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. Author manuscript; available in PMC: 2008 Feb 18.
Published in final edited form as: Mol Cell. 2008 Jan 18;29(1):9–22. doi: 10.1016/j.molcel.2007.11.031

Figure 3.

Figure 3

FAK FERM inhibits p53 activity through enhanced Mdm2-dependent ubiquitination and proteasomal degradation. (A) Epitope-tagged FAK construct schematic. Indicated is the band 4.1, ezrin, radixin, moesin (FERM) and central kinase domains. Filled regions indicate proline-rich motifs. Translation of FAK Δ1-100 starts at Met-101, Myc-tagged FERM encompasses residues 1-402, mutation of Y397 phosphorylation (F397), kinase-dead (KD, R454), Pro-null (Pro 712, 713, 872, 873, 876, and 877 mutated to Ala), and FRNK residues 691-1052 are indicated. (B) FAK FERM but not FAK kinase activity is required for the reduction of steady state p53 levels in FAK-/-p21-/- fibroblasts. Cells were transduced with Ad-Tet transactivator (TA, Mock), or Ad-TA plus the indicated Ad-FAK, Ad-FERM, or Ad-FRNK constructs. After 48 h, lysates were blotted with the indicated antibodies, p53 levels were quantified by densitometry, and mean values +/- SD are expressed as percentage of control (Mock) from 2 experiments. (C) Addition of MG132 (40 μM, 3h) prior to cell lysis prevents Ad-FAK and Ad-FERM-mediated p53 degradation in FAK-/-p21-/- fibroblasts. (D) FAK and FERM but not FRNK inhibit p53 activity as measured by a p21 promoter luciferase assay in FAK-/-p21-/- cells 48h after transfection. Values are means presented as percent of Mock control +/- SD from 3 experiments. (E) FAK and FERM but not FRNK promote enhanced p53 ubiquitination. HEK293 cells were co-transfected with flag-p53 and the indicated FAK constructs. MG132 was added 3h prior to lysis, p53 was isolated by IP, and analyzed by anti-ubiquitin and flag-tag blotting. (F) Mdm2 expression is required for FERM-enhanced p53 ubiquitination. Mdm2-/-p53-/- or Mdm2+/+p53-/- fibroblasts were transfected with flag-p53 and then transduced with Mock or Ad-FAK FERM. MG132 was added 3h prior to lysis, and p53 IPs were analyzed by anti-ubiquitin, anti-p53 (DO-1), and flag-tag blotting. Anti-Myc blotting was used to detect FAK FERM and anti-actin for loading control. Arrows indicate p53-shifted bands induced by FERM expression in Mdm2+/+ cells.