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Volumn 275, Issue 5300, 1997, Pages 661-665

Regulation of neuronal survival by the serine-threonine protein kinase Akt

Author keywords

[No Author keywords available]

Indexed keywords

PROTEIN SERINE THREONINE KINASE;

EID: 0031053586     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.275.5300.661     Document Type: Article
Times cited : (2258)

References (49)
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    • Cerebellar neurons were cultured from Long-Evans rats at postnatal day 6 to 8 (9) on polyornithine-coated plates (Falcon) or polyornithine plus laminin-coated glass cover slips (Bellco). Cells were grown in basal medium Eagle (BME, Sigma) with calf serum (10%, Hyclone), 25 mM KCI, 2 mM glutamine, and penicillin-streptomycin (complete media), which promotes optimal survival. On day 1 after culturing (1 DIV), the antimitotic cytosine-β-D-arabinofuranoside (10 μM) was added to prevent proliferation of non-neuronal cells, Immunostaining with the granule cell-specific antibody Q600 (33) indicated that these cultures were composed predominantly of granule neurons (11). Cells were treated at 6 to 7 DIV with the same treatment protocol for survival or biochemical assays, as follows: Cells were washed twice in medium containing no serum and 5 mM KCI (deprivation medium) and were then placed in the same medium in the absence or presence of stimulants. Media containing growth factor also included bovine serum albumin (BSA, 0.1 mg/ml) as carrier protein, which itself caused no detectable signaling responses. The following concentrations were used: insulin, 10μg/ml; IGF-1, 50ng/ml (except 25ng/ml in Fig.1); BDNF, 100 ng/ml; wortmannin, 100 nM; rapamycin, 100 pM to 20 nM; and LY294002, 10 μM. When inhibitors were used for signaling assays, cells were treated with the inhibitors for 45 min before, and for the duration of, the stimulation. In survival assays, cells that did not receive inhibitors received control vehicle (dimethyl sulfoxide for LY294002, ethanol for rapamycin). The effect of insulin on cerebellar neuron survival in this system most likely reflects cross-reactivity of insulin with the IGF-1 receptor (34), because lower insulin concentrations (for example, 100 ng/ml) promoted survival poorly (11).
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    • 32P]ATP (NEN), and histone H2B (Boehringer Mannheim, 500 ng) or recombinant GSK-3 (New England Biolabs, 200 ng) per 40-μl assay reaction.
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    • note
    • The cytomegalovirus (CMV) promoter-based expression vectors CMV-6, HA-Akt, and HA-Akt(K179M) have been described (6). HA-PH contains the coding region of HA-AH1 (24), including amino acids 1 through 147 of Akt, in CMV-6. The CMV-β-Gal expression vector was pON260 (36). All forms of Akt were tagged with a hemagglutinin (HA) epitope. Transfection of HA-Akt, but not HA(K179M), into COS cells (by DEAE-dextran) resulted in increased Akt activity relative to that in cells transfected with vector (CMV-6) (11). Cerebellar neurons were transfected in 24-well plates (Costar) by the calcium phosphate method on 4 or 5 DIV (or 4 or 6 DIV, in (29)] largely as described (36); cells were transfected in Dulbecco's modified Eagle's medium (DMEM) and then returned to complete medium. For some of the experiments of Fig. 5B, the glutamate receptor inhibitors kynurenate and D(-)-2-amino-5-phosphonovaleric acid (D-APV) were included in the transfection as described (36); however, these proved to be unnecessary and were subsequently omitted. Cells were stained as described (2); Cy-2 coupled antibody was from Biological Detection Systems. Scoring of transfected cells was done blinded with respect to the identity of the transfected plasmids. Transfectants were identified by immunostaining for the cotransfected β-Gal marker. This allowed comparison between empty vector and Akt transfectants. In addition, because β-Gal staining was relatively stable during apoptosis, staining for β-Gal avoids scoring bias toward healthy cells. For all neuronal transfections, a 2:1 DNA ratio of Akt expression vector to β-Gal expression vector was used, and thus all healthy cells expressing β-Gal also expressed transfected Akt (measured by anti-HA immunostaining); all Akt constructs showed similar intensity of staining.
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    • unpublished data
    • T. Franke, Z. Songyang, L. Cantley, D. Kaplan, unpublished data. 3T3 fibroblasts were transfected with HA-Akt, alone or with expression vectors for the two Akt mutants. Cells were lysed, Akt was immunoprecipitated, and immune-complex kinase assays were done with histone H2B as substrate (6). Cotransfection of mutant Akt constructs reduced the activity of wild-type Akt.
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    • note
    • Two days after transfection, the extent of apoptosis of transfected cells in medium containing 25 mM KCI plus serum was as follows (means of two experiments): CMV-6, 22%; HA-Akt, 21%; K179M, 35%; and HA-PH, 28%. The ability of the dominant negative Akt proteins to inhibit survival in KCI plus insulin or serum may indicate that they are more effective than LY294002 at blocking activation of Akt, possibly because the dominant negative Akt proteins also block PI3-K-independent activation of Akt (37). Transfected wild-type Akt may be effective in promoting survival because it is expressed in amounts large enough so that its basal activity is sufficient for survival. Akt is believed to be regulated by dimerization (24), and this may contribute to activation of transfected wild-type Akt.
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    • note
    • We thank J. Blenis, M. Weber, S. Pons, and M. White for antibodies; M. Kozlowski and J. Avruch for 40S ribosomal subunits; Amgen Inc. for BDNF; K. Auger and T. Roberts for assistance with the PI3-K activity assay; S. Vasquez for assistance with cerebellar cultures; L. Lipfert for assistance generating antibodies to Akt; and members of the Greenberg lab for critical reading of the manuscript and for technical assistance. T.F.F. acknowledges L. Cantley for continuing support. Supported by American Cancer Society grant PF4059 (H.D.), NIH grants DK39519 (M.J.B.), R01 CA43855 (M.E.G.), and R01 CA18689 (G.M.C.), Mental Retardation Research Center grant NIH P30-HD18655, a K. M. Hunter fellowship in Cancer Research from the National Cancer Institute of Canada (NCIC) (T.F.F.), NCIC grant 7168 (D.R.K.), and an H. E. Johns and Canadian Cancer Society Research Scientist Award from NCIC (D.R.K.). Animal care was in accordance with institutional guidelines.


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