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. Author manuscript; available in PMC: 2009 Jul 16.
Published in final edited form as: Mol Cell. 2009 Jan 16;33(1):109–116. doi: 10.1016/j.molcel.2008.12.020

Figure 2. Identification of the BimEL degron.

Figure 2

(A) Ser93, Ser94, and Ser98 are required for the interaction of BimEL with βTrCP1. HEK293 cells were transfected with an empty vector (EV), HA-tagged βcatenin (positive control), HA-tagged wild type BimEL, or the indicated HA-tagged BimEL mutants. Twenty-four hours post-transfection, cells were treated for three hours with PMA and MG132. Whole cell extracts (WCE) were subjected to immunoprecipitation (IP) with anti-HA resin (α-HA) and immunobloting for the indicated proteins.

(B) The BimEL degron requires phosphorylation to bind βTrCP1. 35S-labeled, in vitro-translated βTrCP1, Fbxw2, and Fbxw4 were used in binding reactions with beads coupled to the BimEL peptide 88CLSRSSSGYFSFD100 (lane 2) or the phosphopeptide 88CLSRSpSpSGYFpSFD100 (lane 3). Beads were washed with lysis buffer, and bound proteins were eluted and subjected to SDS-PAGE and autoradiography. The first lane shows 10% of the in vitro-translated protein inputs.

(C) In vivo phoshorylation of BimEL on Ser93/94/98 is induced by mitogens. HEK293 cells were serum deprived (SD) for 24 hours (lane 1), and then either serum (S) (lanes 2-3) or PMA (lane 4-5) was added for 20 minutes in the absence or presence of UO126, as indicated. Whole cell extracts (WCE) were subjected to immunoprecipitation (IP) with an anti-Bim antibody and immunobloting for the indicated proteins.

(D) Ser69 promotes phoshorylation of BimEL on Ser93/94/98. HEK293 cells were transfected with HA-tagged wild type (WT) BimEL (lane 1-3), HA-tagged BimEL(S69A) (lane 4), or HA-tagged BimEL(S94/98A) (lane 5). Cells were serum deprived (SD) for 24 hours (lane 1), and then either serum (S) (lane 2) or PMA (lane 3-5) was added for 20 minutes. Whole cell extracts (WCE) were subjected to immunoprecipitation (IP) with anti-HA resin (α-HA) and immunobloting for the indicated proteins.

(E) Cytokines induce phosphorylation of BimEL on Ser93/94/98. Activated primary mouse T cells were deprived of IL2 for 5 hours and Ba/F3 and FL5.12 cells were deprived of IL3 for 5 hours. Then cells were stimulated for 15 minutes with IL2 or IL3, in the absence or presence of UO126, as indicated. Whole cell extracts (WCE) were subjected to immunoprecipitation with an anti-Bim antibody and immunobloting for the indicated proteins.