Figure 1.
(A) ASPP1 and ASPP2 have two conserved putative MAPK2 phosphorylation sites in their C-terminus. (B) The C-terminus fragment of ASPP2 is phosphorylated in vitro by MAPK1 (left panel). The intensity of phosphorylation is quantified (middle panel). The MAPK1 phosphorylated ASPP2 fragment was digested with trypsin and chromatographed and the radioactive peptides were measured by mass spectrometry (right panel). The first peak represents the GST linker region whereas the second presented a region of equal mass to the fragment containing serine 827. (C) An in vitro phosphorylation assay was performed on the ASPP2 C-terminus fragment with recombinant MAPK1 and non-radioactive ATP. The phosphorylation status of ASPP2 was assessed using the purified NGH.S4 phospho-specific ASPP2 antibody (upper panel). Total ASPP2 is shown in the lower panel. (D) Saos2 cells were starved then stimulated with serum and EGF. At the indicated times the cells were harvested and either blotted for phospho/total MAPK (upper panel) or immunoprecipitated for total ASPP2 and blotted with NGH.S4 phospho-ASPP2 antibody. (E) Total cell lysates from HKe3 ER:HRASV12 cells treated with or without 4-OHT were transfected with control siRNA or siRNA against ASPP2. ASPP2 phosphorylation was detected with ES1 phospho-ASPP2 antibody and total ASPP2.
Figure 2.
Activated Raf enhances the transactivation activity of ASPP2 and p53 to the same extent as activated RAS.
(A) Saos2 cells were transfected as indicated with a Bax-luciferase reporter and the luciferase activity shown. * P=0.05 (B) The value of ASPP2+p53 was taken as 1.0 to reflect the fold increase of ASPP2 and p53 in the presence of activated Raf and mutant RAS. ** P=0.0055; **** P=0.0001.
Figure 3.
Phosphorylation of ASPP2 by the Raf/MAPK pathway enhances p53-mediated transactivation.
(A) Saos2 cells were transfected with p53 and either wild-type or mutant ASPP2 as indicated together with a Bax-luciferase reporter. Luciferase activity is shown following harvesting of cells. Values are Relative Light Units (RLU). (B) Saos2 cells were transfected with p53, constitutively active Raf CAAX and either wild-type or mutant ASPP2 as indicated together with a Bax-luciferase reporter. ** P=0.013; **** P=0.0001 (C) Saos2 cells were transfected with a Bax-luciferase reporter, ASPP2 and p53 and treated with 20 µM UO126 or DMSO for 20 hours. Luciferase activity is shown in the left panel and protein expression was verified by Western Blot (right panel). **** P=0.0001.
Figure 4.
Wild-type ASPP2, but not mutant ASPP2 (S827A), translocates to the cytosol and nucleus upon oncogenic RAS activation and this results in an increased interaction with p53.
(A) RAS activation induces cytoplasmic and nuclear translocation of wild-type ASPP2 but not ASPP2 (S827A) in HKe3 ER:HRAS12 cells as detected by immunofluorescence. Arrows indicate cell membrane and stars indicate cytosol. (B) RAS activation enhances the binding of wild-type ASPP2 but not ASPP2 (S827A) to p53. Total cell lysates from HKe3 ER:HRASV12 cells treated with or without 4-OHT were immunoprecipitated with an anti-p53 antibody or control IgG as indicated.
Figure 5.
Diagram summarizes the inter-regulation between ASPP2 and RAS.
ASPP2 binds active RAS at the plasma membrane, thereby increasing RAS signaling to its downstream pathway effectors Raf/MAPK. Activated MAPK phosphorylates ASPP2 which can then relocate to the nucleus and activate p53 pro-apoptotic signaling.