Direct interaction of Gβγ with a C-terminal Gβγ-binding domain of the Ca2+ channel α1 subunit is responsible for channel inhibition by G protein-coupled receptors

Several classes of voltage-gated Ca2+ channels (VGCCs) are inhibited by G proteins activated by receptors for neurotransmitters and neuromodulatory peptides. Evidence has accumulated to indicate that for non-L-type Ca2+ channels the executing arm of the activated G protein is its βγ dimer (Gβγ). We report below the existence of two Gβγ-binding sites on the A-, B-, and E-type α1 subunits that form non-L-type Ca2+ channels. One, reported previously, is in loop 1 connecting transmembrane domains I and II. The second is located approximately in the middle of the ca. 600-aa-long C-terminal tails. Both Gβγ-binding regions also bind the Ca2+ channel β subunit (CCβ), which, when overexpressed, interferes with inhibition by activated G proteins. Replacement in α1E of loop 1 with that of the G protein-insensitive and Gβγ-binding-negative loop 1 of α1C did not abolish inhibition by G proteins, but the exchange of the α1E C terminus with that of α1C did. This and properties of α1E C-terminal truncations indicated that the Gβγ-binding site mediating the inhibition of Ca2+ channel activity is the one in the C terminus. Binding of Gβγ to this site was inhibited by an α1-binding domain of CCβ, thus providing an explanation for the functional antagonism existing between CCβ and G protein inhibition. The data do not support proposals that Gβγ inhibits α1 function by interacting with the site located in the loop I–II linker. These results define the molecular mechanism by which presynaptic G protein-coupled receptors inhibit neurotransmission.

Interaction between replication protein A and p53 is disrupted after UV damage in a DNA repair-dependent manner

Replication protein A (RPA) is required for both DNA replication and nucleotide excision repair. Previous studies have shown that RPA interacts with the tumor suppressor p53. Herein, we have mapped a 20-amino acid region in the N-terminal part of p53 that is essential for its binding to RPA. This region is distinct from the minimal activation domain of p53 previously identified. We also demonstrate that UV radiation of cells greatly reduces the ability of RPA to bind to p53. Interestingly, damage-induced hyperphosphorylated RPA does not associate with p53. Furthermore, down-regulation of the RPA/p53 interaction is dependent upon the capability of cells to perform global genome repair. On the basis of these data, we propose that RPA may participate in the coordination of DNA repair with the p53-dependent checkpoint control by sensing UV damage and releasing p53 to activate its downstream targets.

Interaction of the copper chaperone HAH1 with the Wilson disease protein is essential for copper homeostasis

The delivery of copper to specific sites within the cell is mediated by distinct intracellular carrier proteins termed copper chaperones. Previous studies in Saccharomyces cerevisiae suggested that the human copper chaperone HAH1 may play a role in copper trafficking to the secretory pathway of the cell. In this current study, HAH1 was detected in lysates from multiple human cell lines and tissues as a single-chain protein distributed throughout the cytoplasm and nucleus. Studies with a glutathione S-transferase-HAH1 fusion protein demonstrated direct protein–protein interaction between HAH1 and the Wilson disease protein, which required the cysteine copper ligands in the amino terminus of HAH1. Consistent with these in vitro observations, coimmunoprecipitation experiments revealed that HAH1 interacts with both the Wilson and Menkes proteins in vivo and that this interaction depends on available copper. When these studies were repeated utilizing three disease-associated mutations in the amino terminus of the Wilson protein, a marked diminution in HAH1 interaction was observed, suggesting that impaired copper delivery by HAH1 constitutes the molecular basis of Wilson disease in patients harboring these mutations. Taken together, these data provide a mechanism for the function of HAH1 as a copper chaperone in mammalian cells and demonstrate that this protein is essential for copper homeostasis.

