7.4 The proto-oncogene DEK
7.4.3 DEK in apoptosis
The above mentioned subcellular localisation of DEK (see 7.4.2) is not changed during apoptosis. By immunocytochemistry, a decrease in the intensity of the nuclear DEK-specific signal could be detected concomitantly to alterations of chromatin morphology during apoptosis, but no translocation to other cellular compartments was detected (Fig. 23).
Additionally it could be demonstrated that the DEK protein is neither transcriptionally nor translationally upregulated during apoptosis (Fig. 25 and Fig. 26).
In the proteomic screen two protein spots migrating at the same molecular weight but at distinct isoelectric points were attributed to the DEK protein (Fig. 14). The intensity of the more acidic spot was decreased, whereas the more basic spot was increased. Since DEK is a phosphoprotein with many phosphorylation sites (Kappes et al., 2004a), and such variations in
isoelectric points are most commonly related to changes in protein phosphorylation, the data indicate that the phosphorylation status of DEK might be altered during apoptosis.
Additionally it was shown that the phosphorylated form of DEK migrates at a molecular weight of approximately 60 kDa in SDS-PAGE. Dephosphorylation resulted in a shift of mobility to 43 kDa.
The data shown here indicate that the binding affinity of DEK to DNA is reduced in cells undergoing apoptosis. In intermediate stage apoptosis, when morphological changes and caspase processing have already started, more soluble DEK was extracted at low ionic strength and in parallel, less DEK was observed in the insoluble protein fraction (Fig. 27).
This was observed on Western blots using two DEK-specific antibodies, although the intensity change was more evident when the monoclonal antibody was employed. This was in accordance with experiments on the binding characteristics of the two antibodies (Fig. 22). In contrast to the affinity purified serum, recognizing all forms of DEK, the monoclonal antibody does not recognize the unphosphorylated form migrating at 43 kDa. Further, it is likely that the monoclonal antibody only binds to one specific phosphorylated form, with the phosphorylation site located within the epitope from amino acid 19 to 167 recognised by the antibody, whereas the serum might recognize different phosphorylated forms. This would explain the differences in the signal intensities of the phosphorylated DEK in Western blots depending on the antibody used (Fig. 22). First attempts to identify phosphorylation sites in vivo revealed two putative phosphorylation sites inside the binding epitope of the monoclonal antibody (F. Kappes, unpublished data).
The release of DEK from DNA is not dependent on PKC, but can be abolished when inhibiting CK2, which is known to be the kinase mainly responsible for DEK phosphorylation (Kappes et al., 2004a). However, the CK2 inhibitor itself has major effect on the viability of the cells. Although the cells seemed to be protected from apoptosis, as no caspases were activated and no nuclear fragmentation occurred, the morphology of the cells was altered.
Both unstimulated control and apoptotic cells were shrunk and the nuclei were condensed.
Since it was previously reported that the CK2 inhibitor TBB decreases cell viability (Ruzzene et al., 2002), cell-free apoptosis reactions were performed.
Inhibition of PKC did not change the release of DEK from DNA, whereas CK2-inhibition again significantly impaired it, in line with the results obtained in intact cells. The same
The open question is how the release of DEK from DNA is triggered during apoptosis. It is known that dephosphorylated DEK binds strongly to DNA, whereas the phosphorylated form binds rather weakly. In addition, filter binding experiments demonstrated that the DEK/DNA interaction is dependent on ionic strength. Phosphorylated and unphosphorylated DEK bind equally well to DNA at 50 mM salt. At 100 mM salt the amount of DNA bound to the phosphorylated form of DEK is lower than the amount of DNA bound to the dephosphorylated form (Kappes et al., 2004a).
The results from the proteomic approach in this work indicate that there might be a phosphorylation event during apoptosis, which could lead to the observed release of DEK from DNA. Whether post-translational modification by phosphorylation occurs in apoptosis and which phosphorylation sites are affected, needs to be examined further.
From the data presented and discussed above different hypothetical mechanisms for apoptotic release of DEK from chromatin can be envisaged:
(1) It is clear that CK2 has an effect on DEK release. It is therefore likely that phosphorylation via this kinase triggers the release of DEK, but it remains open how caspases are involved in this phosphorylation event.
(2) Caspases themselves might have a direct influence on DEK. This seems unlikely, since proteolysis of DEK was not observed and no consensus site for caspase cleavage could be identified in DEK’s primary sequence.
(3) Possibly, caspases might target an additional protein which might bridge DEK to chromatin. This hypothetical cleavage could subsequently reduce the interaction between DEK and DNA. However, no protein which binds to DEK was found in yeast two-hybrid screens apart from DEK itself (Kappes et al., 2004b).
(4) It is more likely that caspases interact directly or indirectly with CK2 leading to the activation of the kinase, consequently to a higher phosphorylation of DEK and therefore to a loss of its affinity towards chromatin. Similar influences of caspases on kinases have been described previously, such as the activation of the human serine/threonine kinase, mammalian STE20-like kinase (MST) (Lee et al., 2001) and MEK kinase 1 (MEKK1) (Widmann et al., 1998) by caspase-3. In addition, it was shown that MEKK1 induces mitochondrial permeability transition upon cleavage by caspases resulting in a potentiation of apoptotic execution (Gibson et al., 2002).
8 PERSPECTIVES
Over the last years, there has been growing evidence that nuclear events are crucial steps in the process of apoptosis. Caspases, the major effector proteases in apoptosis were identified to directly or indirectly play a role in the process of chromatin fragmentation. In addition, factors independent of caspase activation were characterised to have influences on the nuclear execution of cell death, such as proteases (cathepsins and calpains), nucleases, and kinases.
In present study it was possible to apply a mass spectrometry-based proteomic approach and to identify 13 nuclear proteins to be altered as a consequence of apoptosis induction. In subsequent biochemical analyses it was possible to validate the observed nuclear alterations of individual candidates, such as hnRNPs, HMGBs and DEK.
DEK is a chromatin-binding protein whose physiological function has not been identified so far. There was no previous evidence which supported the involvement of DEK in apoptotic signalling.
In this work, changes in the binding affinity of DEK to chromatin in the course of apoptosis were observed which seem to depend on the phosphorylation status of the protein. Many questions regarding the apoptosis-specific changes of DEK are still open.
Which is the nature of the signals leading to DEK release from DNA? How are CK2 and caspases linked to each other: is CK2 downstream of caspases or vice versa? What are the molecular mechanisms involved in this process and do other proteins, apart from caspases and CK2, participate?
In order to address these questions, incorporation experiments with radioactive labelled phosphates should be performed. A molecular mapping of the phosphorylation sites affecting DEK-DNA binding affinity in apoptosis together with the analysis of site specific mutant forms of DEK might lead to a better understanding of the role of this post-translational modification in apoptosis.
Additionally, the physiological function of the DEK protein is unknown so far. Although the protein is ubiquitously expressed, it has not been investigated if the cell needs DEK for viability. Further evidences for the role of DEK in chromatin reorganisation and nuclear
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