Abstract #5350: Protein-protein interactions between the multifunctional protein CAD and protein phosphatase 1 in breast cancer
Authors: Elizabeth Masko, Andrea Hortua, Meenal Mhaskar, Deborah Heyl-Clegg, Hedeel Guy-Evans, David Evans
Journal: Cancer Research (2009)

CAD is a multifunctional protein that catalyzes the first three reactions in de novo pyrimidine biosynthesis, a pathway essential for cell division and proliferation. Uncontrolled cell proliferation, a hallmark of cancer, is invariably associated with CAD activation. CAD is up-regulated by Erk1/2 MAP kinase (MAPK), and down-regulated by protein kinase A (PKA). The protein is phosphorylated by MAPK just prior to the S phase of the cell cycle and is simultaneously translocated into the nucleus. Interestingly, CAD found in malignant MCF-7 breast cells is persistently phosphorylated by MAPK, leading to continuous activation of pyrimidine biosynthesis. The levels of CAD in the nucleus are also much higher in MCF-7 cells as compared to cells from the immortalized MCF-10A line. The mechanisms that control the timing of these events are not well understood. For example, it is known that the phosphorylation of CAD by MAPK does not coincide with the activation of the kinase, but is delayed until just before the onset of DNA synthesis when the demand for pyrimidine nucleotides is highest. This study is based on the hypothesis that the timing of the activation and nucleocytoplasmic dynamics of CAD is controlled by signaling complexes with kinases and phosphatases. The formation of a stable complex between CAD and PP1 has been demonstrated by co-immunoprecipation, immunofluorescence, and in vitro binding experiments. Interestingly, the sole consensus sequence for PP1 targeting proteins is located immediately adjacent to Thr456, the residue phosphorylated by MAPK, suggesting that the binding of PP1 would mask the MAPK phosphorylation site. Conversely, phosphorylation of Thr456 might be expected to impair the binding of PP1. To test this idea, a polypeptide was synthesized corresponding to residues 444-460 of CAD that encompasses both the PP1 consensus sequence and Thr456, as well a two mutants peptides in which the targeted Thr residue has been replaced with Ala or Asp. The wild type peptide binds to PP1 while the Asp mutant, which mimics phosphorylated CAD, does not. Moreover, PP1 activity assays have shown that the CAD peptide effectively competes with the PP1 inhibitor, I-2, for a common binding site on PP1. In contrast, the Asp mutant peptide does not impede the I-2 inhibition of PP1. In vitro phosphorylation experiments support the peptide results. A plausible interpretation of these results is that the association with PP1 may block the premature activation of CAD in the cytoplasm until it is translocated into the nucleus whereupon PP1 dissociates and CAD is activated by MAPK. Understanding the control mechanisms that are involved in the up and down regulation of the pathway may lead to the development of more efficacious chemotherapeutic drugs that target specific signaling complexes rather than the individual pathway enzymes.  Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 5350.