Ion chromatographic separation and quantitation of alkyl methylamines and ethylamines in atmospheric gas and particulate matter using preconcentration and suppressed conductivity detection.

Two methods based on ion chromatography (IC) were developed for the detection of methyl and ethyl alkyl amines (methylamine (MA), ethylamine (EA), dimethylamine (DMA), diethylamine (DEA), trimethylamine (TMA) and triethylamine (TEA)) and NH(3)/NH(4)(+) in online atmospheric gas-particle and size-resolved particulate samples. The two IC methods were developed to analyze samples collected with an ambient ion monitor (AIM), an online gas-particle collection system, or with a Micro Orifice Uniform Deposit Impactor (MOUDI) for size-resolved particle samples. These methods enable selective and (semi-) quantitative detection of alkyl amines at ambient atmospheric concentrations (pptv and pgm(-3)) in samples where significant interferences can be expected from Na(+) and NH(4)(+), for example marine and rural air masses. Sample pre-concentration using a trace cation column enabled instrumental detection limits on the order of pmol (sub-ng) levels per sample, an improvement of up to 10(2) over current IC methods. Separation was achieved using a methanesulfonic acid gradient elution on Dionex CS12A and CS17 columns. The relative standard deviations in retention times during 3 weeks continuous (hourly) sampling campaigns ranged from 0.1 to 0.5% and 0.2 to 5% for the CS12A and CS17 across a wide dynamic range of atmospheric concentrations. Resolution of inorganic and organic cations is limited to 25min for online samples. Mass-dependent coelution of NH(4)(+)/MA/EA occurred on the CS12A column and DEA/TMA coeluted on both columns. Calibrations of ammonium show a non-linear response across the entire calibration range while all other analytes exhibit high linearity (R(2)=0.984-0.999), except for EA and TEA on the CS12A (R(2)=0.960 and 0.941, respectively). Both methods have high analytical accuracy for the nitrogenous bases ranging from 9.5 to 20% for NH(3) and <5-15% for the amines. Hourly observations of amines at Egbert, ON in October 2010 showed gaseous DMA and TMA+DEA at 1-10pptv in air, while particulate DMA and TMA+DEA were present at 0.5-4ng m(-3). A size-resolved particulate sample collected over 23h was found to contain DMA, TMA+DEA and MEA at 1.78, 8.15 and 0.03ngm(-3) mass loadings, with the amine mass enhanced in particle sizes between 100 and 1000nm. These results highlight a need for very sensitive and selective detection of methyl and ethyl amines in addition to NH(3) in continuous online monitoring strategies.

A versatile computational pipeline for bacterial genome annotation improvement and comparative analysis, with Brucella as a use case.

We present a bacterial genome computational analysis pipeline, called GenVar. The pipeline, based on the program GeneWise, is designed to analyze an annotated genome and automatically identify missed gene calls and sequence variants such as genes with disrupted reading frames (split genes) and those with insertions and deletions (indels). For a given genome to be analyzed, GenVar relies on a database containing closely related genomes (such as other species or strains) as well as a few additional reference genomes. GenVar also helps identify gene disruptions probably caused by sequencing errors. We exemplify GenVar's capabilities by presenting results from the analysis of four Brucella genomes. Brucella is an important human pathogen and zoonotic agent. The analysis revealed hundreds of missed gene calls, new split genes and indels, several of which are species specific and hence provide valuable clues to the understanding of the genome basis of Brucella pathogenicity and host specificity.

PATRIC: the VBI PathoSystems Resource Integration Center.

