Identification of amino acids in the tetratricopeptide repeat and C-terminal domains of protein phosphatase 5 involved in autoinhibition and lipid activation.

Protein phosphatase 5 (PP5) exhibits low basal activity due to the autoinhibitory properties of its N-terminal and C-terminal domains but can be activated approximately 40-fold in vitro by polyunsaturated fatty acids. To identify residues involved in regulating PP5 activity, we performed scanning mutagenesis of its N-terminal tetratricopeptide repeat (TPR) domain and deletion mutagenesis of its C-terminal domain. Mutating residues in a groove of the TPR domain that binds to heat shock protein 90 had no effect on basal phosphatase activity. Mutation of Glu-76, however, whose side chain projects away from this groove, resulted in a 10-fold elevation of basal activity without affecting arachidonic acid-stimulated activity. Thus, the interface of the TPR domain involved in PP5 autoinhibition appears to be different from that involved in heat shock protein 90 binding. We also observed a 10-fold elevation of basal phosphatase activity upon removing the C-terminal 13 amino acids of PP5, with a concomitant 50% decrease in arachidonic acid-stimulated activity. These two effects were accounted for by two distinct amino acid deletions: deleting the four C-terminal residues (496-499) of PP5 had no effect on its activity, but removing Gln-495 elevated basal activity 10-fold. Removal of a further three amino acids had no additional effect, but deleting Asn-491 resulted in a 50% reduction in arachidonic acid-stimulated activity. Thus, Glu-76 in the TPR domain and Gln-495 at the C-terminus were implicated in maintaining the low basal activity of PP5. While the TPR domain alone has been thought to mediate fatty acid activation of PP5, our data suggest that Asn-491, near its C-terminus, may also be involved in this process.

Regulation of the atrial natriuretic peptide receptor by heat shock protein 90 complexes.

Heat shock protein 90 (hsp90) is a chaperone required for the proper folding and trafficking of many proteins involved in signal transduction. We tested whether hsp90 plays a role as a chaperone for GC-A, the membrane guanylate cyclase that acts as a receptor for atrial natriuretic peptide (ANP). When cultured cells expressing recombinant GC-A were treated with geldanamycin, an inhibitor of hsp90 function, the ANP-stimulated production of cyclic GMP was inhibited. This suggested that hsp90 was required for GC-A processing and/or stability. A physical association between hsp90 and GC-A was demonstrated in coimmunoprecipitation experiments. Treatment with geldanamycin disrupted this association and led to the accumulation of complexes containing GC-A and heat shock protein 70 (hsp70). Protein folding pathways involving hsp70 and hsp90 include several pathway-specific co-chaperones. Complexes between GC-A and hsp90 contained the co-chaperone p50(cdc37), typically found associated with protein kinase.hsp90 heterocomplexes. GC-A immunoprecipitates did not contain detectable amounts of Hop, FKBP51, FKBP52, PP5, or p23, all co-chaperones found in hsp90 complexes with other signaling proteins. The association of hsp90 and p50(cdc37) with GC-A was dependent on the kinase homology domain of this receptor but not on its ANP-binding, transmembrane, or guanylate cyclase domains. The data suggest that GC-A is regulated by hsp90 complexes similar to those involved in the maturation of protein kinases.

Protein phosphatase 5 in signal transduction.

Protein phosphatase 5 (PP5) possesses unique biochemical properties, which include its tetratricopeptide repeat (TPR) targeting/regulatory domain and its ability to be activated by lipids. PP5 has been studied as a paradigm for TPR protein structure and function. Roles for PP5 in signal transduction are emerging: from cell cycle regulation and signaling by nuclear receptors, to possible regulation of membrane receptors and ion channels.

Cyclic nucleotide-gated channels mediate membrane depolarization following activation of store-operated calcium entry in endothelial cells.

