Hsp90-dependent activation of protein kinases is regulated by chaperone-targeted dephosphorylation of Cdc37.

Activation of protein kinase clients by the Hsp90 system is mediated by the cochaperone protein Cdc37. Cdc37 requires phosphorylation at Ser13, but little is known about the regulation of this essential posttranslational modification. We show that Ser13 of uncomplexed Cdc37 is phosphorylated in vivo, as well as in binary complex with a kinase (C-K), or in ternary complex with Hsp90 and kinase (H-C-K). Whereas pSer13-Cdc37 in the H-C-K complex is resistant to nonspecific phosphatases, it is efficiently dephosphorylated by the chaperone-targeted protein phosphatase 5 (PP5/Ppt1), which does not affect isolated Cdc37. We show that Cdc37 and PP5/Ppt1 associate in Hsp90 complexes in yeast and in human tumor cells, and that PP5/Ppt1 regulates phosphorylation of Ser13-Cdc37 in vivo, directly affecting activation of protein kinase clients by Hsp90-Cdc37. These data reveal a cyclic regulatory mechanism for Cdc37, in which its constitutive phosphorylation is reversed by targeted dephosphorylation in Hsp90 complexes.

Long-term, large scale cryopreservation of insect cells at -80 °C.

Standard tissue culture methods advise freezing cells in small aliquots (≤1 × 10(7) cells in 1 mL), and storing in liquid nitrogen. This is inconvenient for laboratories culturing large quantities of insect cells for recombinant baculovirus expression, owing to the length of time taken to produce large scale cultures from small aliquots of cells. Liquid nitrogen storage requires use of specialized cryovials, personal protective equipment and oxygen monitoring systems. This paper describes the long-term, large scale cryopreservation of 8 × 10(8) insect cells at -80 °C, using standard 50 mL conical tubes to contain a 40 mL cell suspension. Sf9, Sf21 and High 5 cells were recovered with a viability > 90 % after storage for one year under these conditions, which compared favorably with the viability of cells stored in liquid nitrogen for the same length of time. Addition of green fluorescent protein encoding baculovirus demonstrated that cells were "expression ready" immediately post thaw. Our method enables large scale cultures to be recovered rapidly from stocks cryopreserved at -80 °C, thus avoiding the inconvenience, hazards and expense associated with liquid nitrogen.

Structure of an Hsp90-Cdc37-Cdk4 complex.

Activation of many protein kinases depends on their interaction with the Hsp90 molecular chaperone system. Recruitment of protein kinase clients to the Hsp90 chaperone system is mediated by the cochaperone adaptor protein Cdc37, which acts as a scaffold, simultaneously binding protein kinases and Hsp90. We have now expressed and purified an Hsp90-Cdc37-Cdk4 complex, defined its stoichiometry, and determined its 3D structure by single-particle electron microscopy. Comparison with the crystal structure of Hsp90 allows us to identify the locations of Cdc37 and Cdk4 in the complex and suggests a mechanism by which conformational changes in the kinase are coupled to the Hsp90 ATPase cycle.

Crystal structure of an activated Akt/protein kinase B ternary complex with GSK3-peptide and AMP-PNP.

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 structure of the cell cycle protein Cdc14 reveals a proline-directed protein phosphatase.

The Cdc14 family of dual-specificity protein phosphatases (DSPs) is conserved within eukaryotes and functions to down-regulate mitotic Cdk activities, promoting cytokinesis and mitotic exit. We have integrated structural and kinetic analyses to define the molecular mechanism of the dephosphorylation reaction catalysed by Cdc14. The structure of Cdc14 illustrates a novel arrangement of two domains, each with a DSP-like fold, arranged in tandem. The C-terminal domain contains the conserved PTP motif of the catalytic site, whereas the N-terminal domain, which shares no sequence similarity with other DSPs, contributes to substrate specificity, and lacks catalytic activity. The catalytic site is located at the base of a pronounced surface channel formed by the interface of the two domains, and regions of both domains interact with the phosphopeptide substrate. Specificity for a pSer-Pro motif is mediated by a hydrophobic pocket that is capable of accommodating the apolar Pro(P+1) residue of the peptide. Our structural and kinetic data support a role for Cdc14 in the preferential dephosphorylation of proteins modified by proline-directed kinases.

Molecular mechanism for the regulation of protein kinase B/Akt by hydrophobic motif phosphorylation.

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.

Structural basis for recruitment of glycogen synthase kinase 3beta to the axin-APC scaffold complex.

Glycogen synthase kinase 3beta (GSK3beta) is a serine/threonine kinase involved in insulin, growth factor and Wnt signalling. In Wnt signalling, GSK3beta is recruited to a multiprotein complex via interaction with axin, where it hyperphosphorylates beta-catenin, marking it for ubiquitylation and destruction. We have now determined the crystal structure of GSK3beta in complex with a minimal GSK3beta-binding segment of axin, at 2.4 A resolution. The structure confirms the co-localization of the binding sites for axin and FRAT in the C-terminal domain of GSK3beta, but reveals significant differences in the interactions made by axin and FRAT, mediated by conformational plasticity of the 285-299 loop in GSK3beta. Detailed comparison of the axin and FRAT GSK3beta complexes allows the generation of highly specific mutations, which abrogate binding of one or the other. Quantitative analysis suggests that the interaction of GSK3beta with the axin scaffold enhances phosphorylation of beta-catenin by >20 000-fold.

Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF.

Over 30 mutations of the B-RAF gene associated with human cancers have been identified, the majority of which are located within the kinase domain. Here we show that of 22 B-RAF mutants analyzed, 18 have elevated kinase activity and signal to ERK in vivo. Surprisingly, three mutants have reduced kinase activity towards MEK in vitro but, by activating C-RAF in vivo, signal to ERK in cells. The structures of wild type and oncogenic V599EB-RAF kinase domains in complex with the RAF inhibitor BAY43-9006 show that the activation segment is held in an inactive conformation by association with the P loop. The clustering of most mutations to these two regions suggests that disruption of this interaction converts B-RAF into its active conformation. The high activity mutants signal to ERK by directly phosphorylating MEK, whereas the impaired activity mutants stimulate MEK by activating endogenous C-RAF, possibly via an allosteric or transphosphorylation mechanism.