The PathoSystems Resource Integration Center (PATRIC) is one of eight Bioinformatics Resource Centers (BRCs) funded by the National Institute of Allergy and Infection Diseases (NIAID) to create a data and analysis resource for selected NIAID priority pathogens, specifically proteobacteria of the genera Brucella, Rickettsia and Coxiella, and corona-, calici- and lyssaviruses and viruses associated with hepatitis A and E. The goal of the project is to provide a comprehensive bioinformatics resource for these pathogens, including consistently annotated genome, proteome and metabolic pathway data to facilitate research into counter-measures, including drugs, vaccines and diagnostics. The project's curation strategy has three prongs: 'breadth first' beginning with whole-genome and proteome curation using standardized protocols, a 'targeted' approach addressing the specific needs of researchers and an integrative strategy to leverage high-throughput experimental data (e.g. microarrays, proteomics) and literature. The PATRIC infrastructure consists of a relational database, analytical pipelines and a website which supports browsing, querying, data visualization and the ability to download raw and curated data in standard formats. At present, the site warehouses complete sequences for 17 bacterial and 332 viral genomes. The PATRIC website (https://patric.vbi.vt.edu) will continually grow with the addition of data, analysis and functionality over the course of the project.

Mutation of Tec family kinases alters T helper cell differentiation.

The Tec kinases Rlk and Itk are critical for full T cell receptor (TCR)-induced activation of phospholipase C-gamma and mitogen-activated protein kinase. We show here that the mutation of Rlk and Itk impaired activation of the transcription factors NFAT and AP-1 and production of both T helper type 1 (TH1) and TH2 cytokines. Consistent with these biochemical defects, Itk-/- mice did not generate effective TH2 responses when challenged with Schistosoma mansoni eggs. Paradoxically, the more severely impaired Rlk-/-Itk-/- mice were able to mount a TH2 response and produced TH2 cytokines in response to this challenge. In addition, Rlk-/-Itk-/- cells showed impaired TCR-induced repression of the TH2-inducing transcription factor GATA-3, suggesting a potential mechanism for TH2 development in these hyporesponsive cells. Thus, mutations that affect Tec kinases lead to complex alterations in CD4+ TH cell differentiation.

Biochemical and genetic analyses of the Tec kinases Itk and Rlk/Txk.

The Tec kinases have been implicated as important components of signalling pathways downstream of lymphocyte antigen receptors. Activation of these kinases requires two steps: (i) phosphorylation by Src family kinases and (ii) plasma membrane localization, which is mediated by interaction between the pleckstrin homology (PH) domains of Tec kinases and the products of phosphoinositide-3 kinase (PI-3K). Itk and Rlk/Txk are Tec kinases expressed in T-lymphocytes. Despite similarity to other Tec kinases, Rlk/Txk lacks a PH domain and instead possesses a palmitoylated cysteine-string motif. We have found that both Rlk/Txk and Itk are phosphorylated in response to T-cell receptor stimulation and can be activated by phosphorylation by Src family kinases. However, consistent with its lack of PH domain, Rlk/Txk is phosphorylated independent of PI-3K activity. Furthermore, we demonstrated that like Itk, Rlk/Txk is associated with lipid RAFTs (detergent-insoluble, cholesterol-rich regions of the membrane), but unlike Itk, Rlk/Txk's RAFT association is independent of PI-3K activity. Despite these differences, Rlk/Txk partially compensates for loss of Itk in gene-targeted animals, suggesting overlapping functions for these kinases.

Altered lymphocyte responses and cytokine production in mice deficient in the X-linked lymphoproliferative disease gene SH2D1A/DSHP/SAP.

We have introduced a targeted mutation in SH2D1A/DSHP/SAP, the gene responsible for the human genetic disorder X-linked lymphoproliferative disease (XLP). SLAM-associated protein (SAP)-deficient mice had normal lymphocyte development, but on challenge with infectious agents, recapitulated features of XLP. Infection of SAP- mice with lymphocyte choriomeningitis virus (LCMV) or Toxoplasma gondii was associated with increased T cell activation and IFN-gamma production, as well as a reduction of Ig-secreting cells. Anti-CD3-stimulated splenocytes from uninfected SAP- mice produced increased IFN-gamma and decreased IL-4, findings supported by decreased serum IgE levels in vivo. The Th1 skewing of these animals suggests that cytokine misregulation may contribute to phenotypes associated with mutation of SH2D1A/SAP.

Tec kinases: modulators of lymphocyte signaling and development.