Calcium agonists induce membrane depolarization in endothelial cells through an unknown mechanism. Present studies tested the hypothesis that pulmonary artery endothelial cells express a cyclic nucleotide-gated (CNG) cation channel activated by store-operated calcium entry to produce membrane depolarization. In the whole-cell configuration, voltage-clamped cells revealed a large non-inactivating, outwardly rectifying cationic current in the absence of extra- or intracellular Ca(2+) that was reduced upon replenishment of Ca(2+). The inward current was non-selective for K(+), Na(+), Cs(+), and Rb(+) and was not inhibited by high tetraethylammonium concentrations. cAMP and cGMP stimulated the current and changed the cation permeability to favor Na(+). Moreover, 8-bromo-cAMP stimulated the current in voltage-clamped cells in the perforated patch mode. The cationic current was inhibited by the CNG channel blocker LY83,583, and reverse transcriptase-polymerase chain reaction cloning identified expression of a CNG channel resembling that seen in olfactory neurons. Activation of store-operated calcium entry using thapsigargin increased a current through the CNG channel. Stimulation of the current paralleled pulmonary artery endothelial cell membrane depolarization, and both the current and membrane depolarization were abolished using LY83,583. Taken together, these data demonstrate activation of store-operated calcium entry stimulates a CNG channel producing membrane depolarization. Such membrane depolarization may contribute to slow feedback inhibition of store-operated calcium entry.

Overlapping sites of tetratricopeptide repeat protein binding and chaperone activity in heat shock protein 90.

The sequential binding of different tetratricopeptide repeat (TPR) proteins to heat shock protein 90 (hsp90) is essential to its chaperone function in vivo. We have previously shown that three basic residues in the TPR domain of PP5 are required for binding to the acidic C-terminal domain of hsp90. We have now tested which acidic residues in this C-terminal domain are required for binding to three different TPR proteins as follows: PP5, FKBP52, and Hop. Mutation of Glu-729, Glu-730, and Asp-732 at the C terminus of hsp90 interfered with binding of all three TPR proteins. Mutation of Glu-720, Asp-722, Asp-723, and Asp-724 inhibited binding of FKBP52 and PP5 but not of Hop. Mutation of Glu-651 and Asp-653 did not affect binding of FKBP52 or PP5 but inhibited both Hop binding and hsp90 chaperone activity. We also found that a conserved Lys residue required for PP5 binding to hsp90 was critical for the binding of FKBP52 but not for the binding of Hop to hsp90. These results suggest distinct but overlapping binding sites on hsp90 for different TPR proteins and indicate that the binding site for Hop, which is associated with hsp90 in intermediate stages of protein folding, overlaps with a site of chaperone activity.

Identification of conserved residues required for the binding of a tetratricopeptide repeat domain to heat shock protein 90.

The sequential binding of heat shock protein 90 (hsp90) to a series of tetratricopeptide repeat (TPR) proteins is critical to its function as a molecular chaperone. We have used site-directed mutagenesis to clarify the structural basis for the binding of hsp90 to the TPR domain of phosphoprotein phosphatase 5 (PP5). This TPR domain was chosen for study because its three-dimensional structure is known. We examined co-immunoprecipitation of hsp90 with wild type and mutant TPR constructs from transfected cells. Only mutations located on one face of the TPR domain affected hsp90 binding. This allowed the identification of a binding groove. Three basic residues that are highly conserved in hsp90-binding TPR proteins extend prominently into this groove. Lys-97 and Arg-101 were absolutely required for hsp90 binding, while mutation of Arg-74 diminished, but did not abrogate, hsp90 binding. Mutation of Lys-32, another conserved basic residue in the binding groove, also blocked hsp90 binding. The TPR domain of PP5 bound specifically to a 12-kDa C-terminal fragment of hsp90. This binding was reduced by mutation of acidic residues in the hsp90 fragment. These data suggest conservation, among hsp90-binding TPR proteins, of a binding groove containing basic residues that interact with acidic residues near the C terminus of hsp90.

High-molecular-weight FK506-binding proteins are components of heat-shock protein 90 heterocomplexes in wheat germ lysate.

