Identification of peripherin as a Akt substrate in neurons
ABSTRACT 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.
- SourceAvailable from: Kyohei Tokizane
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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 "
ABSTRACT: Prolonged exposure to stress elicits profound effects on homeostasis that may lead to cryptogenic disorders such as chronic fatigue syndrome. To investigate the pathophysiology associated with the syndrome, we used a rat continuous stress (CS) model where the pituitary represents one of the most affected organs. Here we found that mRNA for VGF (non-acronymic), a member of the granin family, was induced specifically in the intermediate lobe (IL). This was matched by a concomitant increase at the peptide/protein level assessed by C-terminal antibody. Furthermore, the upregulation of VGF was confirmed by immunohistochemistry in a subset of melanotrophs. VGF expression was altered in the IL of rats receivingthe dopamine D2 receptor agonist bromocriptine or the antagonist sulpiride. In vitro, dopamine dose-dependently decreased the mRNA levels in cultured melanotrophs. These findings suggest that VGF expression under CS is negatively regulated by dopaminergic neurons projecting from the hypothalamus.Molecular and Cellular Endocrinology 03/2013; DOI:10.1016/j.mce.2013.03.012 · 4.24 Impact Factor
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- "The functional characteristics that distinguish peripherin's role during these events may be driven partly by the specific sets of peripherin isoforms expressed by injured or diseased neurons. Recent insights into peripherin expression and assembly have revealed complex intra-isoform associations that are tissue-specific and regulated through a number of different post-transcriptional processes, including alternative splicing and translation , nitrotyrosination, and phosphorylation (Aletta et al. 1989; Huc et al. 1989; Landon et al. 2000; Konishi et al. 2007; Tedeschi et al. 2007; McLean et al. 2008; Xiao et al. 2008). Our previous findings have demonstrated that there are physiological requirements for the production and regulation of different peripherin isoforms. "
ABSTRACT: Peripherin is a type III intermediate filament protein that is up-regulated during neuronal injury and is a major component of pathological inclusions found within degenerating motor neurons of patients with amyotrophic lateral sclerosis (ALS). The relationship between these inclusions and their protein constituents remains largely unknown. We have previously shown that peripherin expression is characterized by tissue-specific, intra-isoform associations that contribute to filament structure; changes to the normal isoform expression pattern is associated with malformed filaments and intracellular inclusions. Here, we profile peripherin isoform expression and ratio changes in traumatic neuronal injury, transgenic mouse models of motor neuron disease, and ALS. Extensive western blot analyses of Triton X-100 soluble and insoluble fractions of neuronal tissue from these conditions revealed significant changes in peripherin isoform content which could be differentiated by electrophoretic banding patterns to produce distinct peripherin biochemical signatures. Significantly, we found that the pattern of peripherin expression in ALS most closely approximates that of peripherin over-expressing mice, but differs with regard to inter-individual variations in isoform-specific expression. Overall, these results provide important insights into complex post-transcriptional processes that may underlie a continuum between peripherin-mediated neuronal repair and its role in the pathogenesis of motor neuron disease.Journal of Neurochemistry 08/2010; 114(4):1177-92. DOI:10.1111/j.1471-4159.2010.06846.x · 4.24 Impact Factor
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- "It is interesting to speculate why Per-45 is the major isoform expressed in the brain. It has recently been found that Akt, a serine/threonine protein kinase critical to the survival and regeneration of injured neurons, interacts with the N-terminal head domain of peripherin, phosphorylating Ser66 (Konishi et al. 2007). Although the functional relevance of phosphorylation of peripherin by Akt is unknown, it is increasingly recognized that IF proteins are important mediators of cell signaling (Paramio and Jorcano 2002; Kim and Coulombe 2007). "
ABSTRACT: Peripherin is a type III neuronal intermediate filament protein detected within the intraneuronal inclusions characteristic of amyotrophic lateral sclerosis. The constitutively expressed peripherin isoform is encoded by all nine exons of the human and mouse peripherin genes to generate a protein species of approximately 58 kDa on sodium dodecyl sulfate-polyacrylamide gels. Expression of this isoform, termed Per-58, generates a filament network in transfected SW13 vim cells. On immunoblots of cell lysates derived from these transfected cells, we have consistently observed a second peripherin species of approximately 45 kDa. In this study, we show that this species is a novel peripherin isoform generated through the use of an in-frame downstream initiation codon. This isoform, that we have designated Per-45, is co-expressed together with Per-58 and, thus, constitutive in both human and mouse. Using mutational analysis, we show that Per-45 is required for normal network formation, with the absence of Per-45 leading to irregular filamentous structures. We further show that peripherin expression in the normal nervous system is characterized by tissue-specific Per-58 : Per-45 isoform ratios. Taken together, these results identify novel processing requirements for peripherin expression and indicate a hitherto unrecognized role for neuronal intermediate filament network formation through intra-isoform associations.Journal of Neurochemistry 04/2008; 104(6):1663-73. DOI:10.1111/j.1471-4159.2007.05198.x · 4.24 Impact Factor