The data provided here show that a phospho-resistant alanine at residue 776 of ATXN1, replacing a serine, destabilizes the protein in Purkinje cells. This finding is consistent with previous studies using transfected cell lines and a Drosophila
model of SCA1 indicating that phosphorylation of ATXN1 at S776 stabilized the protein (Chen et al. 2003
). Chen colleagues also suggested that Akt is the ATXN1-S776 kinase. In contrast, we found that inhibiting Akt activity either in vivo
or in a cerebellar extract failed to decrease the amount of phospho-S776-ATXN1 detected. However, several lines of evidence indicated that PKA is the S776 kinase, including co-fractionation with the peak of S776 phosphorylating activity in cerebellar cytosol (), and reduction of S776 phosphorylation by cerebellar lysates upon addition of two PKA inhibitors () and following PKA immunodepletion (). Although Purkinje cells amply express both Akt and PKA (Allen Brain Atlas – Lein et al. 2007
), the data reported here are consistent with the conclusion that Akt is not the S776 kinase with PKA being a stronger candidate for the ATXN1-S776 kinase active in the cerebellum.
A cerebellar signaling pathway linked to regulating PKA activity is the rebound potentiation, a form of synaptic plasticity at GABAergic synapses between inhibitory interneurons (basket and stellate neurons) and Purkinje cells. In a model described by Kawaguchi and Hirano 2002
; it was assumed that there is a relatively high basal level of PKA activity in Purkinje cells that is reduced with the activation of GABAB
R during depolarization at inhibitory synapses. If this assumption were accurate, a high basal activity of PKA in Purkinje cells would require that the basal intracellular concentration of cAMP be relatively high, perhaps via the activation of the glutamate receptor mGluR1 that activates adenyl-cyclase and increases cAMP (Miyashita and Kubo 2000
). Since phosphorylation of ATXN1 at S776 promotes neurodegeneration (Serra et al. 2004
; Chen et al. 2003
), it is tempting to speculate that perhaps a high basal activity of PKA in Purkinje cells contributes to their enhanced sensitivity to the toxic affects of mutant ATXN1 in SCA1.
Multiple mechanisms regulate the specificity by which proteins are phosphorylated. These include structure of the catalytic site on the kinase, activation state of the kinase, interactions between the kinase and substrate, as well as interactions involving scaffolding and adaptor proteins. Such regulatory mechanisms are required since the number of potential phosphorylation sites in the proteome is on the order of 700,000 for any one kinase (Ubersax and Ferrell 2007
). The data reported by Chen et al. 2003
demonstrate that the peptide sequence in ATXN1 surrounding S776 can function as a recognition motif for Akt. Thus, it is likely that some higher level regulatory mechanism prevents Akt in the cerebellum from phosphorylating ATXN1 at S776 as effectively as it can in the systems used by Chen et al. 2003
. At this time it is unclear why Akt is not effective in phosphorylating S776 of ATXN1 in the cerebellum.
Disparate effects of altering a kinase-mediated signaling pathway between Drosophila
and rodent models of neurological disease are becoming increasingly common, perhaps reflecting that in mammals the kinome is more complex than in the fly (Manning et al. 2002
; Caenepeel et al. 2004
). For example, loss of function mutations in the putative mitochondrial protein PINK1 (PTEN-induced kinase 1) are linked to recessive forms of Parkinson’s disease (Cookson et al. 2007
). In Drosophila
loss of function of the PINK1 homolog leads to dramatic morphological abnormalities of mitochondria (Clark et al. 2006
; Park et al. 2006
; Yang et al. 2006
). While in the mouse, deletion of PINK1
impairs mitochondrial functions in the absence of gross changes in the ultrastructure or the total number of mitochondria (Gautier et al. 2008
). Parkinson’s disease is also associated with the deposition of phospho-S129-α-synuclein into Lewy bodies in brain tissue. In a Drosophila
model of α-synuclein induced neurodegeneration, phosphorylation at S129 was associated with enhanced pathology (Chen and Feany 2005
). However, in a rat model of Parkinson’s disease S129 phosphorylation decreased degeneration (Gorbatyuk et al. 2008
). In closing, the studies reported here on the phosphorylation of ATXN1 in the cerebellum provide further support for the importance of examining findings from Drosophila
and cell culture systems with those from an in vivo
mammalian model of the human neurodegenerative disease.