In animal cell lysates the multiprotein heat-shock protein 90 (hsp90)-based chaperone complexes consist of hsp70, hsp40, and p60. These complexes act to convert steroid hormone receptors to their steroid-binding state by assembling them into heterocomplexes with hsp90, p23, and one of several immunophilins. Wheat germ lysate also contains a hsp90-based chaperone system that can assemble the glucocorticoid receptor into a functional heterocomplex with hsp90. However, only two components of the heterocomplex-assembly system, hsp90 and hsp70, have thus far been identified. Recently, purified mammalian p23 preadsorbed with JJ3 antibody-protein A-Sepharose pellets was used to isolate a mammalian p23-wheat hsp90 heterocomplex from wheat germ lysate (J.K. Owens-Grillo, L.F. Stancato, K. Hoffmann, W.B. Pratt, and P. Krishna [1996] Biochemistry 35: 15249-15255). This heterocomplex was found to contain an immunophilin(s) of the FK506-binding class, as judged by binding of the radiolabeled immunosuppressant drug [3H]FK506 to the immune pellets in a specific manner. In the present study we identified the immunophilin components of this heterocomplex as FKBP73 and FKBP77, the two recently described high-molecular-weight FKBPs of wheat. In addition, we present evidence that the two FKBPs bind hsp90 via tetratricopeptide repeat domains. Our results demonstrate that binding of immunophilins to hsp90 via tetratricopeptide repeat domains is a conserved protein interaction in plants. Conservation of this protein-to-protein interaction in both plant and animal cells suggests that it is important for the biological action of the high-molecular-weight immunophilins.

p50(cdc37) binds directly to the catalytic domain of Raf as well as to a site on hsp90 that is topologically adjacent to the tetratricopeptide repeat binding site.

Several protein kinases (e.g. pp60(src), v-Raf) exist in heterocomplexes with hsp90 and a 50-kDa protein that is the mammalian homolog of the yeast cell cycle control protein Cdc37. In contrast, unliganded steroid receptors exist in heterocomplexes with hsp90 and a tetratricopeptide repeat (TPR) domain protein, such as an immunophilin. Although p50(cdc37) and TPR domain proteins bind directly to hsp90, p50(cdc37) is not present in native steroid receptor.hsp90 heterocomplexes. To obtain some insight as to how v-Raf selects predominantly hsp90.p50(cdc37) heterocomplexes, rather than hsp90.TPR protein heterocomplexes, we have examined the binding of p50(cdc37) to hsp90 and to Raf. We show that p50(cdc37) exists in separate hsp90 heterocomplexes from the TPR domain proteins and that intact TPR proteins compete for p50(cdc37) binding to hsp90 but a protein fragment containing a TPR domain does not. This suggests that the binding site for p50(cdc37) lies topologically adjacent to the TPR acceptor site on the surface of hsp90. Also, we show that p50(cdc37) binds directly to v-Raf, with the catalytic domain of Raf being sufficient. We propose that the combination of exclusive binding of p50(cdc37) versus a TPR domain protein to hsp90 plus direct binding of p50(cdc37) to Raf allows the protein kinase to select for the dominant hsp90.p50(cdc37) composition that is observed with a variety of protein kinase heterocomplexes immunoadsorbed from cytosols.

Protein phosphatase 5 is a major component of glucocorticoid receptor.hsp90 complexes with properties of an FK506-binding immunophilin.

