Identification of Peripherin as a Akt Substrate in Neurons

Graduate School of Engineering, Osaka City University, Ōsaka, Ōsaka, Japan
Journal of Biological Chemistry (Impact Factor: 4.57). 09/2007; 282(32):23491-9. DOI: 10.1074/jbc.M611703200
Source: PubMed


Activation of Akt-mediated signaling pathways is crucial for survival and regeneration of injured neurons. In this study,
we attempted to identify novel Akt substrates by using an antibody that recognized a consensus motif phosphorylated by Akt.
PC12 cells that overexpressed constitutively active Akt were used. Using two-dimensional PAGE, we identified protein spots
that exhibited increased immunostaining of the antibody. Mass spectrometry revealed several major spots as the neuronal intermediate
filament protein, peripherin. Using several peripherin fragments, the phosphorylation site was determined as Ser66 in its head domain in vitro. Furthermore, a co-immunoprecipitation experiment revealed that Akt interacted with the head domain of peripherin in HEK
293T cells. An antibody against phosphorylated peripherin was raised, and induction of phosphorylated peripherin was observed
not only in Akt-activated cultured cells but also in nerve-injured hypoglossal motor neurons. These results suggest that peripherin
is a novel substrate for Akt in vivo and that its phosphorylation may play a role in motor nerve regeneration.

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    • "However, most of nuclear Akt substrates or binding partners are also ubiquitously expressed in various cell types other than neurons, and perhaps involves other cell survival signaling. Neuron-specific substrates of Akt are also identified, such as the intermediate filament protein peripherin [51], but yet we do not know how Akt phosphorylation could affect their functions. Thus, identification of additional neuron-specific targets or binding proteins of nuclear Akt may provide a better understanding of the neuroprotection mechanism that is modulated by Akt signaling in the nucleus. "
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    • "ILs and PLs were obtained from NCS and 5dCS rats (n = 8 each), and lysed in a buffer containing 8 M urea and 2% 3-[(3-Cholamido- propyl dimethylammonio)] propanesulfonate (CHAPS), as described previously (Konishi et al., 2007). The supernatants, containing 27 lg protein, were loaded onto SuperSep™ Ace 5–20% gradient gels (Wako Pure Chemicals, Osaka, Japan) and blots were Fig. 1 "
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    • "In fact, as with other intermediate filament proteins, peripherin is structured as a central coiled-coil α-helical rod domain flanked by a head and a tail domain. Head and tail domains of intermediate filament proteins are phosphorylated at several Ser/Thr sites and this has also been demonstrated for peripherin [3, 51, 55, 59]. Phosphorylation at certain sites has been shown to induce filament disassembly [53]. "
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