Neuronal thread protein regulation and interaction with microtubule-associated proteins in SH-Sy5y neuronal cells.
In Alzheimer's disease (AD), neuronal thread protein (NTP) accumulates in cortical neurons and colocalizes with phospho- tau-immunoreactive cytoskeletal lesions that correlate with dementia. To generate additional information about the potential role of NTP in AD, we characterized its expression and regulation in human SH-Sy5y neuronal cells. Quantitative real-time reverse transcription-polymerase chain reactin and Western blot analysis demonstrated prominent insulin, moderate insulin-like growth factor, type 1 (IGF-1) and minimal nerve growth factor stimulation of NTP expression. In addition, NTP protein was more stable and it progressively accumulated in cells that were stimulated with insulin for 24 or 48 h. Metabolic labeling and phospho-amino acid analysis demonstrated phosphorylation of NTP on Serine residues, 30-60 min after insulin or IGF-1 stimulation, when glycogen synthase kinase 3beta (GSK-3beta) activity would no longer have been suppressed. Kinase inhibitor and in vitro phosphorylation studies demonstrated a role for GSK-3beta in the positive regulation of NTP expression and phosphorylation. Coimmunoprecipitation studies demonstrated physical interactions between NTP and tau or microtubule-associated protein 1b (MAP-1b), and ubiquitin immunoreactivity in NTP immunoprecipitates. In summary, these studies showed that (i) NTP expression is regulated at the level of transcription by insulin and IGF-1 stimulation; (ii) NTP is phosphorylated by GSK-3beta; (iii) NTP can physically interact with tau and MAP-1b and (iv) NTP-MAP complexes are ubiquitinated. The results suggest a functional role for NTP in relation to the turnover or processing of neuronal cytoskeletal proteins, attributes that may be modulated by insulin/IGF-1-mediated signaling.
de la Monte
S M
SM
Department of Medicine, Rhode Island Hospital and Brown Medical School, 55 Claverick Street, Providence, Rhode Island 02903, USA. suzannedelamontemd@brown.edu
Chen
G J
GJ
Rivera
E
E
Wands
J R
JR
D000911:Antibodies, Monoclonal / Q000276:immunology; D001921:Brain / Q000276:immunology / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000378:metabolism; D051847:Lithostathine; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D009419:Nerve Tissue Proteins; D009474:Neurons / Q000276:immunology / Q000378:metabolism*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D015398:Signal Transduction / Q000276:immunology; D016875:tau Proteins / Q000378:metabolism
C497612:AD7c-NTP protein, human; D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; D007328:Insulin; D051847:Lithostathine; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; C085284:microtubule-associated protein 1B; D016875:tau Proteins; D010770:Phosphotransferases