Identification of peripherin as a Akt substrate in neurons.
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 Ser(66) 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.
Konishi
Hiroyuki
H
Department of Anatomy and Neurobiology, Osaka City University, Graduate School of Medicine, Osaka 545-8585, Japan.
Namikawa
Kazuhiko
K
Shikata
Keiji
K
Kobatake
Yuji
Y
Tachibana
Taro
T
Kiyama
Hiroshi
H
D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D007381:Intermediate Filament Proteins / Q000737:chemistry* / Q000502:physiology*; D008297:Male; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000502:physiology*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000502:physiology*; D009474:Neurons / Q000378:metabolism*; D016716:PC12 Cells; D064531:Peripherins; D011485:Protein Binding; D040901:Proteomics / Q000379:methods; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012694:Serine / Q000737:chemistry
D007381:Intermediate Filament Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; C578994:PRPH protein, human; D064531:Peripherins; D012694:Serine; D051057:Proto-Oncogene Proteins c-akt