Unique Ligand and Kinase-Independent Roles of the Insulin Receptor in Regulation of Cell Cycle, Senescence and Apoptosis

Insulin acts through the insulin receptor (IR) tyrosine kinase to exert its classical metabolic and mitogenic actions. Here, using receptors with either short or long deletion of the ß-subunit or mutation of the kinase active site (K1030R), we uncover a second novel IR signaling pathway that is intracellular domain dependent, but ligand and tyrosine kinase-independent (LYK-I). These LYK-I actions of the IR are linked to changes in phosphorylation of a network of proteins involved in the regulation of extracellular matrix organization, cell cycle, ATM signaling and cellular senescence; and result in upregulation of expression of multiple extracellular matrix-related genes and proteins, down-regulation of immune/interferon-related genes and proteins, and increased sensitivity to apoptosis. Thus, in addition to classical ligand and tyrosine kinase-dependent (LYK-D) signaling, the IR regulates a second, novel ligand and tyrosine kinase-independent (LYK-I) pathway which regulates the cellular machinery involved in senescence, matrix interaction and response to extrinsic challenges. Overall design: To define the receptor-dependent but ligand- and kinase-independent signaling for insulin receptor (IR), we have generated preadipocytes in which the endogenous IR and IGF1R have been genetically inactivated to create DKO cells, and then the cells have been reconstituted with the normal wild-type IR, a kinase-dead mutant of IR (K1030R), a truncated IR lacking 79 amino acids from the C-terminus (?CT) or a truncated IR lacking almost the entire intracellular domain including both tyrosine-kinase domain and C-terminus, leaving only the intracellular juxtamembrane 36 amino acids (Juxtamembrane-Domain-Only, JMO). We assessed gene expression in the DKO, IR, K1030R, ?CT and JMO cells using RNAseq after 6 h serum starvation with or without stimulation by 100 nM insulin for 15 min. This was mostly done in triplicate.