(A) Activation loop autophosphorylation requires the activity of the kinase before it is itself phosphorylated on its activation loop. Logic dictates that the autophosphorylation reaction must, therefore, be mechanistically distinct from substrate phosphorylation. (B) Distinct mechanistic aspects of auto- and substrate phosphorylation. Substrate phosphorylation is always an asymmetric trans reaction while autophosphorylation can occur in distinct reaction modes. Sequence recognition can vary, depending on the structural context, such that the activation loop is not recognized as a substrate if supplied as a peptide in trans. The substrate sequence can also vary between the activation loop and downstream substrates. (C) Protein abundance levels were plotted against their RNA transcript levels from a proteome-wide screen in HEK293 cells. The abundance of kinases (black) is in the nanomolar range and relatively low in comparison to the total proteome (gray). Kinases that depend on activation loop autophosphorylation are generally expressed in the nanomolar concentration range, suggesting that trans-autoregulated kinases need a dedicated mechanism for dimerization. (D) Structure of the kinase domain of insulin-like growth factor receptor (IGFR) in a dimeric, face-to-face configuration in which the activation loop (purple-blue) of each protomer makes symmetric interactions with the catalytic site of the opposing protomer, presenting Y1135 of the activation loop in trans. PDB ID: 3d94. (ref = reference molecule). (E) Structure of the kinase domain of fibroblast growth factor receptor (FGFR) in a dimeric, face-to-face configuration in which the activation loop (salmon) of one protomer makes asymmetric contacts with the catalytic site of the opposing protomer, presenting Y647 of the activation loop in trans. PDB ID: 6pnx. (ref = reference molecule). (F) Activation loop trajectories of unphosphorylated insulin receptor kinase (IRK) (magenta), phosphorylated IRK (teal), IGFR in the trans, symmetric conformation (purple-blue), and FGFR in the trans, asymmetric conformation (salmon), displayed on the surface of the phosphorylated IRK kinase domain. PDB IDs: 1irk, 1ir3, 3d94, 6pnx. (G) B-factor plot for IRK kinase domain (apo structure, PDB ID: 1irk). High B-factors for the activation loop indicate that it is the most mobile (and least ordered) region of the kinase domain. (H) Zoom-in on the kinase-activation loop interactions observed in: phosphorylated IRK in complex with a substrate peptide (red), unphosphorylated IRK with its own activation loop in cis (magenta), IGFR with the activation loop of the opposing protomer in trans (symmetric, purple-blue), and FGFR with the activation loop of the opposing protomer in trans (asymmetric, pink). PDB IDs: 1irk, 1ir3, 3d94, 6pnx.