Neuritic/synaptic dysfunction or degeneration are observed in clinical studies of Parkinson disease (PD) patients and in genetic and toxic/environmental models of PD. For example, expression of the autosomal dominant PD-associated G2019S mutant form of LRRK2 (leucine-rich repeat kinase 2) causes shortening of neurite lengths and simplification of branch points as indices of neuritic injury (MacLeod et al., 2006
; Plowey et al., 2008
). In this study, we observed that treatment with db-cAMP or coexpression of a GFP-tagged PKA catalytic subunit prevented neurite injury caused by LRRK2 G2019S in cortical neurons (). As previously observed with LRRK2 (MacLeod et al., 2006
), there were no effects on the mean soma area (). db-cAMP also protected against neuronal injury caused by LRRK2 G2019S and the parkinsonian neurotoxin MPP+
in differentiated SH-SY5Y cells (). Because both injuries are mediated by autophagy (Fig. S1 a
; Zhu et al., 2007
; Plowey et al., 2008
), we investigated whether PKA activity regulated autophagy in neuronal cells. Treatment with db-cAMP reduced the number of autophagic vacuoles (AVs) induced by either LRRK2 G2019S or MPP+
Figure 1. PKA signaling reduces neurite injury and autophagy. (a–d) Mouse cortical neurons coexpressing GFP or GFP-PKA and LRRK2 G2019S or vector 7 d after transfection. (b) Quantification of neurite lengths. *, P = 0.001 versus empty vector/GFP; **, P (more ...)
To determine whether db-cAMP reduced GFP-LC3 puncta by decreasing autophagy induction or by increasing AV maturation, we treated cells with rapamycin to induce autophagy and used the tandem reporter RFP-GFP-LC3 (Kimura et al., 2007
), which labels early AVs with dual red and green fluorescence and late AVs with red only (). Rapamycin increased the number of early AVs at 1 h (Fig. S1 c). At 3 h, there was an increase in both early and late AVs (), which is consistent with maturation of rapamycin-induced AVs. Cotreatment with db-cAMP reduced the number of rapamycin-induced AVs at all time points (; and Fig. S1 c). Because db-cAMP reduced the number of rapamycin-induced early AVs without increasing late AVs, these data suggest that cAMP/PKA signaling suppresses autophagy induction. Treatment with the PKA inhibitor (E)-N
-(2-(3-(4-bromophenyl)allylamino)ethyl)isoquinoline-5-sulfonamide (H89) led to an increase in lipidated LC3-II in SH-SY5Y, HeLa, and 293T cells ( and Fig. S1 b), further indicating that PKA down-regulates autophagy induction.
Figure 2. Cytoplasmic PKA inhibits autophagy. (a) RFP-GFP-LC3–labeled AVs in SH-SY5Y cells treated with rapamycin ± cAMP for 1 h. (b) Quantification of early AVs (GFP+ RFP+ puncta) in SH-SY5Y cells cotreated with rapamycin and cAMP for 3 h. *, P (more ...)
Because PKA has both transcriptional (Chalovich et al., 2006
) and posttranslational (Bok et al., 2003
) neuroprotective effects, we used untargeted, nuclear-targeted (NLS), or cytoplasmic-targeted (nuclear export signal) GFP-tagged PKA (Bok et al., 2003
) to study the effects of PKA subcellular localization on autophagy. Although the targeted PKA constructs elicited similar levels of cAMP response element binding protein phosphorylation (not depicted), nuclear localized PKA was unable to significantly reduce the number of AVs induced by rapamycin or LRRK2 G2019S (), whereas nuclear excluded PKA showed the same potency as untargeted PKA. These results suggest that the effect of PKA on autophagy is mediated by a cytoplasmic target.