The Tec kinases are implicated as important components of the antigen receptor signaling required for proper lymphocyte activation and development. Recent data suggest that these kinases contribute to multiprotein complexes containing LAT and SLP-76 in T cells, and BLNK/SLP-65 in B cells, which are required for activation of PLC-gamma and downstream pathways.

Requirements for activation and RAFT localization of the T-lymphocyte kinase Rlk/Txk.

BACKGROUND: The Tec family kinases are implicated in signaling from lymphocyte antigen receptors and are activated following phosphorylation by Src kinases. For most Tec kinases, this activation requires an interaction between their pleckstrin homology (PH) domains and the products of phosphoinositide 3-Kinase, which localizes Tec kinases to membrane RAFTs. Rlk/Txk is a Tec related kinase expressed in T cells that lacks a pleckstrin homology domain, having instead a palmitoylated cysteine-string motif. To evaluate Rlk's function in T cell receptor signaling cascades, we examined the requirements for Rlk localization and activation by Src family kinases. RESULTS: We demonstrate that Rlk is also associated with RAFTs, despite its lack of a pleckstrin homology domain. Rlk RAFT association requires the cysteine-string motif and is independent of PI3 Kinase activity. We further demonstrate that Rlk can be phosphorylated and activated by Src kinases, leading to a decrease in its half-life. A specific tyrosine in the activation loop of Rlk, Y420, is required for phosphorylation and activation, as well as for decreased stability, but is not required for lipid RAFT association. Mutation of this tyrosine also prevents increased tyrosine phosphorylation of Rlk after stimulation of the T cell receptor, suggesting that Rlk is phosphorylated by Src family kinases in response to T cell receptor engagement. CONCLUSIONS: Like the other related Tec kinases, Rlk is associated with lipid RAFTs and can be phosphorylated and activated by Src family kinases, supporting a role for Rlk in signaling downstream of Src kinases in T cell activation.

Deficiency of PTEN in Jurkat T cells causes constitutive localization of Itk to the plasma membrane and hyperresponsiveness to CD3 stimulation.

Pleckstrin homology (PH) domain binding to D3-phosphorylated phosphatidylinositides (PI) provides a reversible means of recruiting proteins to the plasma membrane, with the resultant change in subcellular localization playing a key role in the activation of multiple intracellular signaling pathways. Previously we found that the T-cell-specific PH domain-containing kinase Itk is constitutively membrane associated in Jurkat T cells. This distribution was unexpected given that the closely related B-cell kinase, Btk, is almost exclusively cytosolic. In addition to constitutive membrane association of Itk, unstimulated JTAg T cells also exhibited constitutive phosphorylation of Akt on Ser-473, an indication of elevated basal levels of the phosphatidylinositol 3-kinase (PI3K) products PI-3,4-P(2) and PI-3,4,5-P(3) in the plasma membrane. Here we describe a defect in expression of the D3 phosphoinositide phosphatase, PTEN, in Jurkat and JTAg T cells that leads to unregulated PH domain interactions with the plasma membrane. Inhibition of D3 phosphorylation by PI3K inhibitors, or by expression of PTEN, blocked constitutive phosphorylation of Akt on Ser-473 and caused Itk to redistribute to the cytosol. The PTEN-deficient cells were also hyperresponsive to T-cell receptor (TCR) stimulation, as measured by Itk kinase activity, tyrosine phosphorylation of phospholipase C-gamma1, and activation of Erk compared to those in PTEN-replete cells. These data support the idea that PH domain-mediated association with the plasma membrane is required for Itk activation, provide evidence for a negative regulatory role of PTEN in TCR stimulation, and suggest that signaling models based on results from Jurkat T-cell lines may underestimate the role of PI3K in TCR signaling.

rlk/TXK encodes two forms of a novel cysteine string tyrosine kinase activated by Src family kinases.