Apoprotein B100 has a prolonged interaction with the translocon during which its lipidation and translocation change from dependence on the microsomal triglyceride transfer protein to independence

When lipid synthesis is limited in HepG2 cells, apoprotein B100 (apoB100) is not secreted but rapidly degraded by the ubiquitin-proteasome pathway. To investigate apoB100 biosynthesis and secretion further, the physical and functional states of apoB100 destined for either degradation or lipoprotein assembly were studied under conditions in which lipid synthesis, proteasomal activity, and microsomal triglyceride transfer protein (MTP) lipid-transfer activity were varied. Cells were pretreated with a proteasomal inhibitor (which remained with the cells throughout the experiment) and radiolabeled for 15 min. During the chase period, labeled apoB100 remained associated with the microsomes. Furthermore, by crosslinking sec61β to apoB100, we showed that apoB100 remained close to the translocon at the same time apoB100–ubiquitin conjugates could be detected. When lipid synthesis and lipoprotein assembly/secretion were stimulated by adding oleic acid (OA) to the chase medium, apoB100 was deubiquitinated, and its interaction with sec61β was disrupted, signifying completion of translocation concomitant with the formation of lipoprotein particles. MTP participates in apoB100 translocation and lipoprotein assembly. In the presence of OA, when MTP lipid-transfer activity was inhibited at the end of pulse labeling, apoB100 secretion was abolished. In contrast, when the labeled apoB100 was allowed to accumulate in the cell for 60 min before adding OA and the inhibitor, apoB100 lipidation and secretion were no longer impaired. Overall, the data imply that during most of its association with the endoplasmic reticulum, apoB100 is close to or within the translocon and is accessible to both the ubiquitin-proteasome and lipoprotein-assembly pathways. Furthermore, MTP lipid-transfer activity seems to be necessary only for early translocation and lipidation events.

Pontin52, an interaction partner of β-catenin, binds to the TATA box binding protein

β-catenin, the vertebrate homolog of the Drosophila Armadillo protein, has been shown to have dual cellular functions, as a component of both the cadherin-catenin cell adhesion complex and the Wnt signaling pathway. At Wnt signaling, β-catenin becomes stabilized in the cytoplasm and subsequently available for interaction with transcription factors of the lymphocyte enhancer factor-1/T-cell factor family, resulting in a nuclear localization of β-catenin. Although β-catenin does not bind DNA directly, its carboxyl- and amino-terminal regions exhibit a transactivating activity still not well understood molecularly. Here we report the identification of an interaction partner of β-catenin, a nuclear protein designated Pontin52. Pontin52 binds β-catenin in the region of Armadillo repeats 2–5 and, more importantly, also binds the TATA box binding protein. We provide evidence for an in vivo multiprotein complex composed of Pontin52, β-catenin, and lymphocyte enhancer factor-1/T-cell factor. Our results suggest involvement of Pontin52 in the nuclear function of β-catenin.

Structural details of an interaction between cardiolipin and an integral membrane protein

Anionic lipids play a variety of key roles in biomembrane function, including providing the immediate environment for the integral membrane proteins that catalyze photosynthetic and respiratory energy transduction. Little is known about the molecular basis of these lipid–protein interactions. In this study, x-ray crystallography has been used to examine the structural details of an interaction between cardiolipin and the photoreaction center, a key light-driven electron transfer protein complex found in the cytoplasmic membrane of photosynthetic bacteria. X-ray diffraction data collected over the resolution range 30.0–2.1 Å show that binding of the lipid to the protein involves a combination of ionic interactions between the protein and the lipid headgroup and van der Waals interactions between the lipid tails and the electroneutral intramembrane surface of the protein. In the headgroup region, ionic interactions involve polar groups of a number of residues, the protein backbone, and bound water molecules. The lipid tails sit along largely hydrophobic grooves in the irregular surface of the protein. In addition to providing new information on the immediate lipid environment of a key integral membrane protein, this study provides the first, to our knowledge, high-resolution x-ray crystal structure for cardiolipin. The possible significance of this interaction between an integral membrane protein and cardiolipin is considered.

