Mitochondria are essential organelles in most eukaryotic cells and function in the maintenance of cellular energy supplies. Mitochondria are responsible for thermoregulation and synthesis of essential molecules . In addition, mitochondria also participate in key signaling events regulating cell proliferation and death . PKB has a critical role in regulating apoptosis [28–30] and directly phosphorylates and interacts with key factors involved apoptosis signaling [30, 31]. PKB also indirectly regulates apoptotic transcription factors such as FOXO3a and NFκB . Additionally, activated PKB is localized to various subcellular compartments including the mitochondria and nucleus . Therefore, there is a complex interplay between PKB signaling and mitochondria-mediated apoptosis.
We provide the first evidence that CTMP, a negative regulator of PKB, localizes to the mitochondria in a MTS-dependent manner (Figure 3A and 3B). We found CTMP is located at the mitochondrial intermembrane space and/or matrix (Figure 2D–2F). In addition, we discovered CTMP is phosphorylated on Ser37/Ser38 (Figure 1) and phosphorylation on these residues inhibited CMTP mitochondrial localization (Figure 3C). Furthermore, adenovirus-mediated CTMP overexpression sensitized cells to staurosporine-stimulated apoptosis (Figure 3D–3F). Interestingly CTMP interacts with Hsp70, resulting the sequestration of Hsp70 from Apaf-1 (Figure 4). Taken together, these data suggest CTMP is a novel mitochondrial protein and is involved in apoptosis (Figure 4D).
Evidence suggest that CTMP negatively regulates PKB activity in v-Akt transformed cells , ciliary ganglion neurons , and K-ras-induced lung cancer model . This observation is further supported by a recent report showing epigenetic down-regulation of CTMP transcription in malignant glioblastomas . As previously suggested , CTMP is phosphorylated in vivo in response to pervanadate stimulation (Figure 1). In the current study, two serine residues on CTMP that are phosphorylated in response to pervanadate in vivo were identified (Figure 1). Ser37/Ser38 is located at the amino-terminus of CTMP, close to the putative MTS, as predicted by MitoProt II 1.0a4 . These two seine residues are conserved in CTMP homologues from mouse (NP_03707.1), rat (NP_001020188.1) and dog (NP_001074103.1) but lower organisms such as D. melanogaster, S. cerevisiae, S. pombe or C. elegans do not appear to contain a gene for CTMP, suggesting CTMP is a relatively recent protein during evolution and the two identified serine residues might be important for its function. In addition, Ser37 was predicted by the motif prediction program (Prosite) to be phosphorylated by Casein kinase II. However, this possibility needs to be experimentally evaluated in the context of apoptosis and will provide detailed insight into CTMP regulation.
CTMP was detected in the membrane and cytosol fraction of LN229 cells . Furthermore, two molecular species corresponding to CTMP were observed in different cell lines, suggesting post-translational modifications. In addition, CTMP associated with intracellular structures similar to membrane ruffles . Recently, a nuclear pool of CTMP was detected in human pancreatic duct epithelial cells . Consistent with recent reports , a mitochondrial pool of CTMP from HEK 293 cells dependent on its MTS was detected in our experiments (Figure 2 and 3). Furthermore, this mitochondrial CTMP localization was inhibited by phosphorylation (Figure 3C). Therefore, CTMP may have distinct roles in various subcellular compartments.
Mitochondrial localization is mediated by the MTS. In most cases, the MTS is present as a cleavable sequence at the N terminus, also called a pre-sequence . Bioinformatic analysis of the CTMP sequence indicated that a potential MTS exists in CTMP at the N-terminus of protein. This predicted N-terminal MTS appears to be functional since an N-terminal 31-amino acid deletion-mutant of CTMP did not localize to the mitochondria (Figure 3A and 3B). N-terminal MTS of mitochondrial precursors are in most cases cleaved by the mitochondrial-processing peptidase (MPP) as soon as the cleavage sites reach the mitochondrial matrix [34, 35]. Mutational studies show the R-2 or R-3 motif are important but not sufficient to direct cleavage by MPP . Site-directed mutagenesis of the -2 or -3 arginine in different precursor molecules completely or partially inhibits processing [36, 37]. Further studies are needed to interrogate if the R-2 motif of CTMP is important in MTS-mediated mitochondria localization.
The molecular pathways that mediate apoptosis are tightly regulated by a series of positive and negative signals, the balance of which determines whether or not cells commit suicide . Recent evidence indicates that the coordinated interaction between Hsps and the components of apoptosis machinery may determine cellular susceptibility to damaging stresses . Indeed, CTMP associate specifically Hsp70, but not Hsp90, in HeLa cells (Figure 4A and 4B). Furthermore, complex formation of Hsp70 and Apaf-1 was decreased in CTMP-infected cell compared to controls (Figure 4C), indicating why CTMP overexpression sensitized the cell to apoptosis induced by staurosporine (Figure 4D). Indeed, Miyawaki and colleagues recently reported that upregulation of CTMP in hippocampal neurons is required for ischemia-induced neuronal death . Based on previous studies ([10, 12, 40], CTMP phosphorylation event occurs at the plasma membrane in response to activation of growth factor signaling, leading to the release of PKB. In the case of apoptosis, phosphorylation of the mitochondria pool of CTMP by as-yet-unidentified kinase will promote the exclusive cytoplasmic localization of CTMP, resulting the sequestration of Hsp70 from Apaf-1. One can also proposed that 14-3-3 may bind the phosphorylated CTMP and maintain it's cytoplasmic localization. In this regard, it will be of course interesting to discover how phosphorylation affects the interaction between CTMP and Hsp70. Further studies are required to elucidate mechanisms of CTMP-dependent apoptosis under pathophysiological conditions.