Steroid receptors are recovered from hormone-free cells in multiprotein complexes containing hsp90, p23, an immunophilin, and often some hsp70. The immunophilin, which can be of the FK506- or cyclosporin A-binding class, binds to hsp90 via its tetratricopeptide repeat (TPR) domain, and different receptor heterocomplexes exist depending upon which immunophilin occupies the TPR-binding region of hsp90. We have recently reported that a protein serine/threonine phosphatase that is designated PP5 and contains four TPRs binds to hsp90 and is co-purified with the glucocorticoid receptor (GR) (Chen, M.-S., Silverstein, A. M., Pratt, W. B., and Chinkers, M. (1996) J. Biol. Chem. 271, 32315-32320). In this work, we show that PP5 is recovered with both GR that is nuclear and GR that is cytoplasmic in hormone-free cells. Approximately one-half of the GR.hsp90 heterocomplexes in L cell cytosol contains an immunophilin with high affinity FK506 binding activity, such as FKBP51 or FKBP52, and approximately 35% contains PP5. Only a small (but undetermined) fraction of the native GR.hsp90 heterocomplexes contain the cyclosporin A-binding immunophilin CyP-40. PP5, FKBP52, and CyP-40 exist in separate heterocomplexes with hsp90, and competition binding experiments with the PP5 TPR domain suggest that the three proteins occupy a common binding site on hsp90. A 55-residue connecting region between the N-terminal TPR domain of human PP5 and its C-terminal phosphatase domain has 50% amino acid homology and 22% identity with the central portion of the peptidylprolyl isomerase domain of human FKBP52. Of the 9 residues in this portion of FKBP52 involved in high affinity interactions with FK506, 3 residues are retained and 4 have homologous substitutions in PP5. Although immunoadsorbed PP5 did not bind [3H]FK506, we found that both rabbit PP5 in reticulocyte lysate and purified rat PP5 were specifically retained by an FK506 affinity matrix. Thus, we propose that PP5 possesses properties of an immunophilin with low affinity FK506 binding activity and that it determines a major portion of the native GR heterocomplexes in L cell cytosol.

The tetratricopeptide repeat domain of protein phosphatase 5 mediates binding to glucocorticoid receptor heterocomplexes and acts as a dominant negative mutant.

We previously identified a protein-serine phosphatase designated PP5, based on the binding of its tetratricopeptide repeat (TPR) domain to the atrial natriuretic peptide receptor (Chinkers, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11075-11079). We have now identified another protein complex to which PP5 is targeted through its TPR domain. A 90-kDa protein, identified as heat shock protein 90 (hsp90) by immunoblotting, specifically co-immunoprecipitated from COS-7 cell lysates with the FLAG-tagged TPR domain of PP5. hsp90 also co-immunoprecipitated with full-length FLAG-tagged PP5 overexpressed in COS-7 cells and with endogenous PP5 from untransfected COS-7 cells or rat brain. During gel filtration, PP5 and hsp90 comigrated in a high molecular weight complex. Since glucocorticoid receptors (GR) exist as large heterocomplexes containing hsp90 bound to TPR proteins, we hypothesized that PP5 might be associated with these complexes. Consistent with this hypothesis, PP5 specifically co-immunoprecipitated with GR from mouse L cell lysates. To test the functional importance of this TPR-mediated association in living cells, we used a dominant negative PP5 mutant consisting only of its TPR domain. The mutant inhibited GR-mediated transactivation by approximately 70% in transfected CV-1 cells. This is the first evidence that the TPR proteins in steroid receptor heterocomplexes may be required for signaling in vivo.

Identification of sequences mediating guanylyl cyclase dimerization.

Deletion mutagenesis was used to identify sequences required for dimerization and enzymatic activity of the intracellular domain of the membrane guanylyl cyclase, GC-A. The intracellular domain of GC-A contains a protein kinase-like domain near its amino terminus, a guanylyl cyclase catalytic domain near its carboxyl terminus, and, between these domains, a region of unknown function predicted to form an amphipathic alpha-helix. Gel filtration analysis of deletion mutants of the GC-A intracellular domain suggested that a 43 amino acid sequence within the interdomain region was both necessary and sufficient for dimerization and was required for guanylyl cyclase catalytic activity. The ability of this sequence to mediate protein dimerization was confirmed in the yeast two-hybrid system, in which its fusion to the lexA DNA-binding domain and to the VP16 transcriptional activation domain led to their dimerization and consequent activation of a lexA-HIS3 gene. Thus, we have identified sequences responsible for dimerization of the intracellular domain of a guanylyl cyclase and shown that they are required for enzyme activity. Modulation of their interaction may be important in guanylyl cyclase activation.