Rlk/Txk is a member of the BTK/Tec family of tyrosine kinases and is primarily expressed in T lymphocytes. Unlike other members of this kinase family, Rlk lacks a pleckstrin homology (PH) domain near the amino terminus and instead contains a distinctive cysteine string motif. We demonstrate here that Rlk protein consists of two isoforms that arise by alternative initiation of translation from the same cDNA. The shorter, internally initiated protein species lacks the cysteine string motif and is located in the nucleus when expressed in the absence of the larger form. In contrast, the larger form is cytoplasmic. We show that the larger form is palmitoylated and that mutation of its cysteine string motif both abolishes palmitoylation and allows the protein to migrate to the nucleus. The cysteine string, therefore, is a critical determinant of both fatty acid modification and protein localization for the larger isoform of Rlk, suggesting that Rlk regulation is distinct from the other Btk family kinases. We further show that Rlk is phosphorylated and changes localization in response to T-cell-receptor (TCR) activation and, like the other Btk family kinases, can be phosphorylated and activated by Src family kinases. However, unlike the other Btk family members, Rlk is activated independently of the activity of phosphatidylinositol 3-kinase, consistent with its lack of a PH domain. Thus, Rlk has two distinct isoforms, each of which may have unique properties in signaling downstream from the TCR.

Geldanamycin, a heat shock protein 90-binding benzoquinone ansamycin, inhibits steroid-dependent translocation of the glucocorticoid receptor from the cytoplasm to the nucleus.

When they are translated, steroid receptors are assembled into a multiprotein complex containing hsp90, p23, an immunophilin, and often some hsp70. Some of the receptors, such as that for progesterone, have nuclear localization signals that are functional in the absence of hormone, and they move into the nucleus where they exist in the same multiprotein heterocomplex with hsp90. Other receptors, such as the glucocorticoid receptor, are localized predominantly in the cytoplasm in the absence of hormone and move into the nucleus in a hormone-dependent fashion. We have previously proposed that hsp90 and the immunophilin play a role in receptor trafficking [Pratt, W. B. (1993) J. Biol. Chem. 268, 21455-21458]. In this work, we show that treatment of L cells with geldanamycin, a benzoquinone ansamycin that binds to hsp90 and disrupts its function, impedes dexamethasone-dependent trafficking of the glucocorticoid receptor from the cytoplasm to the nucleus. Because geldanamycin treatment of hormone-free cells causes a rapid loss of steroid binding activity, receptors were prebound with dexamethasone by incubating cells with hormone at 0 degrees C prior to shifting the temperature to 37 degrees C for 20 min to permit receptor transformation and translocation in the presence or absence of geldanamycin. Geldanamycin does not cause steroid to dissociate from prebound receptors, and it does not inhibit hormone-mediated receptor transformation assayed by conversion to the DNA-binding state. However, as reported previously for the progesterone receptor, geldanamycin blocks assembly of the glucocorticoid receptor-hsp90 heterocomplex at an intermediate state of assembly where the receptor is bound to hsp70 and p60, both of which are required components in the assembly mechanism. Our observations support the proposal that dynamic association of receptors with hsp90 is required for receptor translocation from the cytoplasm to the nucleus.

Perceived learning needs of patients with coronary artery disease using a questionnaire assessment tool.

OBJECTIVE: To determine the perceived learning needs and their level of importance in patients with angina pectoris or myocardial infarction at hospitalization and follow-up clinic visit and to assess the reliability of a self-administered questionnaire. DESIGN: Longitudinal, exploratory. SETTING: West Coast university-affiliated medical center and clinics that serve primarily a veteran population. PATIENTS: Twenty-eight adults who were admitted with a diagnosis of angina pectoris or myocardial infarction. Age range was 31 to 80 years (mean 61 years). OUTCOME MEASURES: Learning needs, questionnaire. INTERVENTION: Data collection was initiated at hospitalization and continued at the first clinic visit after discharge. A self-administered questionnaire and personal data sheet were completed by the patients. RESULTS: Matched-pair t-tests and Pearson's correlation were used for data analysis. No statistical differences were demonstrated between learning needs at hospitalization and clinic visit. Three content areas including symptom recognition, cardiac anatomy and physiology, and medications were ranked as the most important learning needs of patients at hospitalization and follow-up clinic visit. The least important learning needs at both times were content areas of smoking, work, and sex. No correlation was found between the importance of perceived learning needs and age, occupation, smoking, and marital status. The questionnaire contains 38 items and is self-administered. It was developed on the basis of a previous tool and preliminary study results. Content and validity were supported by clinical experts. The internal consistency reliability alpha coefficients of the questionnaire were 0.96 at hospitalization and 0.93 at the clinic visit. CONCLUSIONS: The findings of this study suggest that the most important perceived learning needs of patients at hospitalization and follow-up clinic visit are those that affect survival. A self-administered questionnaire can be used as a practical and reliable tool to determine the perceived learning needs of patients with coronary artery disease during the recovery phase of illness. Cardiac educational programs for patients with coronary artery disease can focus on the content areas that patients consider most important.

