We have shown previously that phosphomimetic mutants of HtrA2 at S400 result in increased proteolytic activity and contribute to enhanced resistance to mitochondrial stress.9
Here we show that one of the kinases responsible for phosphorylation of HtrA2 at S400 is Cdk5. The interaction between HtrA2 and Cdk5 occurs in both mitochondria and cytosol. Activation of Cdk5 activity increases its interaction with HtrA2, the phosphorylation of HtrA2 at S400 and translocation of Cdk5 to mitochondria. Δψm
is compromised in cells where the p38 pathway is activated and HtrA2 is silenced. Overexpression of WT HtrA2 but not the non-phosphorylatable S400A HtrA2 mutant rescues this phenotype, suggesting that phosphorylation of HtrA2 at S400 by Cdk5 is important for mitochondrial health.
HtrA2 is phosphorylated at S400 upon stimulation of the p38 stress signalling pathway. We have shown previously that activation of this pathway induced the phosphorylation of HtrA2 at another site, S142, also adjacent to an amino acid found mutated in patients with PD.9
Taken together, these findings suggest that HtrA2 phosphorylation at both of these sites is important for the cellular stress response. HtrA2 is also known to be phosphorylated at S212 by Akt, which reduces its serine protease activity and proapoptotic function, thereby promoting cell survival.29
By phosphorylating S400, Cdk5 could also regulate the proapoptotic function of HtrA2 once it is released into the cytosol from mitochondria. Phosphorylation at S400 was shown previously to increase HtrA2 serine protease activity in vitro
HtrA2 promotes cell death by its ability to bind to IAPs but also through its serine protease activity1, 3
and might, therefore, also modulate its proapoptotic function, especially as the interaction between Cdk5 and HtrA2 is partly detected in the cytosolic fractions.
In mitochondria, HtrA2 is thought to function in a similar manner to that of its bacterial homologues DegS and DegP, which are involved in protection against cell stress.5
Loss of HtrA2 results in the accumulation of unfolded proteins in mitochondria, defective mitochondrial respiration and increased oxidative stress.28
Increase in HtrA2 protease activity by Cdk5 phosphorylation might be important in removing unfolded/damaged proteins in mitochondria. Indeed, both HtrA2 and Cdk5 have been linked recently to autophagy and mitochondrial fission,30, 31, 32
two processes that can be initiated by damage to mitochondrial proteins and which are critical for mitochondrial health. Whether HtrA2 phosphorylation at S400 occurs in the cytosol and/or in mitochondria remains to be determined. However, we show that Cdk5 partially localises with mitochondria in human SH-SY5Y neuroblastoma cells, and that the mitochondrial pool of Cdk5 and its activators p35/p25 is increased following glutamate treatment, suggesting that Cdk5 is available at the mitochondria for phosphorylating HtrA2 under stress conditions where increased protease activity is required.
Certain kinases have an increasingly recognised role in mitochondrial processes,24
and some kinases that are required in mitochondria, including Cdk5, do not contain an MTS. How they gain entry into the mitochondria remains unclear. It is possible that they could associate with scaffold proteins imported to the mitochondria through an MTS. In the case of Cdk5, it is possible that the distribution of the active protein is dictated by its association with p35. p35 contains an N-terminal myristoylation signal motif,20
which targets the protein complex to cellular membranes, where many substrates of Cdk5 reside.
Mitochondrial dysfunction is increasingly recognised as having a crucial role in neurodegenerative diseases; in particular PD. The PD-associated proteins PINK1, parkin and HtrA2 all have important roles at the mitochondria. Contrary to PINK1 and HtrA2, parkin does not include an MTS,33
yet parkin was shown recently to be recruited by PINK1 to depolarised mitochondria for initiating their clearance from the cell by autophagy.34, 35
PINK1, parkin and HtrA2 have been proposed to be components of inter-connected molecular pathways that ultimately lead to mitochondrial dysfunction in PD,6, 10
but the necessity for HtrA2 in these pathways has been disputed, in particular in Drosophila
Similarly, in humans, whether or not HtrA2 contributes to PD pathogenesis is an matter of debate.8
We show here that the PD-associated kinase PINK1, previously shown to bind to HtrA2,9
is not required for the interaction between HtrA2 and Cdk5 (Supplementary Figure S2D
). Although importantly, Cdk5 also phosphorylates the PD-associated protein parkin and regulates its E3 ubiquitin ligase activity,38
emphasising cellular cross-talk between the pathways associated with neurodegeneration.
Strikingly, we show that HtrA2 phosphorylation at S400 is significantly reduced in the brain of mice carrying the G399S HtrA2 mutation, although total levels of HtrA2 protein remain unchanged. This could indicate that HtrA2 phosphorylation (and therefore, increase of its proteolytic activity) is important for mitochondrial function, known to be deregulated in neurodegenerative disorders. We also found that mice overexpressing the Cdk5 activator associated with neurodegeneration, p25, have increased interaction between Cdk5 and HtrA2 in the midbrain, the brain region preferably destroyed in PD. These mice have a well-characterised neurodegenerative phenotype with age-related deficits in learning and memory.39
Our data suggest that active Cdk5 interacts with HtrA2 and that this is important for HtrA2 protective function in mitochondria. This modulation of HtrA2 may reflect the fine tuning necessary to respond to cellular signals accordingly. Although we suggest that activation of Cdk5 under stress conditions increases the interaction between HtrA2 and Cdk5, this appears to promote mitochondrial health as a stress response. One could hypothesise that the interaction of HtrA2 and Cdk5 could also be proapoptotic, in particular if Cdk5 and HtrA2 interact in the cytosol. Deregulation of Cdk5 is widely reported to promote neurodegeneration; however, it is the constitutive activation of Cdk5 rather than activation of Cdk5 that is thought to promote neurodegeneration (Patrick et al.20
; for a review see Dhavan and Tsai12
). Therefore, we propose that transient phosphorylation of HtrA2 by Cdk5 under normal conditions does not contribute to neurodegeneration directly; rather it is a key process responsible for modulating HtrA2 function to fine tune the correct response to the cellular environment.
The exact mechanism of HtrA2 function in mitochondria awaits further characterisation. HtrA2 may be a key player in maintaining mitochondrial protein quality control through its ability to clear damaged or misfolded proteins. We have shown that loss of Δψm
in HtrA2-silenced cells under stress induced by the activation of the p38 pathway was rescued by expressing WT and S142A HtrA2 but not the non-phosphorylatable S400A or S142/400A HtrA2 mutants. These data suggest that HtrA2 phosphorylation at S400, and likely subsequent increase of its protease activity, may be important for HtrA2 mitochondrial function. Indeed, previous work indicated that phosphorylation of HtrA2 S400 conferred protection from cell death.9
As mitochondria do not have a ubiquitin proteasome system, clearance of damaged mitochondrial proteins or regulation of the levels of proteins involved in mitochondrial dynamics is an essential process, which in mitochondrial compartments is thought to be heavily reliant on mitochondrial proteases such as HtrA2. Indeed, modulation of the levels of the intra-mitochondrial fusion protein OPA1 by HtrA2 protease activity has been shown recently.40
Mitochondrial function is of critical importance in neurons and mitochondrial dysfunction is well documented to be a major factor underlying the pathogenesis of neurodegenerative diseases.31, 32
We have defined a mitochondrial stress response pathway involving Cdk5 and HtrA2, two proteins associated with neurodegeneration. These events may be relevant for neurodegenerative diseases and further characterisation of the pathway involving HtrA2 and Cdk5 that lead to mitochondrial dysfunction may allow better understanding of disease pathogenesis.