To identify potential autophagy-modulating cytoplasmic targets of PKA, we performed isoelectric focusing/SDS-PAGE 2D gel analysis on untreated cortical neurons () and SH-SY5Y cells (not depicted). 2D immunoblots for LC3 revealed the presence of distinct species differing in isoelectric points, which is consistent with multiple phosphorylation states. Furthermore, treatment with forskolin, a known regulator of adenylate cyclase, increased the intensity of species migrating at more acidic isoelectric points, which is consistent with increased phosphorylation (). In living cells, we found that forskolin elicited increased 32P incorporation into HA-tagged LC3 (), which was prevented by cotreatment with the PKA inhibitor H89 (). Using in vitro kinase assays, we determined that PKA was capable of directly phosphorylating recombinant rat LC3 ().
Figure 3. PKA directly phosphorylates LC3. (a) 2D immunoblot probed for LC3 shows distinct species differing by isoelectric points. Forskolin increases abundance of species with a more acidic isoelectric point. (b) Metabolic labeling reveals greater 32P incorporation (more ...)
We used matrix-assisted laser desorption/ionization (MALDI) time of flight (TOF) mass spectrometry (MS) to identify the PKA phosphorylation site on LC3. Analysis of the MS1 spectra revealed a singly charged peptide [M+
ion signal at m/z 821.33 () that was lost from samples treated with PKA, accompanied by the appearance of a new peptide [M+
ion signal at m/z 901.41, corresponding to the predicted 80 D gain of a phosphate group (). Alkaline phosphatase treatment of PKA-phosphorylated LC3 eliminated the ion signal at m/z 901.41 (unpublished data). Tandem MS/MS analysis identified the m/z 901.41 ion as residues 8–13 containing a phosphorylation at serine 12 (Fig. S2 a
). To confirm that serine 12 was the PKA phosphorylation site, mutagenesis of serine 12 to alanine (S12A) abolished LC3 phosphorylation by PKA (). Custom antibodies generated to the phosphorylated peptides effectively labeled PKA-phosphorylated recombinant LC3 (Fig. S2 b) and endogenously phosphorylated LC3 in SH-SY5Y and cortical neurons but not in the presence of the immunizing peptide (Fig. S2 c). Endogenously phosphorylated LC3 was further increased by treatment with forskolin to stimulate PKA ().
We investigated whether endogenous LC3 phosphorylation was regulated by rapamycin or MPP+
, two stimuli that induce increased autophagic flux (Budovskaya et al., 2005
; Zhu et al., 2007
). Treatment with either rapamycin or MPP+
led to a reduction in phospho-LC3 as compared with vehicle-treated cells () with no significant change in levels of LC3 probed using an antibody raised against a C-terminal region epitope of mature LC3 distant to the phosphorylation site (LC3). These results suggest that LC3 is dephosphorylated during autophagy induction. Blots reprobed using the N-terminal antibody for LC3 revealed the expected increase in LC3-II caused by rapamycin. Endogenously phosphorylated LC3 comigrated with nonlipidated forms of LC3 ( and Fig. S2 c). To determine whether loss of phosphorylation was sufficient to increase the number of GFP-LC3–positive AVs, we transfected cells with a nonphosphorylatable mutant, GFP-LC3–S12A. Under basal conditions, GFP-LC3–S12A migrated predominantly as a lipidated LC3-II band, forming more puncta than GFP-LC3–wild type (WT; ), indicating that phosphorylation of LC3 regulates its incorporation into AVs.
Figure 4. LC3 dephosphorylation is associated with enhanced recruitment to autophagosomes. (a and b) Immunoblots of SH-SY5Y cells treated with rapamycin for 1 h (a) or MPP+ for 24 h (b) were probed using the phospho-specific antibody, a C-terminal LC3 antibody (more ...)
We determined whether dephosphorylation contributed to autophagy induction using the S12D phosphomimetic mutant in cells treated with rapamycin. The S12D mutation reduced the number of GFP-LC3 puncta in rapamycin-treated cells (). Similarly, the S12D mutant formed significantly fewer puncta in cells expressing LRRK2 G2019S or in cells treated with MPP+ (). Furthermore, the autophagy-related neurite shortening caused by LRRK2 G2019S or MPP+ was attenuated by the S12D mutation in SH-SY5Y cells and cortical neurons (). Collectively, the data indicate that LC3 phosphorylation by PKA suppresses induced autophagy and autophagic neurite degeneration elicited by LRRK2 G2019S and MPP+.