Interaction of the Ras-related protein associated with diabetes Rad and the putative tumor metastasis suppressor NM23 provides a novel mechanism of GTPase regulation

Rad is the prototypic member of a new class of Ras-related GTPases. Purification of the GTPase-activating protein (GAP) for Rad revealed nm23, a putative tumor metastasis suppressor and a development gene in Drosophila. Antibodies against nm23 depleted Rad-GAP activity from human skeletal muscle cytosol, and bacterially expressed nm23 reconstituted the activity. The GAP activity of nm23 was specific for Rad, was absent with the S105N putative dominant negative mutant of Rad, and was reduced with mutations of nm23. In the presence of ATP, GDP⋅Rad was also reconverted to GTP⋅Rad by the nucleoside diphosphate (NDP) kinase activity of nm23. Simultaneously, Rad regulated nm23 by enhancing its NDP kinase activity and decreasing its autophosphorylation. Melanoma cells transfected with wild-type Rad, but not the S105N-Rad, showed enhanced DNA synthesis in response to serum; this effect was lost with coexpression of nm23. Thus, the interaction of nm23 and Rad provides a potential novel mechanism for bidirectional, bimolecular regulation in which nm23 stimulates both GTP hydrolysis and GTP loading of Rad whereas Rad regulates activity of nm23. This interaction may play important roles in the effects of Rad on glucose metabolism and the effects of nm23 on tumor metastasis and developmental regulation.

The Putative “Switch 2” Domain of the Ras-related GTPase, Rab1B, Plays an Essential Role in the Interaction with Rab Escort Protein

Posttranslational modification of Rab proteins by geranylgeranyltransferase type II requires that they first bind to Rab escort protein (REP). Following prenylation, REP is postulated to accompany the modified GTPase to its specific target membrane. REP binds preferentially to Rab proteins that are in the GDP state, but the specific structural domains involved in this interaction have not been defined. In p21 Ras, the α2 helix of the Switch 2 domain undergoes a major conformational change upon GTP hydrolysis. Therefore, we hypothesized that the corresponding region in Rab1B might play a key role in the interaction with REP. Introduction of amino acid substitutions (I73N, Y78D, and A81D) into the putative α2 helix of Myc-tagged Rab1B prevented prenylation of the recombinant protein in cell-free assays, whereas mutations in the α3 and α4 helices did not. Additionally, upon transient expression in transfected HEK-293 cells, the Myc-Rab1B α2 helix mutants were not efficiently prenylated as determined by incorporation of [3H]mevalonate. Metabolic labeling studies using [32P]orthophosphate indicated that the poor prenylation of the Rab1B α2 helix mutants was not directly correlated with major disruptions in guanine nucleotide binding or intrinsic GTPase activity. Finally, gel filtration analysis of cytosolic fractions from 293 cells that were coexpressing T7 epitope-tagged REP with various Myc-Rab1B constructs revealed that mutations in the α2 helix of Rab1B prevented the association of nascent (i.e., nonprenylated) Rab1B with REP. These data indicate that the Switch 2 domain of Rab1B is a key structural determinant for REP interaction and that nucleotide-dependent conformational changes in this region are largely responsible for the selective interaction of REP with the GDP-bound form of the Rab substrate.

Nuclear Import of Sterol Regulatory Element–binding Protein-2, a Basic Helix-Loop-Helix–Leucine Zipper (bHLH-Zip)–containing Transcription Factor, Occurs through the Direct Interaction of Importin β with HLH-Zip

The sterol regulatory element–binding protein-2 (SREBP-2) is produced as a large precursor molecule attached to the endoplasmic reticulum membrane. In response to the sterol depletion, the N-terminal segment of the precursor, which contains a basic helix-loop-helix–leucine zipper domain, is released by two sequential cleavages and is translocated to the nucleus, where it activates the transcription of target genes. The data herein show that released SREBP-2 uses a distinct nuclear transport pathway, which is mediated by importin β. The mature form of SREBP-2 is actively transported into the nucleus when injected into the cell cytoplasm. SREBP-2 binds directly to importin β in the absence of importin α. Ran-GTP but not Ran-GDP causes the dissociation of the SREBP-2–importin β complex. G19VRan-GTP inhibits the nuclear import of SREBP-2 in living cells. In the permeabilized cell in vitro transport system, nuclear import of SREBP-2 is reconstituted only by importin β in conjunction with Ran and its interacting protein p10/NTF2. We further demonstrate that the helix-loop-helix–leucine zipper motif of SREBP-2 contains a novel type of nuclear localization signal, which binds directly to importin β.

