2, Akt and Raf panels).

Together, the results of these experiments suggest that, at least in hematopoietic cells, activation of mitochondrial Raf-1 is important in transducing Akt-dependent antiapoptotic signals.

Potential mechanisms of Akt-dependent Raf-1 activation were assessed by investigating the effects of Akt on known activators of Raf-1. The Akt-dependent activation of Raf-1 was apparently Ras-independent, because the active, GTP-bound Ras was undetectable in clones expressing constitutively active Akt mutants (Fig. 4,A), and transient expression of the dominant-negative N17Ras mutant did not inhibit Akt-dependent Raf-1 activation (Fig. 4,B) but suppressed IL-3-dependent activation of Ras (Fig. 4 C).

On the basis of the postulated role of BAG-1 (19) and PKCs (20, 21, 22, 23, 24) in the activation of Raf-1, we asked whether they might be involved in the Akt-dependent activation of Raf-1. BAG-1 did not coimmunoprecipitate with Raf-1 in cells expressing constitutively active forms of Akt (data not shown), suggesting that it may not be involved in the process of Raf-1 activation. On the other hand, the enzymatic activity of four (α, δ, ε, and ζ) of the six PKC isoforms known to activate Raf-1 (20, 21, 22, 23, 24) was elevated in cells expressing E40KAkt and MyrAkt (Fig. 5,A). To assess the role of PKC in Akt-dependent Raf-1 activation and inhibition of apoptosis, serum and/or IL-3-starved cells expressing c-Akt, E40KAkt, or MyrAkt were incubated with PKC-specific peptide inhibitors, and apoptosis susceptibility and enzymatic activity of Raf-1, Akt, and PKC were examined. At concentrations that blocked PKC activation, the PKC inhibitors had no effect on Akt activity but abrogated Akt-dependent Raf-1 activation (Fig. 5,B). Thus, Akt appears to activate Raf-1 in a Ras-independent but PKC-dependent manner. PKC inhibitors did not alter Raf-1 activation in 32Dcl3 cells expressing BCR/ABL (data not shown), indicating that, in addition to Akt (7), alternative pathways of Raf-1 activation (i.e., Ras) are stimulated by BCR/ABL. In addition, the PKC inhibitors, at the concentration used, did not affect Raf-1 directly. PKC activity was important in Akt-dependent protection from apoptosis, because treatment of IL-3-starved E40KAkt- or MyrAkt-expressing cells with the specific PKC inhibitors induced apoptosis in these cells (Fig. 6).

To identify potential Raf-1 downstream effectors involved in Akt-dependent antiapoptotic signaling, we assessed whether BAD, which can be phosphorylated by both Akt and Raf-1 (8, 9, 12) and which is involved in the regulation of apoptosis (8, 9), underwent posttranslation modifications indicative of changes in its activation status. As expected, expression of the constitutively active E40KAkt mutant induced phosphorylation of BAD in IL-3- and serum-starved 32Dcl3 cells (Fig. 7). Expression of MM-Raf-1 DNM in these cells was associated with a marked decrease in BAD phosphorylation; however, this mutant did not affect Akt-dependent phosphorylation of MAP kinase (Fig. 7), supporting the specificity of the effects. Moreover, BAD, but not MAP kinase, was phosphorylated in IL-3- and serum-starved 32Dcl3 cells transfected with MM-Raf-1 DAM (Fig. 7). Together, these results are consistent with the existence of an Akt-dependent pathway of Raf-1 activation which, through mitochondria Raf-1, leads to BAD phosphorylation and increased resistance to apoptosis.

