Weingartner, K. A. | Search bioRxiv

bioRxiv2026

A conserved dimerization element is required for protein kinase activation by trans-autophosphorylation

Trans-autophosphorylation is the most common mode of protein kinase activation and involves two copies of the same kinase dimerizing so that one can phosphorylate the activation loop of the other. The diversity among structures of trans-autophosphorylation dimers supported the view that each kinase evolved a unique mode of recognition. We screened all human kinase crystal structures and identified an expanded set of dimers compatible with trans-autophosphorylation (655 dimers from 143 kinases). These dimers share no conserved structural arrangement, but 85% bury the same helix, G, at the dimer interface. We validate G-mediated dimerization by mutagenesis in kinases from each group of the kinome activated by trans-autophosphorylation. G substitution impaired or abolished activation of full-length proteins, in cells, in every case. In purified kinase domains, G substitution disrupted dimerization and autophosphorylation. These data establish that dimerization during trans-autophosphorylation is conserved and is mediated by a common structural element that, surprisingly, does not impose a specific arrangement of the two kinase domains relative to each other. G is the least conserved element in the kinase fold, yet is required for activation across both the human kinome and other species, suggesting an ancestral function of the kinase fold. Significance StatementProtein kinases are the largest enzyme family in the human genome and common pharmaceutical targets. Most are activated by trans-autophosphorylation, during which two copies of the same kinase dimerize and one phosphorylates the activation loop of the other. How this is achieved remains poorly understood. We find dimerization during activation is conserved, but in an unexpected way. An unbiased structural screen of all human kinase crystal structures reveals kinases across the kinome bury the same helix, G, at the dimer interface. G-mediated dimerization extends to other species suggesting an ancestral function of the kinase fold. Substitution of G disrupts activation of every kinase tested. Despite this conservation, G-mediated dimers share no common arrangement, representing an unusual mode of protein-protein interaction.

biochemistry↗

bioRxiv2023

Dimerization and autophosphorylation of the MST family of kinases are controlled by the same set of residues

The Hippo pathway controls tissue growth and regulates stem cell fate through the activities of core kinase cassette that begins with the Sterile 20-like kinase MST1/2. Activation of MST1/2 relies on trans-autophosphorylation but the details of the mechanisms regulating that reaction are not fully elucidated. Proposals include dimerization as a first step and include multiple models for potential kinase-domain dimers. Efforts to verify and link these dimers to trans-autophosphorylation were unsuccessful. We explored the link between dimerization and trans-autophosphorylation for MST2 and the entire family of MST kinases. We analyzed crystal lattice contacts of structures of MST kinases and identified an ensemble of kinase-domain dimers compatible with trans-autophosphorylation. These dimers share a common dimerization interface comprised of the activation loop and G-helix while the arrangements of the kinase-domains within the dimer varied depending on their activation state. We then verified the dimerization interface and determined its function using MST2. Variants bearing alanine substitutions of the G-helix prevented dimerization of the MST2 kinase domain both in solution and in cells. These substitutions also blocked autophosphorylation of full-length MST2 and its Drosophila homolog Hippo in cells. These variants retain the same secondary structure as wild-type and capacity to phosphorylate a protein substrate, indicating the loss of MST2 activation can be directly attributed to a loss of dimerization rather than loss of either fold or catalytic function. Together this data functionally links dimerization and autophosphorylation for MST2 and suggests this activation mechanism is conserved across both species and the entire MST family.

biochemistry↗