14-3-3gamma is upregulated by in vitro ischemia and binds to protein kinase Raf in primary cultures of astrocytes.
The 14-3-3 protein family comprises critical regulatory molecules involved in signaling during cell division, proliferation, and apoptosis. Despite extensive study, the functions of the 14-3-3 proteins in brain remain unclear. 14-3-3gamma, a subtype of the 14-3-3 family of proteins, was thought to be brain- and neuron-specific. Using RNA arbitrarily primed PCR, we identified an upregulated cDNA fragment of the 14-3-3gamma gene in primary cultures of astrocytes. Using Northern blot analysis, we confirmed this fragment was brain-specific. In cultures of astrocytes, 14-3-3gamma genes and proteins were differentially expressed at different ages and the proteins were distributed only in the cytoplasm. These results indicated that 14-3-3gamma was not neuron-specific but also expressed in astrocytes. The function of this protein in brain is unclear. Northern and Western blot analyses demonstrated that 14-3-3gamma mRNA and protein were upregulated in cultured astrocytes in an anaerobic chamber-induced ischemia model. The induction of 14-3-3gamma proteins was neither suppressed by an MAP kinase inhibitor (U0126) nor a PI-3 kinase inhibitor (LY294002). These data indicated that induction of 14-3-3gamma might not involve PI-3 and MAP kinase-dependent pathways. Using coimmunoprecipitation, we demonstrated that endogenous 14-3-3gamma bound to c-Raf-1 and p-Raf 259. As Raf is one of the critical serine/threonine kinases controlling cell growth, differentiation, and death, the binding of 14-3-3gamma to Raf indicates the critical role of this protein in ischemia-induced apoptosis and the changes in signal transduction in astrocytes in culture.
Neuroscience Research Institute, Peking University and Department of Neurobiology, Peking University Health Science Center, Beijing, China.
Xiao Qian
XQ
Chen
Jian Guo
JG
Chen
Yun
Y
Zhang
Wendy Wen Luan
WW
Hsiao
Albert Cheung Hoi
AC
Yu
D048948:14-3-3 Proteins; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000166:cytology / Q000201:enzymology*; D001483:Base Sequence; D002545:Brain Ischemia / Q000378:metabolism*; D002070:Butadienes / Q000494:pharmacology; D016923:Cell Death / Q000502:physiology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D002867:Chromones / Q000494:pharmacology; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D066298:In Vitro Techniques; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D009025:Morpholines / Q000494:pharmacology; D009570:Nitriles / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding / Q000502:physiology; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation / Q000502:physiology
D048948:14-3-3 Proteins; D002070:Butadienes; D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; D009570:Nitriles; C113580:U 0126; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D014446:Tyrosine 3-Monooxygenase; D019908:Proto-Oncogene Proteins c-raf