High-Copy Suppressor Analysis Reveals a Physical Interaction between Sec34p and Sec35p, a Protein Implicated in Vesicle Docking

A temperature-sensitive mutant, sec34-2, is defective in the late stages of endoplasmic reticulum (ER)-to-Golgi transport. A high-copy suppressor screen that uses the sec34-2 mutant has resulted in the identification of the SEC34 structural gene and a novel gene called GRP1. GRP1 encodes a previously unidentified hydrophilic yeast protein related to the mammalian Golgi protein golgin-160. Although GRP1 is not essential for growth, the grp1Δ mutation displays synthetic lethal interactions with several mutations that result in ER accumulation and a block in the late stages of ER-to-Golgi transport, but not with those that block the budding of vesicles from the ER. Our findings suggest that Grp1p may facilitate membrane traffic indirectly, possibly by maintaining Golgi function. In an effort to identify genes whose products physically interact with Sec34p, we also tested the ability of overexpressed SEC34 to suppress known secretory mutations that block vesicular traffic between the ER and the Golgi. This screen revealed that SEC34 specifically suppresses sec35-1. SEC34 encodes a hydrophilic protein of ∼100 kDa. Like Sec35p, which has been implicated in the tethering of ER-derived vesicles to the Golgi, Sec34p is predominantly soluble. Sec34p and Sec35p stably associate with each other to form a multiprotein complex of ∼480 kDa. These data indicate that Sec34p acts in conjunction with Sec35p to mediate a common step in vesicular traffic.

TGF-β–induced Phosphorylation of Smad3 Regulates Its Interaction with Coactivator p300/CREB-binding Protein

Smads are intermediate effector proteins that transduce the TGF-β signal from the plasma membrane to the nucleus, where they participate in transactivation of downstream target genes. We have shown previously that coactivators p300/CREB-binding protein are involved in TGF-β–mediated transactivation of two Cdk inhibitor genes, p21 and p15. Here we examined the possibility that Smads function to regulate transcription by directly interacting with p300/CREB-binding protein. We show that Smad3 can interact with a C-terminal fragment of p300 in a temporal and phosphorylation-dependent manner. TGF-β–mediated phosphorylation of Smad3 potentiates the association between Smad3 and p300, likely because of an induced conformational change that removes the autoinhibitory interaction between the N- and C-terminal domains of Smad3. Consistent with a role for p300 in the transcription regulation of multiple genes, overexpression of a Smad3 C-terminal fragment causes a general squelching effect on multiple TGF-β–responsive reporter constructs. The adenoviral oncoprotein E1A can partially block Smad-dependent transcriptional activation by directly competing for binding to p300. Taken together, these findings define a new role for phosphorylation of Smad3: in addition to facilitating complex formation with Smad4 and promoting nuclear translocation, the phosphorylation-induced conformational change of Smad3 modulates its interaction with coactivators, leading to transcriptional regulation.

14–3-3 Inhibits the Dictyostelium Myosin II Heavy-Chain-specific Protein Kinase C Activity by a Direct Interaction: Identification of the 14–3-3 Binding Domain

Myosin II heavy chain (MHC) specific protein kinase C (MHC-PKC), isolated from Dictyostelium discoideum, regulates myosin II assembly and localization in response to the chemoattractant cyclic AMP. Immunoprecipitation of MHC-PKC revealed that it resides as a complex with several proteins. We show herein that one of these proteins is a homologue of the 14–3-3 protein (Dd14–3-3). This protein has recently been implicated in the regulation of intracellular signaling pathways via its interaction with several signaling proteins, such as PKC and Raf-1 kinase. We demonstrate that the mammalian 14–3-3 ζ isoform inhibits the MHC-PKC activity in vitro and that this inhibition is carried out by a direct interaction between the two proteins. Furthermore, we found that the cytosolic MHC-PKC, which is inactive, formed a complex with Dd14–3-3 in the cytosol in a cyclic AMP-dependent manner, whereas the membrane-bound active MHC-PKC was not found in a complex with Dd14–3-3. This suggests that Dd14–3-3 inhibits the MHC-PKC in vivo. We further show that MHC-PKC binds Dd14–3-3 as well as 14–3-3ζ through its C1 domain, and the interaction between these two proteins does not involve a peptide containing phosphoserine as was found for Raf-1 kinase. Our experiments thus show an in vivo function for a member of the 14–3-3 family and demonstrate that MHC-PKC interacts directly with Dd14–3-3 and 14–3-3ζ through its C1 domain both in vitro and in vivo, resulting in the inhibition of the kinase.