Immunofluorescence localization of the 90-kDa heat-shock protein to cytoskeleton.

The 90-kDa heat-shock protein, hsp90, is an abundant and conserved protein located predominantly in the cytoplasm. Previous reports have localized a portion of the hsp90 to microtubules and to microfilaments. Here, we show that colocalization of hsp90 with microtubules is seen after long-term but not short-term fixation of rat pulmonary endothelial cells with formaldehyde. Under conditions of methanol fixation, a significant portion of both hsp90 and the hsp90-associated protein p23 is present on fibrillar structures extending throughout the cytoplasm of endothelial cells, however, when cells are treated with colcemid under conditions that eliminate microtubules, the fibrils condense into bright rope-like bundles located in the immediate perinuclear area and extending toward the cell periphery. Identical images are observed with an antibody against vimentin and the pattern seen after colcemid treatment is classical for intermediate filaments. Preabsorption of the anti-hsp90 antibody with purified hsp90 prevents the immunofluorescence but preabsorption with purified p23 does not, and the reverse is the case for immunofluorescence produced by the anti-p23 antibody. These results suggest that hsp90 is able, either directly or indirectly via other proteins, to associate with both microtubules and microfilaments.

A model of protein targeting mediated by immunophilins and other proteins that bind to hsp90 via tetratricopeptide repeat domains.

We have shown recently that the immunophilins CyP-40 and FKBP52/hsp56 bind to a common site on hsp90 and that they exist in separate heterocomplexes with the glucocorticoid receptor (GR). FKBP52/hsp56 binds to hsp90 via its tetratricopeptide repeat (TPR) domains, it is not required for GR.hsp90 heterocomplex assembly, and it is thought to play a role in targeted movement of the GR. In this work we examine the hsp90 binding of four proteins (FKBP52/hsp56, CyP-40, p50, Mas70p) thought to be involved in targeted protein trafficking. FKBP52/hsp56 and CyP-40 (each with three TPRs), localize to the nucleus and nucleoli, respectively, and form relatively weak complexes with hsp90 that are competed by a CyP-40 fragment containing its three TPRs. The p50 component of the Src.hsp90 and Raf.hsp90 heterocomplexes localizes to cytoskeletal fibers extending from the perinuclear region to the plasma membrane and forming a rim under the plasma membrane of endothelial cells. p50, Mas70p (seven TPRs), which is a receptor for mitochondrial import, and the p60 (six to eight TPRs) component of the steroid receptor.hsp90 heterocomplex assembly system bind very tightly to hsp90 in a manner that is not competed by the CyP-40 fragment. However, bacterially expressed p60 blocks the binding of p50, Mas70p, FKBP52/hsp56, and CyP-40 to purified hsp90. The data are consistent with binding of all of these proteins to a site on hsp90 that is a general TPR domain acceptor. Our localization and binding data are used to develop a model in which proteins that are chaperoned by hsp90 move as dynamic complexes to their cellular sites of action, with the TPR-containing protein participating in targeting the movement of the complexes.

Evidence that the FK506-binding immunophilin heat shock protein 56 is required for trafficking of the glucocorticoid receptor from the cytoplasm to the nucleus.

