The protein kinase Akt/PKB is stimulated by the phosphorylation of two regulatory residues, Thr 309 of the activation segment and Ser 474 of the hydrophobic motif (HM), that are structurally and functionally conserved within the AGC kinase family. To understand the mechanism of PKB regulation, we determined the crystal structures of activated kinase domains of PKB in complex with a GSK3beta-peptide substrate and an ATP analog. The activated state of the kinase was generated by phosphorylating Thr 309 using PDK1 and mimicking Ser 474 phosphorylation either with the S474D substitution or by replacing the HM of PKB with that of PIFtide, a potent mimic of a phosphorylated HM. Comparison with the inactive PKB structure indicates that the role of Ser 474 phosphorylation is to promote the engagement of the HM with the N-lobe of the kinase domain, promoting a disorder-to-order transition of the alphaC helix. The alphaC helix, by interacting with pThr 309, restructures and orders the activation segment, generating an active kinase conformation. Analysis of the interactions between PKB and the GSK3beta-peptide explains how PKB selects for protein substrates distinct from those of PKA.

The anaphase-promoting complex (APC/C) is a multimeric RING E3 ubiquitin ligase that controls chromosome segregation and mitotic exit. Its regulation by coactivator subunits, phosphorylation, the mitotic checkpoint complex, and interphase inhibitor Emi1 ensures the correct order and timing of distinct cell cycle transitions. Here, we used cryo-electron microscopy to determine atomic structures of APC/C-coactivator complexes with either Emi1 or a UbcH10-ubiquitin conjugate. These structures define the architecture of all APC/C subunits, the position of the catalytic module, and explain how Emi1 mediates inhibition of the two E2s UbcH10 and Ube2S. Definition of Cdh1 interactions with the APC/C indicates how they are antagonized by Cdh1 phosphorylation. The structure of the APC/C with UbcH10-ubiquitin reveals insights into the initiating ubiquitination reaction. Our results provide a quantitative framework for the design of experiments to further investigate APC/C functions in vivo.The activities of the diverse proteins that orchestrate the sequential biochemical and morphological changes intrinsic to the cell division cycle are controlled through the integration of protein phosphorylation, proteolysis and changes in gene expression. Two cullin-RING E3 ubiquitin ligases, the APC/C and SCF, catalyze the ubiquitination of multiple cell cycle proteins to regulate their proteasome-mediated proteolysis. By regulating the ordered degradation of substrates such as cyclins, securin, mitotic kinases, and microtubule motors and assembly factors, the APC/C controls sister chromatid segregation, cytokinesis and the initiation of chromosome duplication [1][2][3] .The APC/C is a large assembly comprising 19 subunits 4 . Its activity depends on the association of one of two coactivator subunits (either Cdc20 or Cdh1) that specify substrate recognition and stimulate the catalytic activity of APC/C -E2 complexes [4][5][6][7] . Coactivators engage the APC/C through a conserved N-terminal C box motif and a C-terminal Ile-Arg Correspondence should be addressed to D.B. (dbarford@mrc-lmb.cam.ac.uk). Author contributions. L.C. prepared grids, collected and analyzed EM data and determined the 3D reconstructions, fitted coordinates and built models, prepared figures, co-wrote the paper. Z.Z. designed and made constructs, performed biochemical analysis and purified proteins. J.Y. prepared and purified the complexes and performed biochemical analysis. S.H.McL. performed and analyzed SPR experiments. D.B. directed the project, built models and co-wrote the paper.Author information. EM maps are deposited with the EM-DB with accession codes: 2924 (APC/C Cdh1.Emi1 ), 2925 (APC/ C Cdh1.Hsl1.UbcH10-Ub) , 2926 (APC/C Cdh1.Hsl1.Apc11-UbcH10) . APC/C Cdh1.Emi1 coords have accession code 4ui9.The authors declare no competing financial interests. Europe PMC Funders GroupAuthor Manuscript Nature. Author manuscript; available in PMC 2015 December 25. Published in final edited form as:Nature. 2,3 . Cyclin-dependent kinase (CDK) phosphorylates the...

Protein kinase B/Akt plays crucial roles in promoting cell survival and mediating insulin responses. The enzyme is stimulated by phosphorylation at two regulatory sites: Thr 309 of the activation segment and Ser 474 of the hydrophobic motif, a conserved feature of many AGC kinases. Analysis of the crystal structures of the unphosphorylated and Thr 309 phosphorylated states of the PKB kinase domain provides a molecular explanation for regulation by Ser 474 phosphorylation. Activation by Ser 474 phosphorylation occurs via a disorder to order transition of the alphaC helix with concomitant restructuring of the activation segment and reconfiguration of the kinase bilobal structure. These conformational changes are mediated by a phosphorylation-promoted interaction of the hydrophobic motif with a channel on the N-terminal lobe induced by the ordered alphaC helix and are mimicked by peptides corresponding to the hydrophobic motif of PKB and potently by the hydrophobic motif of PRK2.

The ubiquitination of cell cycle regulatory proteins by the anaphase-promoting complex/ cyclosome (APC/C) controls sister chromatid segregation, cytokinesis and the establishment of G1. The APC/C is an unusually large multimeric cullin-RING ligase. Its activity is strictly dependent on regulatory coactivator subunits that promote APC/C -substrate interactions and stimulate its catalytic reaction. Because the structures of many APC/C subunits and their organization within the assembly are unknown, the molecular basis for these processes is poorly understood. Here, from a cryo-EM reconstruction of a human APC/C-coactivator-substrate complex at 7.4 Å resolution, we have determined the complete secondary structural architecture of the complex. With this information we identified protein folds for structurally uncharacterized subunits, and the definitive location of all 20 APC/C subunits within the 1.2 MDa assembly. Comparison with apo APC/C shows that coactivator promotes a profound allosteric transition involving displacement of the cullin-RING catalytic subunits relative to the degron recognition module of coactivator and Apc10. This transition is accompanied by increased flexibility of the cullin-RING subunits and enhanced affinity for UbcH10~ubiquitin, changes which may contribute to coactivator-mediated stimulation of APC/C E3 ligase activity.Regulation of cell division by reversible protein phosphorylation and ubiquitination involves the coordinated interplay of protein kinases and phosphatases, and ubiquitin ligases and deubiquitinases 1 . The anaphase-promoting complex/cyclosome (APC/C) is an E3 cullin-RING ligase that mediates ubiquitin-dependent proteolysis of specific regulatory proteins to control chromosome segregation in mitosis, the events of cytokinesis and mitotic exit,