Identifying three-dimensional structures of autophosphorylation complexes in crystals of protein kinases

Abstract

Protein kinase autophosphorylation is a common regulatory mechanism in cell signaling pathways. Crystal structures of several homomeric protein kinase complexes have a serine, threonine, or tyrosine autophosphorylation site of one kinase monomer located in the active site of another monomer, a structural complex that we call an “autophosphorylation complex.” We developed and applied a structural bioinformatics method to identify all such autophosphorylation kinase complexes in X-ray crystallographic structures in the Protein Data Bank (PDB). We identified 15 autophosphorylation complexes in the PDB, of which 5 complexes had not previously been described in the publications describing the crystal structures. These 5 consist of tyrosine residues in the N-terminal juxtamembrane regions of colony stimulating factor 1 receptor (CSF1R, Tyr561) and EPH receptor A2 (EPHA2, Tyr594), tyrosine residues in the activation loops of the SRC kinase family member LCK (Tyr394) and insulin-like growth factor 1 receptor (IGF1R, Tyr1166), and a serine in a nuclear localization signal region of CDC-like kinase 2 (CLK2, Ser142). Mutations in the complex interface may alter autophosphorylation activity and contribute to disease; therefore we mutated residues in the autophosphorylation complex interface of LCK and found that two mutations impaired autophosphorylation (T445V and N446A) and mutation of Pro447 to Ala, Gly, or Leu increased autophosphorylation. The identified autophosphorylation sites are conserved in many kinases, suggesting that, by homology, these complexes may provide insight into autophosphorylation complex interfaces of kinases that are relevant drug targets.

An electrostatic selection mechanism controls sequential kinase signaling downstream of the T cell receptor

“…We identified one crystal structure of the insulin-like growth factor-1 receptor (IGF1-R) kinase domain in which two of the four molecules in the asymmetric unit interact in a manner that appears compatible with activation loop phosphorylation, with one kinase taking the role of the enzyme and the other that of the substrate (PDB code 3LVP) (Nemecek et al, 2010). Recently, this same autophosphorylation complex was identified independently in an automated survey of the Protein Data Bank (Xu et al, 2015). We built homology models for Lck and c-Src autophosphorylation complexes based on the IGF1-R structure (PDB code 3LVP).…”

“…Several other trans -autophosphorylation complexes have been proposed for tyrosine kinases, including potential structures for activation loop phosphorylation (Wu et al, 2008; Xu et al, 2015) and C-terminal tail phosphorylation (Chen et al, 2008). We considered these alternative proposals for trans -autophosphorylation complexes but favored the model based on PDB code 3LVP as it was consistent with our mutagenesis experiments (noted below, in this section and the following one).…”

Switch-like activation of Bruton’s tyrosine kinase by membrane-mediated dimerization

“…Indeed, the adventitious dimerization of tyrosine kinases leads to improper activation and cancer, as exemplified by the BCR-Abl fusion protein . The ability of non-receptor tyrosines kinases such as Src and Abl to activate through dimerization and trans-autophosphorylation is well established, but the natural mechanisms that promote dimerization are still not well understood (Lamontanara et al, 2014;Xu et al, 2015). In this regard, the unexpected discovery that Btk can be activated in solution by the binding of IP 6 to the PH-TH module in a way that is consistent with the crystallographically observed Saraste dimer was intriguing, but there was no direct evidence that dimerization was in fact responsible for the observed activation (Wang et al, 2015).…”

Chemically reprogramming the phospho‐transfer reaction to crosslink protein kinases to their substrates

“…The transient nature of kinase–substrate complexes not only poses a challenge to their de novo identification but also to their high‐resolution structural characterization. Relatively few structures of kinase–substrate phosphorylation complexes exist in the literature, and even fewer depict protein rather than peptide substrates . A recent structure of the kinase PINK1 in complex with its substrate P‐Ubiquitin required mutation of the kinase to stabilize binding to Ubiquitin as well as a llama antibody raised against the chemically crosslinked PINK1/Ub complex to aid in stabilization for X‐ray structure determination.…”