The data presented herein establish that the Hsp90-Cdc37 chaperone complex regulates mitophagy by modulating the stability and function of Ulk1 and one of its downstream targets, Atg13. Specifically, the interaction with the Hsp90-Cdc37 complex stabilizes Ulk1 by preventing proteasome-mediated degradation and this interaction is required for Ulk1 autophosphorylation and phosphorylation of Atg13 at S318. Strikingly, Ulk1 activation promotes the release of Atg13 from the Hsp90-Cdc37-Ulk1 complex and the localization of Atg13 to depolarized mitochondria, where it plays an essential role in Parkin-dependent mitophagy. Although Hsp90, Cdc37 and Ulk1 regulate the phosphorylation of Atg13 at S318, this modification alone does not appear to be sufficient for promoting Atg13 release, as increased amounts of Ulk1 were not detected in immunoprecipitates of Atg13-S318A versus wild type Atg13, nor did we observe enhanced co-localization of Ulk1 and Atg13-S318A (data not shown). Rather, the release of Atg13 from Ulk1 may depend on a kinase-dependent change in conformation of Ulk1 and/or phosphorylation of Atg13 at sites other than S318. Regardless, the finding that the non-phosphorylatable Atg13-S318A mutant dominantly inhibits mitochondrial clearance highlights the functional significance of Atg13 phosphorylation by Ulk1. Since Hsp90, Cdc37, and Ulk1 together regulate phosphorylation of Atg13 at S318, and all are required for efficient autophagy-mediated clearance of mitochondria, these findings define a new pathway linking mitochondrial homeostasis with the cellular stress response coordinated by Hsp90-Cdc37.
In contrast to the prevailing view that Ulk1 and Atg13 function as a complex, our data indicate that Ulk1 kinase activity promotes the release of Atg13 from Ulk1, and that released Atg13 then localizes to ring-like structures around damaged mitochondria, and thereby contributes to their degradation. Indeed silencing of Atg13 or enforced expression of the non-phosphorylatable Atg13-S318A mutant impairs mitochondrial clearance. Curiously, phosphorylation of Atg13 at S318 is not required for efficient LC3 conversion and degradation (under basal conditions or following amino acid deprivation), suggesting S318 phosphorylation of Atg13 may influence cargo selection during autophagy.
The Hsp90-Cdc37 complex is involved in two distinct paradigms of autophagy-mediated mitochondrial clearance: Parkin-mediated clearance of depolarized mitochondria, and the BNIP3L-dependent developmental clearance of mitochondria in erythroid cells. Although these pathways differ in the mechanism by which mitochondria are targeted for degradation (Parkin versus BNIP3L), both rely on Ulk1-mediated activation of the autophagy pathway, which is regulated by Hsp90 and Cdc37. It is interesting to note that Pink1, a serine-threonine kinase that recruits Parkin to depolarized mitochondria and promotes clearance (Geisler et al., 2010
; Narendra et al., 2010
; Vives-Bauza et al., 2010
), was identified as a client of the Hsp90-Cdc37 chaperone complex (Lin and Kang, 2008
; Moriwaki et al., 2008
; Weihofen et al., 2008
). While the primary defect in mitochondrial clearance occurs after the recruitment of Parkin to mitochondria in ulk1−/−
MEFs, and in ulk1+/+
MEFS treated with 17AAG or having knockdown of cdc37
, we cannot exclude the possibility that Pink1 function is also affected. In fact, the diffuse localization of Parkin in ulk1−/−
MEFs treated with 17AAG suggests that another client of Hsp90, perhaps Pink1, recruits Parkin to mitochondria and maintains the minimal levels of mitochondrial clearance observed the absence of Ulk1. That serine-threonine kinases involved in autophagy (Ulk1) and the mitochondrial targeting pathway (Pink1) are both clients of the Hsp90-Cdc37 chaperone complex suggests that coordinated regulation of mitochondrial turnover is an important homeostatic response that must be preserved, even under adverse conditions.
It has been suggested that Hsp90 inhibition promotes autophagy (Qing et al., 2006
; Siegelin et al., 2010
). Indeed, under normal growth conditions Hsp90 inhibition triggers an initial Ulk1-dependent increase in autophagy-dependent LC3 degradation (Dorsey and Kundu, unpublished observations), a response consistent with the Hsp90-dependent nature of kinases in the PI3K-mTOR pathway (Basso et al., 2002
; Gray et al., 2007
; Ohji et al., 2006
) that inhibit autophagy (Ravikumar et al., 2004
). However, this initial increase in autophagy is self-limiting and is rapidly cancelled by the inactivation of Hsp90-dependent Ulk1 activity, the primary autophagy-related target of mTOR (Hosokawa et al., 2009
; Jung et al., 2009
). In turn, inactivation of Ulk1 cripples the cells ability to respond to stimuli that induce autophagy, such as starvation or mitochondrial damage.
The exquisite dependence of Ulk1-mediated autophagy and mitochondrial clearance on Hsp90 function has important implications for cancer pathogenesis and treatment. First, it has been proposed that the destabilization of proteins associated with growth in the hypoxic, nutrient-deprived tumor milieu, is compensated by increased expression of Hsp90 and its co-chaperones (Whitesell and Lindquist, 2005
). Since autophagy promotes survival under these adverse conditions, the stabilization and activation of Ulk1 by Hsp90 may be an important component of the survival response coordinated by Hsp90 and exploited by certain tumors. Indeed, since many tumors rely on autophagy for survival under stressed conditions (Amaravadi et al., 2007
; Livesey et al., 2009
; Maclean et al., 2008
), the efficacy of Hsp90 inhibitors such as 17AAG may rely on their effects on Ulk1-mediated autophagy.
Collectively, the findings presented herein support a model (see Graphical Abstract) whereby interactions of Ulk1 with the Hsp90-Cdc37 complex stabilizes an active form of Ulk1 that promotes flux through specific arms of the autophagy pathway in response to metabolic or proteotoxic cues. Starvation, mitochondrial depolarization and other stimuli that trigger autophagy activate Ulk1, promoting Ulk1-mediated phosphorylation and release of its essential interacting partner, Atg13, and increased flux through the autophagy pathway. Phosphorylation of Atg13 at specific sites, for example S318, may favor selective degradation of mitochondria. As Ulk1 stability and activity rely on the Hsp90-Cdc37 complex, the function of both Ulk1 and downstream targets, including Atg13, is also subject to regulation by environmental and intracellular cues that alter Hsp90 activity. The Hsp90-Cdc37-to-Ulk1-to-Atg13 pathway thus coordinates and integrates autophagy and mitochondrial homeostasis with the cellular stress response.