Threonine 22 phosphorylation attenuates Hsp90 interaction with cochaperones and affects its chaperone activity

Heat shock protein 90 (Hsp90) is an essential molecular chaperone whose activity is regulated not only by cochaperones but also by distinct posttranslational modifications. We report here that casein kinase 2 phosphorylates a conserved threonine residue (T22) in α helix-1 of the yeast Hsp90 N-domain both in?vitro and in?vivo. This α helix participates in?a hydrophobic interaction with the catalytic loop in Hsp90's middle domain, helping to stabilize the chaperone's ATPase-competent state. Phosphomimetic mutation of this residue alters Hsp90 ATPase activity and chaperone function and impacts interaction with the cochaperones Aha1 and Cdc37. Overexpression of Aha1 stimulates the ATPase activity, restores cochaperone interactions, and compensates for the functional defects of these Hsp90 mutants.     

Balanol analogues probe specificity determinants and the conformational malleability of the cyclic 3',5'-adenosine monophosphate-dependent protein kinase catalytic subunit

The protein kinase family is a prime target for therapeutic agents, since unregulated protein kinase activities are linked to myriad diseases. Balanol, a fungal metabolite consisting of four rings, potently inhibits Ser/Thr protein kinases and can be modified to yield potent inhibitors that are selective-characteristics of a desirable pharmaceutical compound. Here, we characterize three balanol analogues that inhibit cyclic 3',5'-adenosine monophosphate-dependent protein kinase (PKA) more specifically and potently than calcium- and phospholipid-dependent protein kinase (PKC). Correlation of thermostability and inhibition potency suggests that better inhibitors confer enhanced protection against thermal denaturation. Crystal structures of the PKA catalytic (C) subunit complexed to each analogue show the Gly-rich loop stabilized in an "intermediate" conformation, disengaged from important phosphoryl transfer residues. An analogue that perturbs the PKA C-terminal tail has slightly weaker inhibition potency. The malleability of the PKA C subunit is illustrated by active site residues that adopt alternate rotamers depending on the ligand bound. On the basis of sequence homology to PKA, a preliminary model of the PKC active site is described. The balanol analogues serve to test the model and to highlight differences in the active site local environment of PKA and PKC. The PKA C subunit appears to tolerate balanol analogues with D-ring modifications; PKC does not. We attribute this difference in preference to the variable B helix and C-terminal tail. By understanding the details of ligand binding, more specific and potent inhibitors may be designed that differentiate among closely related AGC protein kinase family members.     

High-throughput screening assay for inhibitors of heat-shock protein 90 ATPase activity

The molecular chaperone heat-shock protein 90 (HSP90) plays a key role in the cell by stabilizing a number of client proteins, many of which are oncogenic. The intrinsic ATPase activity of HSP90 is essential to this activity. HSP90 is a new cancer drug target as inhibition results in simultaneous disruption of several key signaling pathways, leading to a combinatorial approach to the treatment of malignancy. Inhibitors of HSP90 ATPase activity including the benzoquinone ansamycins, geldanamycin and 17-allylamino-17-demethoxygeldanamycin, and radicicol have been described. A high-throughput screen has been developed to identify small-molecule inhibitors that could be developed as therapeutic agents with improved pharmacological properties. A colorimetric assay for inorganic phosphate, based on the formation of a phosphomolybdate complex and subsequent reaction with malachite green, was used to measure the ATPase activity of yeast HSP90. The Km for ATP determined in the assay was 510+/-70 microM. The known HSP90 inhibitors geldanamycin and radicicol gave IC(50) values of 4.8 and 0.9 microM respectively, which compare with values found using the conventional coupled-enzyme assay. The assay was robust and reproducible (2-8% CV) and used to screen a compound collection of approximately 56,000 compounds in 384-well format with Z' factors between 0.6 and 0.8.     