Figure 5. LC3 phosphomimic shows reduced recruitment and suppresses neurite degeneration. (a and b) SH-SY5Y cells expressing GFP-LC3–WT or –S12D treated with rapamycin or vehicle for 1 h. Quantification of GFP-LC3 puncta. *, P = 2.31 × 10 (more ...)
This study identifies a new phosphorylation site on LC3 that reduces its recruitment and participation in autophagy. To our knowledge, this represents the first example of phosphorylation-based regulation of this important autophagy effector, whose recruitment represents the convergence point for several pathways of autophagy induction (He and Klionsky, 2009
). As autophagy has been implicated in a growing number of human diseases, it has become clear that a fine balance of autophagy underlies healthy mammalian physiology. Phosphorylation-based regulation of autophagy has thus far centered on the nutrient-sensing target of rapamycin–Atg1 regulatory axis, whereas loss of inhibitory mechanisms in the phosphoinositide 3-kinase–beclin 1 pathway have been implicated in hereditary myopathies and pathological cell death (Zhu et al., 2007
; Scarlatti et al., 2008
; Vergne et al., 2009
). This study implicates components of the ubiquitin-like lipidation system itself as a third potential point of autophagy down-regulation capable of suppressing autophagy induced by metabolic or pathological stimuli. Other autophagy effector proteins may also be subject to phosphoregulation, linking autophagic responses to cellular injury and stress signals.
In yeast, recent studies indicate a nutrient-sensing role for PKA as a parallel pathway to target of rapamycin in suppressing autophagy, with phosphorylation of the kinase Atg1 as a convergence point (Yorimitsu et al., 2007
; Stephan et al., 2009
). There was no effect on Atg1 kinase activity, but phosphorylation inhibited localization to the preautophagosomal structure (Budovskaya et al., 2005
). Although a corresponding preautophagosomal structure has not been identified in mammals, phosphorylation of LC3 reduces its recruitment to rapamycin or MPP+
Interestingly, yeast and Drosophila melanogaster
Atg8 lack this particular PKA consensus site, although it is conserved in all mammalian forms of LC3 (Fig. S3
) but not in the other mammalian Atg8 homologues, GABARAP and GATE16. The crystal structures of LC3, GABARAP, and GATE16 demonstrate significant charge differences in the N-terminal helices, which underlie the differences in their biological functions (Sugawara et al., 2004
). Phosphorylation-related decreases in the net-positive charge of the LC3 N-terminal region may affect its interactions with phospholipids, proteins involved in target recognition, lipidation (Yamada et al., 2007
), cytoskeleton, or other functions (Kouno et al., 2005
; Wang et al., 2006
Although PKA is a major nutrient-sensing pathway in yeast, it plays additional important roles in the mammalian nervous system. Among these is the ability of PKA to promote neuronal differentiation and neurite outgrowth, which has been predominantly studied in terms of transcriptional regulation. However, recent studies indicate that autophagy plays a direct role in mediating neurite retraction in neurons subjected to trophic factor withdrawal, physical axotomy, or toxicity because of chemical or genetic factors (Yang et al., 2007
; Plowey et al., 2008
). As neurite extension/retraction likely reflects a balance of biosynthetic and degradative processes, the ability of cytoplasmically expressed PKA catalytic subunit to phosphorylate LC3 and suppress autophagy-dependent neurite retraction represents a novel mechanism contributing to effects of PKA in maintaining neuronal function.
These data implicate LC3 phosphorylation as a novel switch that modulates its biological function in mammalian cells. Both neuronal and nonneuronal cells exhibit endogenous levels of phosphorylated LC3. These observations support the concept of a reserve pool of phosphorylated LC3 that can be rapidly recruited for autophagy in response to external stimuli such as nutrient deprivation or mitochondrial injury. Dephosphorylation of LC3 would allow cells to rapidly switch from basal to induced autophagy, allowing the compensatory expansion of the autophagic compartment that is observed after injury or stress in mammalian cells (Yue et al., 2009