The FK506-binding immunophilin hsp56 (FKBP52) is one of several chaperone proteins associated with untrasformed steroid receptors in a multiprotein heterocomplex. The function of heat shock protein 56 (hsp56) with respect to receptor action is unknown. hsp56 is not required for glucocorticoid receptor heterocomplex assembly or for proper folding of the receptor hormone-binding domain into a high affinity steroid-binding conformation. In intact cells, the majority of the hsp56 is located in the nucleus, with a minority colocalizing with microtubules in the cytoplasm. hsp56 contains a conserved negatively charged domain that we speculate might serve as a nuclear localization signal recognition sequence. Here we show that injection of an antibody raised against this negative sequence into intact L cells impedes subsequent dexamethasone-mediated shift of the glucocorticoid receptor into the nucleus. Nonimmune rabbit serum and an antibody raised against another site on hsp56 do not affect receptor movement. Inhibition of receptor movement by the 419 antibody against the negative sequence is blocked by preincubation with purified hsp56, but not by preincubation with purified hsp90, hsp70, or BSA. These observations are consistent with the possibility that hsp56 is involved in receptor trafficking to the nucleus, possibly functioning as the nuclear localization signal recognition protein. Receptor trafficking to the nucleus is not affected by FK506, indicating that the peptidylprolyl isomerase activity of hsp56 is not involved.

The hsp56 immunophilin component of untransformed steroid receptor complexes is localized both to microtubules in the cytoplasm and to the same nonrandom regions within the nucleus as the steroid receptor.

In their unliganded state, mouse glucocorticoid receptors (GR) that are overexpressed in the WCL2 line of Chinese hamster ovary cells are distributed in a nonrandom manner throughout all planes of the nucleus. These untransformed nuclear receptors exist in a heterocomplex containing three heat shock proteins, hsp90, hsp70, and hsp56, the latter being an immunophilin of the FK506 binding type whose cellular function is unknown. Because a knowledge of the cellular distribution of hsp56 could provide important clues to its function in steroid-receptor heterocomplexes, we have examined hsp56 localization in intact cells by indirect immunofluorescence using the UPJ56 antibody. The majority of hsp56 is located in the nucleus, with substantial amounts also visualized in the cytoplasm of intact cells. The cytoplasmic hsp56 was examined in rat pulmonary endothelial cells where the protein was found to colocalize with microtubules. The nuclear hsp56 was examined in the WCL2 cells, where the protein was found by confocal imaging to colocalize throughout all planes of the nucleus in the same mottled pattern as the overexpressed GR. Like the GR, the nuclear hsp56 is recovered largely in the cytosolic fraction after hypotonic rupture of WCL2 cells. An observation potentially related to the microtubule-associated fraction of hsp56 is that immunoadsorption of hsp56 from WCL2 cytosol is accompanied by coadsorption of the microtubule-associated protein-1C complex. These observations are discussed with respect to the possible biological functions of hsp56 in the folding and/or cytoplasmic-nuclear trafficking of the receptor.

Characterization of the protein-protein interactions determining the heat shock protein (hsp90.hsp70.hsp56) heterocomplex.

We have reported previously that the three heat shock proteins hsp56, hsp70, and hsp90 exist together in a heterocomplex in human lymphocyte cytosol (Sanchez, E. R., Faber, L. E., Henzel, W. J., and Pratt, W. B. (1990) Biochemistry 29, 5145-5152). All three of these proteins also exist in the native glucocorticoid receptor heterocomplex isolated from WCL2 cell cytosol and we have recently shown that the three heat shock proteins are present when immunopurified mouse glucocorticoid receptor is reconstituted into a heterocomplex by rabbit reticulocyte lysate (Hutchison, K. A., Scherrer, L. C., Czar, M. J., Ning, Y., Sanchez, E. R., Leach, K. L., Deibel, M. R., Jr., and Pratt, W. B. (1993) Biochemistry 32, 3953-3957). In this work, we show that highly purified mouse hsp90 binds in a reversible equilibrium to immunopurified rabbit hsp56, but hsp56 does not bind to purified mouse hsp70. In contrast to the equilibrium binding of hsp90 to hsp56, purified hsp90 binds poorly or not at all to purified hsp70 unless a third factor from reticulocyte lysate is present to permit complex formation. This hsp70.hsp90 complex-forming factor is heat-labile, and in the presence of this factor and ATP, a heat shock protein heterocomplex can be reconstituted from purified mouse hsp90 and hsp70 and rabbit hsp56 that is present in the factor preparation. Our data are consistent with a model in which hsp56 and hsp70 bind to different sites on hsp90 but do not interact with each other. The presence of hsp56 in the heat shock protein heterocomplex is not stabilized by molybdate but hsp56 is stabilized if the glucocorticoid receptor is present in addition to hsp90 and hsp70.

