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.

Cancer therapy depends heavily on the ability to effectively control the activity of oncogenic kinases. Autophosphorylation is a common regulatory mechanism of kinases in signaling pathways, and commonly elevated in cancer. Several autophosphorylation complexes have been identified from within crystals of protein kinases, with a known autophosphorylation site of one kinase monomer sitting in the active site of another monomer of the same protein in the crystal. We have utilized a structural bioinformatics method to identify all such autophosphorylation complexes in X-ray crystal structures in the Protein Data Bank by generating all unique kinase/kinase interfaces within and between asymmetric units of each crystal and measuring the distance between the hydroxyl oxygen of the autophosphorylation sites and the oxygen atoms of the active site aspartic acid residue side chain. With this approach, we have identified 15 autophosphorylation complexes in the PDB, of which 5 complexes have not previously been described. Of greatest interest are five structures of activation loop autophosphorylation - PAK1 (T423), IRAK4 (T345), IGF1R (Y1165 and Y1166), and LCK (Y394), two of which we have identified for the first time (IGF1R-Y1166 and LCK). We show that 269 human kinases have potential S/T or Y phosphorylation sites at positions analogous to the PAK1, IGF1R-Y1165/LCK, and IGF1R-Y1166 structures, and that there are 182 such positions that are annotated as phosphorylation sites in Uniprot. To assess the functional importance of the LCK dimer, we performed mutational analysis of residues in the autophosphorylation complex interface of LCK and found that mutations disrupting the interface either severely impaired autophosphorylation (T445D and N446D) or increased it (P447L,A,G). The P447L mutation has been previously found in a T-cell leukemia cell line and associated with activation of LCK. Three structures of receptor tyrosine kinases contain autophosphorylation complexes of the juxtamembrane segment just N-terminal to the kinase domain, two of which are identified for the first time. One of these, CSF1R (Y561) is a homologous site to a known c-KIT (Y568) autophosphorylation structure. The other is in EPHA2 (Y594), which is homologous to Y570 in c-KIT, which is also an autophosphorylation site. Twenty receptor tyrosine kinases contain autophosphorylation sites at one or both of these positions. Phosphorylation at these sites is associated with interaction with SRC family kinases and other downstream effectors of receptor tyrosine kinase signaling. These structures provide critical information on domain-domain interactions and substrate specificity in autophosphorylation, as well as opportunities for understanding the role of certain cancer driver mutations and the development of non-ATP-competitive inhibitors that block dimerization shown in these structures. Note: This abstract was not presented at the meeting. Citation Format: Qifang Xu, Kimberly Malecka, Jeffrey Peterson, Roland L. Dunbrack. Identification of novel autophosphorylation structures in crystals of protein kinases. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-034. doi:10.1158/1538-7445.AM2015-LB-034