The recently discovered antiapoptotic function of the Akt serine/threonine kinase appears to be of pivotal importance for the survival of many cell types (3, 4); however, the understanding of the mechanisms involved in the antiapoptosis effects of Akt remains limited. Because there are multiple downstream effectors of Akt (25), there may be multiple mechanisms whereby Akt delivers its survival signal. For example, Akt inhibits the activity of Ced3/interleukin 1β converting enzyme-like proteases that specifically cleave the poly(ADP-ribose)polymerase (3) and phosphorylates directly pro-caspase 9, a process that has been proposed to inhibit cytochrome c-dependent activation of caspase 9 in cells overexpressing Akt (10). Akt directly phosphorylates BAD (8, 9), a proapoptotic protein the death-inducing effects of which are suppressed by phosphorylation (11). Moreover, it induces an increase in the expression of Bcl-2 (6, 7), an antiapoptotic protein that can target Raf-1 to mitochondria (12). Mitochondrial Raf-1 has also been shown to induce phosphorylation of BAD (12). Thus, Akt can functionally inactivate BAD directly (8, 9) or indirectly via Raf-1. In the present study, we assessed the antiapoptosis and growth-promoting effects of constitutively active Akt in myeloid precursor 32Dcl3 cells. We found that Akt did not promote growth factor-independent proliferation but enhanced cell survival. The enhanced survival was accompanied by the activation of Raf-1. Interestingly, suppression of the activity of the MAP kinase cascade regulated by plasma membrane Raf-1 did not abrogate the survival-promoting effects of Akt, whereas inhibition of mitochondria Raf-1 function reverted such effects. Mechanistically, Akt was able to activate Raf-1 in living cells in a Ras-independent but PKC-dependent manner.

In a recent study (26), stimulation of PKC by 12-O-tetradecanoylphorbol-13-acetate led to Raf-1 activation, which was blocked by expression of a Raf-1 mutant (R89L Raf-1) that prevents the association of Ras with Raf-1 (26). That finding, which suggests the requirement for Ras in PKC-dependent Raf-1 activation, conflicts with the observation that PKC-dependent Raf-1 activation was not blocked by expression of the dominant-negative N17Ras, which, instead, suppressed epidermal growth factor-dependent Raf-1 activation (26). Thus, the role of RAS in PKC-dependent Raf-1 activation remains unclear; possibly, Akt-dependent activation of PKC leads to direct effects on Raf-1, whereas 12-O-tetradecanoylphorbol-13-acetate exerts more pleiotropic effects, mediated in part by RAS.

Our data indicate that the Akt-dependent activation of Raf-1 and its accumulation in mitochondria are important for the Akt-mediated antiapoptotic effect. Blocking mitochondrial Raf-1 by exposure of a mitochondria-targeted, dominant-negative Raf-1 mutant or inhibition of Raf-1 activation by treatment with PKC inhibitors abrogated the protection from growth factor withdrawal-induced apoptosis in Akt-expressing 32Dcl3 myeloid precursor cells. The biochemical consequences of suppressing mitochondrial Raf-1 function are only partially understood; interestingly, mitochondria-targeted, dominant-negative Raf-1 mutant markedly reduced the Akt-dependent phosphorylation of BAD, supporting the role of Raf-1 in BAD phosphorylation (12).

The death-promoting effect of BAD depends on its interaction with Bcl-2 and Bcl-X_L (27). Such interaction is regulated by phosphorylation because serine-phosphorylated BAD interacts with 14-3-3 protein and is sequestered in the cytoplasm (11, 28). Whereas direct Akt phosphorylation of BAD might be the primary mechanism whereby BAD is functionally inactivated, it appears that there are independent mechanisms that lead to disruption of BAD activity. One such mechanism may be the enhanced expression of active Raf-1 on the mitochondria membrane (12), which would depend on the activation of Raf-1 and its transport to the mitochondria. PKC-mediated activation of Raf-1, in conjunction with Bcl-2-dependent (or independent) mechanisms whereby Raf-1 is transported to the mitochondria, might exemplify a mechanism whereby proapoptotic BAD is inactivated in an Akt-dependent manner. Mitochondrial Raf-1-dependent phosphorylation of BAD might involve residues distinct from those phosphorylated by Akt, yet result in the inhibition of the proapoptotic function of BAD; alternatively, the serine residues of BAD that are phosphorylated by Akt may also undergo Raf-1-dependent phosphorylation in the mitochondria.

In summary, the identification of an Akt-regulated pathway leading to the accumulation of activated Raf-1 in mitochondria supports the essential role of Akt as an apoptosis regulator that uses multiple mechanisms to suppress apoptotic cell death.