Proof that hsp70 is required for assembly of the glucocorticoid receptor into a heterocomplex with hsp90.

Incubation of immunopurified glucocorticoid receptor with rabbit reticulocyte lysate forms a complex between the receptor and hsp90, with simultaneous conversion of the receptor from a non-steroid binding but DNA binding state typical of the transformed receptor back to the steroid binding, non-DNA binding state typical of the untransformed receptor (Scherrer, L. C., Dalman, F. C., Massa, E., Meshinchi, S., and Pratt, W. B. (1990) J. Biol. Chem. 265, 21397-21400). The receptor heterocomplex formed by the lysate also contains hsp70 and is formed in an ATP-dependent and cation-selective manner (Hutchison, K. A., Czar, M.J., Scherrer, L. C., and Pratt, W.B. (1992) J. Biol. Chem. 267, 14047-14053). In this work, we selectively depleted reticulocyte lysate of hsp70 by passing it through a column of ATP-agarose. The hsp70-depleted lysate contains hsp90, but it cannot form a receptor-hsp90 heterocomplex. hsp70 purified from mouse L cells binds to immunopurified glucocorticoid receptor but does not convert it to the steroid binding state. Addition of purified hsp70 to the hsp70-depleted lysate reactivates the heterocomplex assembly system, permitting formation of a receptor-hsp90-hsp70 complex, with the receptor being returned to the high affinity steroid-binding conformation. These data are consistent with a model in which the protein-unfolding activity of hsp70 is required for hsp90 binding to the hormone binding domain of the glucocorticoid receptor. The hsp56 immunophilin component of the native receptor heterocomplex is also present in the reconstituted receptor heterocomplex in an hsp70-dependent manner. In addition to hsp70, other as yet unidentified factors in reticulocyte lysate are required for receptor heterocomplex assembly.

The hsp56 immunophilin component of steroid receptor heterocomplexes: could this be the elusive nuclear localization signal-binding protein?

In many cells, the glucocorticoid receptor undergoes rapid steroid-mediated translocation from the cytoplasm to the nucleus, and this receptor is an excellent model for studying the mechanism of targeted protein movement through the cytoplasm. For such unidirectional movement to occur, the receptor must attach to a retrograde movement system in a manner that involves the nuclear localization signal. It is improbable that such attachment occurs via a direct protein-protein interaction between the receptor and the movement system; rather, one or more linker proteins are likely to be involved. As with other steroid receptors, the glucocorticoid receptor is associated with several other proteins in a heterocomplex. Two of these receptor-associated proteins are the heat shock proteins hsp90 and hsp56, and a third heat shock protein, hsp70, is required for assembly of the receptor heterocomplex. The hormone binding domain of the steroid receptors determines the interaction with both hsp90 and hsp70. Hsp56 is known to bind to hsp90, but its potential site, or sites, of interaction with the receptor are undefined. Hsp56 has recently been cloned and demonstrated to be an immunophilin of the FK506/rapamycin binding class. The immunophilins have peptidyl-prolyl isomerase activity but their cellular functions are unknown. Herein, we review the literature on the hsp56 immunophilin component of the receptor heterocomplex and present a rationale for hsp56 being the protein that determines the direction of receptor movement via a direct protein-protein interaction with the nuclear localization signal.