Apoptosis commitment and activation of mitochondrial Bax during anoikis is regulated by p38MAPK.

Most cells undergo apoptosis through the intrinsic pathway. This is dependent on mitochondrial outer membrane permeabilisation (MOMP), which is mediated by the pro-apoptotic Bcl-2 family proteins, Bax and Bak. During apoptosis, Bax translocates from the cytosol to the outer mitochondrial membrane (OMM), wherein it contributes to the formation of pores to release cytochrome-c. However, it remains unclear whether Bax translocation is sufficient to bring about MOMP or whether Bax requires further signals on the OMM to be fully activated. We have previously shown that during mammary epithelial cell anoikis, Bax translocation does not commit cells to MOMP and detached cells are rescued if survival signals from the extracellular matrix (ECM) are restored. These findings implied that a second signal is required for mitochondrial Bax to fully activate and cause MOMP. We now identify p38MAPK (mitogen-activated protein kinase) as this necessary signal to activate Bax after its translocation to mitochondria. The inhibition of p38MAPK did not prevent Bax translocation, but its activity was required for mitochondrial Bax to bring about MOMP. p38MAPK was activated and recruited to a high molecular weight mitochondrial complex after loss of ECM attachment. Artificially targeting p38MAPK to the OMM increased the kinetics of anoikis, supporting a requirement for its mitochondrial localisation to regulate Bax activation and drive commitment to apoptosis.

Bax targeting to mitochondria occurs via both tail anchor-dependent and -independent mechanisms.

Bax is a member of the Bcl-2 family that, together with Bak, is required for permeabilisation of the outer mitochondrial membrane (OMM). Bax differs from Bak in that it is predominantly cytosolic in healthy cells and only associates with the OMM after an apoptotic signal. How Bax is targeted to the OMM is still a matter of debate, with both a C-terminal tail anchor and an N-terminal pre-sequence being implicated. We now show definitively that Bax does not contain an N-terminal import sequence, but does have a C-terminal anchor. The isolated N terminus of Bax cannot target a heterologous protein to the OMM, whereas the C terminus can. Furthermore, if the C terminus is blocked, Bax fails to target to mitochondria upon receipt of an apoptotic stimulus. Zebra fish Bax, which shows a high degree of amino-acid homology with mammalian Bax within the C terminus, but not in the N terminus, can rescue the defective cell-death phenotype of Bax/Bak-deficient cells. Interestingly, we find that Bax mutants, which themselves cannot target mitochondria or induce apoptosis, are recruited to clusters of activated wild-type Bax on the OMM of apoptotic cells. This appears to be an amplification of Bax activation during cell death that is independent of the normal tail anchor-mediated targeting.

The N-terminal conformation of Bax regulates cell commitment to apoptosis.

The Bcl-2 protein Bax normally resides in the cytosol, but during apoptosis it translocates to mitochondria where it is responsible for releasing apoptogenic factors. Using anoikis as a model, we have shown that Bax translocation does not commit cells to apoptosis, and they can be rescued by reattachment to extracellular matrix within a specific time. Bax undergoes an N-terminal conformational change during apoptosis that has been suggested to regulate conversion from its benign, cytosolic form to the active, membrane bound pore. We now show that the Bax N-terminus regulates commitment and mitochondrial permeabilisation, but not the translocation to mitochondria. We identify Proline 13 within the N-terminus of Bax as critical for this regulation. The subcellular distribution of Proline 13 mutant Bax was identical to wild-type Bax in both healthy and apoptotic cells. However, Proline 13 mutant Bax induced rapid progression to commitment, mitochondrial permeabilisation and death. Our data identify changes in Bax controlling commitment to apoptosis that are mechanistically distinct from those controlling its subcellular localisation. Together, they indicate that multiple regulatory steps are required to activate the proapoptotic function of Bax.