Protein-Protein Interactions Between The Multifunctional Protein Cad And Protein Phosphatase 1 In Breast Cancer

2732 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, and down-regulated by protein kinase A (PKA). The protein is phosphorylated by MAP kinase just prior to the S phase of the cell cycle and is simultaneously translocated into the nucleus. Interestingly, CAD found in malignant MCF-7 cells is persistently phosphorylated by ERK1/2 and is resistant to phosphorylation by PKA, 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, 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. Our hypothesis is 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 far-western analysis, co-immunoprecipation and pull down experiments. Interestingly, the sole consensus sequence for PP1 targeting proteins is located immediately adjacent to Thr456, the residue phosphorylated by MAP kinase, suggesting that the binding of PP1 would mask the MAP kinase 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 as two mutant peptides in which Thr456 was replaced with Ala or Asp. The wild type peptide and Ala mutant bind to PP1 while the Asp mutant, which mimics phosphorylated CAD, does not. Moreover, PP1 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 binding of I-2 to PP1. Fluorescence microscopy of mammalian cells transfected with full length CAD, Thr456Ala, or Thr456Glu mutants suggest that PP1 colocalizes with wild type CAD and the Ala mutant but not with the Glu mutant. 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 MAP kinase. 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.