Identification, characterization, and developmental regulation of a receptor guanylyl cyclase expressed during early stages of Drosophila development.

Membrane forms of guanylyl cyclase are single-transmembrane proteins that are activated by the binding of specific peptide ligands to their extracellular domains. In this report, we describe the identification and characterization of a Drosophila cDNA clone encoding a protein, DrGC-1, with high sequence identity to members of this family of receptor proteins. The protein contains a single, hydrophobic domain predicted to represent a transmembrane segment separating an extracellular domain with significant sequence identity (30%) to sea urchin egg peptide receptors from intracellular domains containing a protein kinase-like domain followed by a region with high sequence identity (65%) to cyclase catalytic domains found in receptor guanylyl cyclases from both vertebrates and invertebrates. In contrast to other members of this family, DrGC-1 is predicted to contain a carboxyl-terminal extension of 430 residues that has no homology to any described protein. Northern analysis indicates that DrGC-1 transcripts are present at variable levels in all stages of development. In situ hybridization demonstrates that high levels of uniformly distributed transcript are present in 0-2-h embryos. Later in embryogenesis (14-18 h), elevated levels of hybridization appear to be preferentially associated with muscle fibers.

Targeting of a distinctive protein-serine phosphatase to the protein kinase-like domain of the atrial natriuretic peptide receptor.

Protein kinase-related domains of unknown function are present in the JAK family of protein tyrosine kinases and in receptor/guanylyl cyclases. I used the yeast two-hybrid system to screen for proteins interacting with the kinase-like domain of the atrial natriuretic peptide (ANP) receptor/guanylyl cyclase. A yeast strain was constructed expressing a fusion of this kinase-like domain to the lexA DNA-binding domain and containing a HIS3 gene under the control of lexA upstream activating sequences. These yeast cells were transformed with a plasmid library of mouse embryo cDNA fragments fused to the VP16 transcriptional activation domain. Cells containing VP16-fusion proteins interacting with the lexA-kinase-like domain fusion protein were selected by growth in the absence of histidine. A partial-length cDNA clone isolated by using this approach encoded a protein that interacted specifically with the ANP-receptor protein kinase-like domain both in yeast cells and in vitro. Tissue-specific expression of a 2.2-kb mRNA hybridizing to this cDNA paralleled the known pattern of ANP-receptor mRNA expression. A full-length cDNA clone isolated from a rat lung library was predicted to encode a 55-kDa protein containing at its amino terminus a targeting domain that binds to the ANP-receptor kinase-like domain and containing at its carboxyl terminus a putative protein-serine phosphatase domain. This protein is a possible candidate for the phosphatase involved in desensitizing the ANP receptor. Targeting of regulatory proteins may be an important function of protein kinase-like domains.

Ligand-independent oligomerization of natriuretic peptide receptors. Identification of heteromeric receptors and a dominant negative mutant.

Activation of many single-transmembrane receptors requires ligand-induced receptor oligomerization. We have examined the oligomerization of the atrial natriuretic peptide receptor, NPR-A, using epitope-tagged receptor in a co-immunoprecipitation assay. Unlike other single-transmembrane receptors, NPR-A oligomerized in a ligand-independent fashion. Extracellular receptor sequences were both necessary and sufficient for oligomer formation. NPR-A was also able to oligomerize with the related natriuretic peptide receptor, NPR-B. A truncated NPR-A lacking most of the cytoplasmic domain blocked activation of the full-length receptor, presumably through formation of an inactive heteromer. These results indicate that oligomerization of this single-transmembrane receptor is important for the transduction of a conformational change across the plasma membrane but are not consistent with models in which natriuretic peptide receptor oligomerization serves merely to bring intracellular domains together.

