Although mutations in the putative kinase PINK1 are a common cause of autosomal recessive PD, very little is known about the biological function of PINK1 and how loss of PINK1 function leads to neurodegeneration. The present study demonstrates that PINK1 is a mitochondrial serine/threonine kinase with a critical role in promoting cell survival. We have identified what is to our knowledge the first substrate for the PINK1 kinase: the mitochondrial molecular chaperone TRAP1. Our findings reveal a hitherto unexplored pathway by which PINK1 phosphorylates downstream effector TRAP1 to protect cells against apoptosis, and suggest a link between the impairment of this novel mitochondrial pathway and neurodegeneration in PD.
It has become increasingly clear that molecular chaperones not only facilitate protein folding, but also regulate a number of cellular processes, including cell survival and apoptosis [24
]. Despite the critical importance of mitochondria in cell function and survival, the current knowledge about mitochondrial molecular chaperones is limited. TRAP1 is a poorly characterized mitochondrial protein with significant sequence homology to the HSP90AA1 family of molecular chaperones [23
]. Our finding that siRNA-mediated depletion of TRAP1 sensitizes cells to oxidative-stress-induced cytochrome c release and cell death suggests a role for TRAP1 in the modulation of the mitochondrial apoptotic cascade. Consistent with this role, TRAP1 knockdown has been shown to enhance cytochrome c release and apoptosis induced by other stressors, such as protein-tyrosine kinase inhibitor β-hydroxyisovalerylshikonin and topoisomerase II inhibitor VP16 [26
]. The molecular basis of the anti-apoptotic function of TRAP1 remains unknown. Although we find that TRAP1 and cytochrome c colocalize in the mitochondrial intermembrane space, our results indicate that TRAP1 does not physically associate with cytochrome c in cells either in the absence or presence of oxidative stress. Thus, TRAP1 may inhibit the release of cytochrome c from mitochondria through a mechanism other than direct interaction/retention. Future studies are required to elucidate the mechanism by which TRAP1 regulates cytochrome c release and apoptosis.
Our data reveal that endogenous TRAP1 is constitutively phosphorylated at low levels, and the TRAP1 phosphorylation is significantly increased in response to oxidative stress induced by H2O2. Both constitutive and oxidative-stress-induced TRAP1 phosphorylation are dramatically enhanced by PINK1 overexpression and are reduced by siRNA-mediated depletion of PINK1, indicating that PINK1 is a major kinase responsible for phosphorylating TRAP1 in cells. These results, together with the in vitro phosphorylation data, provide compelling evidence that TRAP1 is a bona fide substrate for PINK1 kinase. We find that overexpression of PINK1 protects cells against oxidative-stress-induced apoptosis by suppressing cytochrome c release from mitochondria, and this protective action of PINK1 depends on its kinase activity to phosphorylate TRAP1. Conversely, PINK1 depletion causes increased cytochrome c release from mitochondria and sensitizes cells to oxidative-stress-induced cell death, and the pro-apoptotic effect of PINK1 depletion correlates with reduced TRAP1 phosphorylation. Moreover, our studies using TRAP1 siRNAs have provided direct evidence that TRAP1 is required for PINK1-mediated protection against oxidative-stress-induced cytochrome c release and cell death. Collectively, these findings point to a specific pathway in which PINK1 exerts its cytoprotective function by phosphorylating downstream effector TRAP1 to inhibit the release of cytochrome c from mitochondria.
Our work indicates that the mitochondrial PINK1–TRAP1 anti-apoptotic pathway is disrupted by PD-linked PINK1 G309D, L347P, and W437X mutations. Previous studies of PINK1 mutations using in vitro autophosphorylation assays and artificial kinase substrates have led to conflicting results. For example, analyses with recombinant GST-fused PINK1 proteins purified from Escherichia coli
showed that PINK1 autophosphorylation activity was slightly decreased by the G309D mutation [11
] and was significantly increased by the W437X mutation [12
] and that neither of these mutations affected the activity of PINK1 to phosphorylate artificial substrate α-casein [12
]. In contrast, a recent study using recombinant PINK1 proteins isolated from baculovirus-infected sf9
insect cells failed to detect PINK1 autophosphorylation activity, but showed that PINK1 C-terminal truncation reduced the activity of PINK1 to phosphorylate artificial substrate histone H1 [27
]. These discrepancies underscore the importance of knowing physiological substrate(s) for characterizing the kinase function of wild-type and mutant PINK1. Our analyses reveal that single G309D and L347P mutations abolish the kinase activity of PINK1 to phosphorylate its physiological substrate TRAP1 both in vitro and in vivo. By comparison, the W437X mutation has a weaker effect on PINK1-mediated TRAP1 phosphorylation. Our findings that PD-linked mutations impair the ability of PINK1 to promote TRAP1 phosphorylation and cell survival provide strong support for a loss-of-function pathogenic mechanism in familial PD patients carrying homozygous PINK1 mutations.
Recently, an increasing number of PD cases with heterozygous mutations affecting only one PINK1
allele have been reported, including single heterozygous G309D and W437X mutations [6
]. Our results that PD-linked PINK1 mutations specifically inhibit PINK1 kinase function without affecting the substrate binding or subcellular localization of PINK1 raise an interesting possibility that these mutants may act in a dominant negative manner to inhibit the function of the normal PINK1
allele by competing with endogenous wild-type PINK1 for binding its substrate TRAP1. In support of this possibility, we find that PD-linked PINK1 G309D and L347P mutations have a dominant negative effect on in vivo TRAP1 phosphorylation and cell survival. These results suggest a dominant negative pathogenic mechanism by which heterozygous PINK1 mutations lead to Parkinsonian symptoms [6
]. By comparison, overexpression studies show that the W437X mutation has a smaller but significant inhibitory effect on in vivo TRAP1 phosphorylation and cell survival, providing evidence supporting a pathogenic role of the heterozygous W437X mutation. Our finding of a mitochondrial PINK1–TRAP1 anti-apoptotic pathway and its impairment by PD-linked mutations provides new insights into the pathogenic mechanisms of PD and suggests novel targets for therapeutic intervention.