14‐3‐3γ Is upregulated by in vitro ischemia and binds to protein kinase Raf in primary cultures of astrocytes

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Abstract
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. 14‐3‐3γ, 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 14‐3‐3γ gene in primary cultures of astrocytes. Using Northern blot analysis, we confirmed this fragment was brain‐specific. In cultures of astrocytes, 14‐3‐3γ genes and proteins were differentially expressed at different ages and the proteins were distributed only in the cytoplasm. These results indicated that 14‐3‐3γ 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 14‐3‐3γ mRNA and protein were upregulated in cultured astrocytes in an anaerobic chamber‐induced ischemia model. The induction of 14‐3‐3γ proteins was neither suppressed by an MAP kinase inhibitor (U0126) nor a PI‐3 kinase inhibitor (LY294002). These data indicated that induction of 14‐3‐3γ might not involve PI‐3 and MAP kinase‐dependent pathways. Using coimmunoprecipitation, we demonstrated that endogenous 14‐3‐3γ 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 14‐3‐3γ to Raf indicates the critical role of this protein in ischemia‐induced apoptosis and the changes in signal transduction in astrocytes in culture. GLIA 42:315–324, 2003.