We have found an activating mutation (M207T) in the early cell-cycle regulator cyclin dependent kinase 4 (CDK4) in a patient with severely disturbed adipose tissue distribution and metabolic syndrome. To understand the underlying mechanisms, we overexpressed the mutant CDK4 or pharmacologically inhibited the kinase, and conducted proteomic, phosphoproteomic and metabolic analyses during the first two days of adipogenic differentiation of human mesenchymal stem cells. Together, our data covering over 8000 proteins, 20000 phosphosites and 400 metabolites suggest that the mutant CDK4 induces major changes in the flux of glucose, including altered use of the TCA cycle. Here, we present two distinct computational approaches to dissect how CDK4 activity controls signal propagation during early adipogenesis. First, we portray conventional multi-omics integration methods, including matrix factorization and constrained based reconstruction to derive the downstream effectors of active CDK4. Second, we identify possible signal propagation pathways that explain how the observed metabolic changes are controlled by CDK4, by utilizing the SIGnaling Network Open Resource (SIGNOR) database to search for potential shortest paths starting from an activated CDK4 and leading to the observed effectors. We follow the binary activation/deactivation rules of the database to identify the expected activation of proteins along a path and match these expectations to our observed data at both protein and phosphorylation levels. Our approach shows the potential of integrating longitudinal multi-omics data with curated databases to reveal dynamic, intracellular signal propagation pathways that control the early steps of cellular differentiation.

Co-authors: Maarit Holtta, Kari Moisio, Liisa Sarkio, Ville Hietakangas, Elina Ikonen