Phosphorylation of the alpha 2(1) procollagen promoter binding proteins is required for promoter activity

We have previously identified transcription factors binding to the proximal promoter region of the human COL1A2 gene that are essential for promoter activity. Two regions in the proximal promoter, the CCAAT-element and the CME bind the CCAAT-binding factor and an as yet uncharacterized DNA-binding protein, respectively. In this study we show that phosphorylation of both the CCAAT-binding and CME-binding proteins are essential for DNA-binding activity. While the binding of the CCAAT-binding proteins to the COL1A2 proximal promoter was inhibited after treatment of nuclear proteins with calf intestinal phosphatase, PP2A treatment had a significant inhibitory effect on the binding of the CME-binding proteins. The treatment of type I collagen producing fibroblasts, CT-1, with the kinase inhibitor, staurosporine, interfered with DNA-protein interactions on both elements on the COL1A2 proximal promoter. This inhibition was associated with a significant decrease in both endogenous COL1A2 gene expression as observed by northern blot analysis and proximal promoter activity as assayed in transient transfection assays. Our results suggest that the phosphorylation of transcription factors interacting with the COL1A2 proximal promoter is essential for both DNA binding and for the expression of this gene.