Adenine nucleotides are required for activation of rat atrial natriuretic peptide receptor/guanylyl cyclase expressed in a baculovirus system.

Atrial natriuretic peptide (ANP) binds to a transmembrane receptor having intrinsic guanylyl cyclase activity; this receptor has been designated GC-A. Binding of ANP to GC-A stimulates its catalytic activity, resulting in increased production of the second messenger, cyclic GMP. Here we show that GC-A can be expressed in insect cells using a recombinant baculovirus and that the expressed protein retained its abilities to bind ANP and to function as an ANP-activated guanylyl cyclase. In addition, GC-A produced in insect cells was absolutely dependent on the presence of adenine nucleotides for activation by ANP. Millimolar concentrations of ATP were required for optimal activation. The relative potencies of various nucleotides for activation was adenosine 5'-O-(thiotriphosphate) greater than ATP greater than ADP, adenosine 5'-(beta, gamma-imino)triphosphate greater than ADP beta S. AMP had no effect. These studies suggest that binding of an adenine nucleotide, most likely to the protein kinase-like domain of GC-A, is absolutely required for ANP activation. Regulation of guanylyl cyclase activation by adenine nucleotides represents a novel mechanism for the modulation of signal transduction, possibly analogous in some respects to the role of guanine nucleotides and G proteins in the regulation of adenylyl cyclase activity.

The protein kinase domain of the ANP receptor is required for signaling.

A plasma membrane form of guanylate cyclase is a cell surface receptor for atrial natriuretic peptide (ANP). In response to ANP binding, the receptor-enzyme produces increased amounts of the second messenger, guanosine 3',5'-monophosphate. Maximal activation of the cyclase requires the presence of adenosine 5'-triphosphate (ATP) or nonhydrolyzable ATP analogs. The intracellular region of the receptor contains at least two domains with homology to other proteins, one possessing sequence similarity to protein kinase catalytic domains, the other to regions of unknown function in a cytoplasmic form of guanylate cyclase and in adenylate cyclase. It is now shown that the protein kinase-like domain functions as a regulatory element and that the second domain possesses catalytic activity. When the kinase-like domain was removed by deletion mutagenesis, the resulting ANP receptor retained guanylate cyclase activity, but this activity was independent of ANP and its stimulation by ATP was markedly reduced. A model for signal transduction is suggested in which binding of ANP to the extracellular domain of its receptor initiates a conformational change in the protein kinase-like domain, resulting in derepression of guanylate cyclase activity.

The guanylate cyclase/receptor family of proteins.

Guanylate cyclase, which catalyzes the formation of cGMP from GTP, exists in both the soluble and particulate fractions of cells. At least two different cellular compartments for the particulate enzyme exist: the plasma membrane and cytoskeleton. The enzyme form found in the soluble fraction is a heterodimer that can be regulated by free radicals and nitrovasodilators, whereas the membrane form exists as a single-chain polypeptide that can be regulated by various peptides. These peptides include resact and speract obtained from eggs and atrial natriuretic peptides (ANP). The species of guanylate cyclase present in cytoskeletal fractions resists solubilization with non-ionic detergents; its structural properties are not yet known. cDNAs encoding the membrane form of guanylate cyclase have been isolated from different tissues and species, and in all cases the DNA sequences predict a protein containing a single transmembrane domain. The carboxyl (intracellular) domain is highly conserved from sea urchins through mammals, whereas the extracellular domain (amino terminus) varies considerably. The predicted amino acid sequences demonstrate that the membrane form of guanylate cyclase is a member of a diverse and complex family of proteins that includes a low molecular weight ANP receptor, protein kinases, and the cytoplasmic form of guanylate cyclase. cDNA encoding a membrane form of the enzyme from mammalian tissues has been expressed in cultured cells, and the expressed guanylate cyclase specifically binds ANP and is activated by ANP. The membrane form of guanylate cyclase, then, serves as a cell surface receptor, representing the first recognized protein to directly catalyze formation of a low molecular weight second messenger in response to ligand binding.