The present study reveals a novel protective compensatory signaling mechanism via PKCδ-PKD1 molecular interaction in dopaminergic neuronal cells. Through our collective results, we report for the first time four key findings in a dopaminergic neuronal model pertinent to oxidative stress-mediated neurodegenerative processes: (i) A proteolytically activated catalytic PKCδ fragment (PKCδ-CF) phosphorylates and activates protein kinase D1 (PKD1); (ii) PKD1 activation counteracts early stage oxidative damage and protects dopaminergic neuronal cells from cytotoxicity; (iii) PKCδ-dependent phosphorylation of ser 916 residue precedes ser 744/ser 748; (iv) PKCδ - PKD1 crosstalk tightly regulates cell survival and cell death to maintain cellular homeostasis in response to oxidative damage. The elucidation of this compensatory signal transduction mechanism in neurodegenerative diseases may enhance understanding of degenerative processes and lead to development of novel treatment modalities.
H2O2-induced cytotoxicity causes apoptosis in neuronal and non-neuronal cells [15, 43, 44, 50]. Generally, oxidative stress-induced apoptosis can be classified into early and late stages. DNA fragmentation occurs in the late stage of apoptosis and is preceded by ROS generation, mitochondrial dysfunction and caspase-3 activation and membrane phosphatidyl exposure [10, 47]. In neurodegenerative disorders, especially PD, the signaling mechanisms that contribute to increased vulnerability of dopaminergic neurons to oxidative damage are still under investigation. Most current research focuses on cell death mechanisms in dopaminergic neurons. Some of the signaling kinases responsible for cell death mechanisms in PD include JNK, MLK, MAPK, LRRK2, etc. [51–54]. Earlier, the involvement of a novel biochemical mechanism for cell death in dopaminergic neurons through caspase-mediated proteolytic activation of PKCδ was demonstrated [15–19]. The high levels of persistently active PKCδ catalytic fragment mediate apoptosis during oxidative stress in both cell culture and animal models of PD [15–19]. We also have shown in our earlier study that a positive feedback loop exists during the late stages of oxidative stress, where the persistently active PKCδ catalytic fragment translocates to the mitochondria to promote cytochrome C release and apoptosis [16, 17, 55].
We previously demonstrated proteolytic activation of PKCδ occurs during the early stages of oxidative stress, even before cell death can occur, and coincides with the initiation of mitochondrial ROS generation/caspase-3 activation in dopaminergic neurons [15, 17]. Thus, we speculated that proteolytically activated PKCδ might play a regulatory role during the early stages of apoptosis. Previous research suggests the presence of a variety of protective compensatory mechanisms that counteract the early oxidative insult [8–10]. Since we observed in our present study a significant lag time before induction of cell death during the early stages of oxidative stress (Figure 1B), we hypothesize that proteolytically activated PKCδ might sense the extent of oxidative damage and act as a homeostatic regulator in response to oxidative stress, modulating cell survival and cell death mechanisms through interactions with protective signaling molecules.
Protein kinase D1 (PKD1) is emerging as an important signaling molecule associated with oxidative stress in non-neuronal cell lines [31, 35, 36]. Studies have shown that oxidative stress increases PKD1 activation loop phosphorylation (pS744/pS748) via full length PKCs, including PKCδ, in non-neuronal models [37, 56–59]. However, the functions of PKD1 during oxidative stress-induced neurodegeneration have not been studied previously. In the present study, we report that cleaved active PKCδ phosphorylates the activation loop of PKD1 and activates the kinase during the early stages of H2O2 -induced oxidative stress in dopaminergic neuronal cells. We also observed a similar activation pattern for PKD1 and PKCδ during oxidative stress caused by the parkinsonian-specific toxicant 6-OHDA (Figure 9). To our knowledge, this is the first report of a novel cell survival/cell death signal regulation by the cleaved catalytic fragment of PKCδ at two different stages of apoptosis based on the extent of oxidative damage.
PKD1 is mainly activated by a diacylglycerol-dependent PKCs mechanism [22, 60] or by PKD1 cleavage [61–63]. A recent study shows that PKD1 auto-inhibition is released through phosphorylation at the Y463 site in the regulatory domain, leading to the activation loop phosphorylation by PKCδ full length (PKCδ-FL) in Hela cells . PKD1 is in a closed conformation during the resting stage, with the regulatory fragment having an autoinhibitory effect on the catalytic fragment [46, 64]. Multiple phosphorylation sites on PKD1 seem to be important for its activation loop phosphorylation, depending on the cell types and stimuli. In human cancer cell lines, PKD1 can be phosphorylated at multiple sites including Y463, S910 (corresponding to murine Y469, S916) [24, 65]. Phosphorylation of Ser 916 (murine) autophosphorylation site correlated with PKD1 activation loop phosphorylation [58, 66]. During oxidative stress in non-neuronal models, Tyr 469 is phosphorylated by upstream kinases, which results in release of the Pleckstrin homology (PH) domain autoinhibition prior to activation loop phosphorylation; this mechanism does not involve C-terminus Ser 916 phosphorylation [24, 37]. In our dopaminergic neuronal models, oxidative stress failed to induce PKD1 Tyr 469 phosphorylation (Figure 6A), whereas PKD1 Tyr 469 phosphorylation was induced by the positive control desmopressin (Figure 6B). Our results demonstrate that the mechanism of PKD1 activation in dopaminergic neurons is distinct from the mechanisms in other non-neuronal models. We demonstrate that S916 phosphorylation, but not Tyr 469 phosphorylation, is a preceding event that occurs and is required for PKD1ser744/Ser748 activation loop phosphorylation (Figure 6). Our data suggest that Ser 916 phosphorylation on the C-terminal of PKD1 may open the conformation for full activation of the kinase through activation loop phosphorylation during oxidative stress in dopaminergic neurons. A detailed comparative analysis of PKCδ proteolytic activation, PKD1 activation loop phosphorylation and the extent of cell death during oxidative stress revealed an interesting functional relationship between activation of kinases and regulation of cell death. Comparison of PKD1 activation and cytotoxicity shows that PKD1 activation is maximal during the early oxidative stress stage when no measurable cytotoxicity is noted (Figure 7A). Interestingly, when PKD1 activation begins to decline at the end of the early stage, cell death begins to occur. Also, the level of PKCδ proteolytic activation directly correlates with the extent of cell death at the later stage of oxidative stress. When the constitutively active PKD1 mutant (PKD1S744E/S748E) is overexpressed, dopaminergic cells are resistant to H2O2 -induced neurotoxicity, even during the late stages of oxidative stress (Figure 7D), which is consistent with our hypothesis that PKD1 activation protects against oxidative damage. The downstream signaling mechanisms of PKD1 activation in dopaminergic neuronal cells are not known. PKD1 translocates to the nucleus and regulates phosphorylation of HDACs and various transcription factors in various non-neuronal cell lines including B cell, cardiomyocytes & oestoblasts [31, 23, 40, 67]. PKD1 translocation to the nucleus after activation in dopaminergic neurons is also noted in the present study (Figure 8), suggesting that nuclear translocation of PKD1 may activate key cell survival transcription factors and genes. Thus, we suggest that PKD1 functions as a cell survival switch and turns 'ON' a protective compensatory mechanism in dopaminergic neurons. Studies are underway to characterize the downstream protective response of PKD1 signaling in nigral dopaminergic neurons.