Suppression of endogenous estrogen during development affects porcine epididymal sperm maturation

Estrogen plays an important role in male reproduction, critical for sustained fertility in some species. Reducing estrogen's interaction with its receptor(s) in monkey and mouse models is associated with reduced sperm motility and, in some cases, documented elimination of sperm fertilizing ability, suggesting that normal epididymal function may be estrogen dependent. The objective of these experiments was to evaluate the effects of reduced endogenous estrogen on development of epididymal function in the pig, a species in which males have very high levels of endogenous estrogen. Letrozole, a potent inhibitor of estrogen synthesis, was administered to neonatal boars from 1 week of age and markedly suppressed estrogen production. Epididymal function assessed as acquisition of sperm fertilizing ability (in vitro fertilization of zona-free oocytes) was reduced in Letrozole-treated animals at 24 and 28 weeks of age (23% and 30% fertilization, respectively compared with 37% and 54% in vehicle controls) but had recovered by 32 weeks of age. Cauda epididymal sperm numbers were reduced in treated animals (35% of control values at 20 weeks of age) but appeared to be recovering at 32 weeks of age. Reduction of endogenous estrogen had no effect on other aspects of epididymal function (percentage of motile sperm, sperm motion parameters, sperm head morphometrics, or ability of sperm to undergo an acrosome reaction). Reducing endogenous estrogen during postnatal development appears to have transient effects on porcine epididymal function. These transient effects suggest that the pig, with its high endogenous estrogen, may respond differently than other species to reduced estrogen synthesis.     

Developmentally regulated, combinatorial RNA processing modulates AMPA receptor biogenesis

The subunit composition determines AMPA receptor (AMPA-R) function and trafficking. Mechanisms underlying channel assembly are thus central to the efficacy and plasticity of glutamatergic synapses. We previously showed that RNA editing at the Q/R site of the GluR2 subunit contributes to the assembly of AMPA-R heteromers by attenuating formation of GluR2 homotetramers. Here we report that this function of the Q/R site depends on subunit contacts between adjacent ligand binding domains (LBDs). Changes of LBD interface contacts alter GluR2 assembly properties, forward traffic, and expression at synapses. Interestingly, developmentally regulated RNA editing within the LBD (at the R/G site) produces analogous effects. Our data reveal that editing to glycine reduces the self-assembly competence of this critical subunit and slows GluR2 maturation in the endoplasmic reticulum (ER). Therefore, RNA editing sites, located at strategic subunit interfaces, shape AMPA-R assembly and trafficking in a developmentally regulated manner.     

MyD88 adapter-like (Mal) is phosphorylated by Bruton's tyrosine kinase during TLR2 and TLR4 signal transduction

Members of the Toll-like receptor (TLR) family are essential players in activating the host innate immune response against infectious microorganisms. All TLRs signal through Toll/interleukin 1 receptor domain-containing adapter proteins. MyD88 adapter-like (Mal) is one such adapter that specifically is involved in TLR2 and TLR4 signaling. When overexpressed we have found that Mal undergoes tyrosine phosphorylation. Three possible phospho-accepting tyrosines were identified at positions 86, 106, and 187, and two mutant forms of Mal in which tyrosines 86 and 187 were mutated to phenylalanine acted as dominant negative inhibitors of NF-kappaB activation by lipopolysaccharide (LPS). Activation of THP-1 monocytic cells with the TLR4 agonist LPS and the TLR2 agonist macrophage-activating lipopeptide-2 induced phosphorylation of Mal on tyrosine residues. We found that the Bruton's tyrosine kinase (Btk) inhibitor LFM-A13 could block the endogenous phosphorylation of Mal on tyrosine in cells treated with macrophage-activating lipopeptide-2 or LPS. Furthermore, Btk immunoprecipitated from THP-1 cells activated by LPS could phosphorylate Mal. Our study therefore provides the first demonstration of the key role of Mal phosphorylation on tyrosine during signaling by TLR2 and TLR4 and identifies a novel function for Btk as the kinase involved.