The Kit receptor promotes cell survival via activation of PI 3-kinase and subsequent Akt- mediated phosphorylation of Bad on Ser136  Peter Blume-Jensen, Ralf Janknecht, Tony Hunter  Current Biology  Volume 8, Issue 13, Pages 779-785 (June 1998) DOI: 10.1016/S0960-9822(98)70302-1

Figure 1 Activation of Akt by different Kit/SCF-R mutants parallels their activation of PI 3-kinase. HEK 293 cells were transiently cotransfected with DNA constructs expressing wild-type (WT) or mutant forms of HA–Akt together with wild-type or mutant forms of Kit. (a) HA–Akt proteins were immunoprecipitated and subjected to an in vitro kinase assay using histone H2B as a substrate. Proteins were immunoblotted with an anti-HA monoclonal antibody (upper panel), and relative phosphorylation of histone H2B (lower panel) was quantitated by phosphorimager analysis. (b) Cells were stimulated with SCF, where indicated, with or without preincubation with 100 nM wortmannin for 30 min. HA–Akt proteins were immunoprecipitated and used in an in vitro kinase assay with histone H2B as a substrate. Current Biology 1998 8, 779-785DOI: (10.1016/S0960-9822(98)70302-1)

Figure 2 SCF stimulates phosphorylation of Ser136 on Bad in a manner dependent on both PI 3-kinase and Akt. (a) U2-OS cells and (b) BHK-21 cells were transfected with cDNA constructs for wild-type (WT). Bad or S112/136A-Bad and WT Kit, Y721F-Kit or S741/746A-Kit. After incubation with SCF and wortmannin where indicated, Bad proteins were immunoprecipitated and then immunoblotted with an anti-Bad antibody. (c) U2-OS cells cotransfected with WT Bad and either p110∗ or E40K-Akt were stimulated and lysed, and Bad detected as above. The phosphorylation-dependent shift of Bad is seen as a broadening of the Bad protein band in this experiment due to a high degree of phosphorylation. (d) Bad-transfected HEK 293 T cells were labeled with [32P]orthophosphate, stimulated with SCF, and Bad was immunoprecipitated. (e) HEK 293 cells were transfected with constructs encoding HA–Akt and p110∗, as indicated, and Akt subjected to an in vitro kinase assay using GST–Bad fusion proteins as substrate (see Supplementary material published with this paper on the internet for methodological details). Current Biology 1998 8, 779-785DOI: (10.1016/S0960-9822(98)70302-1)

Figure 3 Bad and Akt colocalize in vivo. HeLa cells were cotransfected with constructs encoding wild-type versions of HA–Akt and Bad, stimulated with SCF where indicated, and then stained with rabbit anti-Bad (N-20) and mouse anti-HA (12CA5) antibodies. After mounting, cells were viewed under a confocal microscope. Colocalization of Bad and Akt is seen in yellow. Current Biology 1998 8, 779-785DOI: (10.1016/S0960-9822(98)70302-1)

Figure 4 SCF-stimulated suppression of Bad-induced apoptosis via PI 3-kinase/Akt depends on Ser136 in Bad. U2-OS cells were transfected with the indicated Kit and Bad constructs together with a plasmid encoding GFP. After serum starvation, cells were fixed, permeabilized, and cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). The upper row in each panel shows DAPI-stained nuclei, the lower row shows transfected GFP-positive cells. Arrowheads identify apoptotic cells; full arrows identify cells undergoing mitosis. See Supplementary material for a quantitation of the extent of apoptosis. Current Biology 1998 8, 779-785DOI: (10.1016/S0960-9822(98)70302-1)