Figure 4. A3B inhibits PFD5-mediated degradation of cMyc.

(A) Left: Diagram of tumor suppressor PFD5 recruiting Cullin-2 RING ubiquitin ligase for cMyc ubiquitylation and degradation. Right: A3B interacts with PFD5 leading to the hypothesis that A3B can disrupt this degradative pathway and lead to A3B-mediated, but deamination independent, dysregulation of the cell cycle. (B-C) Co-IP experiments from HEK293T cells treated with 12.5 μM MG132 for 16 hr demonstrated that A3B-HA and cMyc each interact with PFD5-FLAG. Using combinations of transfections of low A3B (1μg, +), high A3B (2μg, ++), low cMyc (1μg, +), or high cMyc (4μg, ++) demonstrated that the presence of A3B resulted in less co-purification of cMyc with PFD5 in a concentration dependent manner. (C) is the quantification of (B). (D-E) This interaction was functional and resulted in more cMyc in cells in the presence of A3B-HA, when PFD5-FLAG was also present. (E) is the quantification of three independent blots, with a representative blot shown in (D). (F-G) A3B-HA regulation of endogenous PFD5 occurs in the MCF7 breast cancer cell line. MCF7 cells were transfected with increasing amounts of A3B-HA. The endogenous cMyc detected increased with the amount of A3B-HA protein in cells. The cMyc was quantified relative to the amount of α-tubulin in each lane and normalized to the no A3B condition (value of 1). Quantification is plotted in (G) with error bars that represent the standard deviation from two independent experiments. A representative blot shown in (F). (H-I) MCF7 cells with doxycycline inducible A3B-FLAG were transfected with siRNA scramble or siRNA PFD5 to knock down endogenous PFD5 in the absence or presence of doxycycline. (H) The effect on the endogenous cMyc was determined by immunoblotting. (I) The intensity of the cMyc and PFD5 was quantified and is shown with the error bars that represent the standard deviation from two independent experiments.