AB122. Profiling the serine threonine kinase phosphorylation of TGF-β1 stimulated fibroblast using peptide microarray

Background: Transforming growth factor (TGF)-β is involved in various physiological roles from cell growth, cell differentiation, apoptosis, and some disease development. The TGF-β is tightly regulated and involves a myriad of molecules. Moreover, signal cross talk is prominent in the pathway, making it very complex and very difficult to study. Serine-threonine kinases play important role in TGF-β signaling. TGF-β ligand activates its serine-threonine kinase receptors (TGF-βRI and TGF-βRII). The activated receptor phosphorylates R-Smad proteins Smad2/3 and recruits CoSmad4. The Smad complexes are then translocated into the nucleus to regulate the transcription of the target genes in cooperation with other co-factors. How the TGF-β signaling, particularly the Smad signaling, can control different physiological roles remains poorly understood. It is not known whether TGF-β via its secondary messenger signaling molecules activates various physiological processes simultaneously or each process in an independent fashion activated by a previous process. We seek to further understand how TGF-β regulates various physiological phenomenons through the workings of its secondary messenger signaling using state-of-the-art serine-threonine kinase microarray.

Methods: Normal fibroblast cell lines were grown in Ham’s F-10 Nut Mix and serum-starved overnight. The fibroblast were then separated into two groups, stimulated and non-stimulated. The stimulated group were added TGF-β1 and incubated at room temperature for 25 minutes. The cells were then lysed and processed using PamGene commercial serine-threonine kinase microarray and analyzed using the PamStation12 at 200 ms exposure. The serine-threonine kinase chip contains 144 peptides immobilized into a 12×12 porous aluminum oxide substrates. Functional readout of the chip was based on the phosphorylation occurring on the array using labeled anti-phospho-antibodies.

Results: Out of 144 peptides on the serine-threonine kinase microarray, 95 peptides were found to have higher phosphorylation level and 6 peptides were found to have lower phosphorylation level after stimulation. Stimulation with TGF-β1 activated various cellular processes simultaneously such as cell division, cell proliferation, cytoskeleton remodeling, ions transport channels, cell-to-cell adhesion, apoptosis, cellular metabolism, carbohydrate and lipid metabolism.

Conclusions: The ability to determine the function and involvement of ligands and their secondary messenger response during cellular process can help examine the regulation of certain changes induced not only by TGF-β ligands but also other ligands and helps understand their involvement in many diseases such as cancer and various genetic diseases. Current high-throughput methods for the analysis of protein interaction such as peptide microarrays can yield more information and are taking the modeling of signal transduction processes to a new level.

Keywords: Transforming growth factor-β; serine-threonine kinase; microarray; cellular process