A green fluorescent protein-reporter mammalian two-hybrid system with extrachromosomal maintenance of a prey expression plasmid: Application to interaction screening

An improved mammalian two-hybrid system designed for interaction trap screening is described in this paper. CV-1/EBNA-1 monkey kidney epithelial cells expressing Epstein–Barr virus nuclear antigen 1 (EBNA-1) were stably transfected with a reporter plasmid for GAL4-dependent expression of the green fluorescent protein (GFP). A resulting clone, GB133, expressed GFP strongly when transfected transiently with transcriptional activators fused to GAL4 DNA-binding domain with minimal background GFP expression. GB133 cells maintained plasmids containing the OriP Epstein–Barr virus replication origin that directs replication of plasmids in mammalian cells in the presence of the EBNA-1 protein. GB133 cells transfected stably with a model bait expressed GFP when further transfected transiently with an expression plasmid for a known positive prey. When the bait-expressing GB133 cells were transfected transiently with an OriP-containing expression plasmid for the positive prey together with excess amounts of empty vector, cells that received the positive prey were readily identified by green fluorescence in cell culture and eventually formed green fluorescent microcolonies, because the prey plasmid was maintained by the EBNA-1/Ori-P system. The green fluorescent microcolonies were harvested directly from the culture dishes under a fluorescence microscope, and total DNA was then prepared. Prey-encoding cDNA was recovered by PCR using primers annealing to the vector sequences flanking the insert-cloning site. This system should be useful in mammalian cells for efficient screening of cDNA libraries by two-hybrid interaction.

Distinct roles of the NH2- and COOH-terminal domains of the protein inhibitor of activated signal transducer and activator of transcription (STAT) 1 (PIAS1) in cytokine-induced PIAS1–Stat1 interaction

STATs are activated by tyrosine phosphorylation on cytokine stimulation. A tyrosine-phosphorylated STAT forms a functional dimer through reciprocal Src homology 2 domain (SH2)–phosphotyrosyl peptide interactions. IFN treatment induces the association of PIAS1 and Stat1, which results in the inhibition of Stat1-mediated gene activation. The molecular basis of the cytokine-dependent PIAS1–Stat1 interaction has not been understood. We report here that a region near the COOH terminus of PIAS1 (amino acids 392–541) directly interacts with the NH2-terminal domain of Stat1 (amino acids 1–191). A mutant PIAS1 lacking the Stat1-interacting domain failed to inhibit Stat1-mediated gene activation. By using a modified yeast two-hybrid assay, we demonstrated that PIAS1 specifically interacts with the Stat1 dimer, but not tyrosine-phosphorylated or -unphosphorylated Stat1 monomer. In addition, whereas the NH2-terminal region of PIAS1 does not interact with Stat1, it serves as a modulatory domain by preventing the interaction of the COOH-terminal domain of PIAS1 with the Stat1 monomer. Thus, the cytokine-induced PIAS1–Stat1 interaction is mediated through the specific recognition of the dimeric form of Stat1 by PIAS1.

Mycoparasitic interaction relieves binding of the Cre1 carbon catabolite repressor protein to promoter sequences of the ech42 (endochitinase-encoding) gene in Trichoderma harzianum

The fungus Trichoderma harzianum is a potent mycoparasite of various plant pathogenic fungi. We have studied the molecular regulation of mycoparasitism in the host/mycoparasite system Botrytis cinerea/T. harzianum. Protein extracts, prepared from various stages of mycoparasitism, were used in electrophoretic mobility-shift assays (EMSAs) with two promoter fragments of the ech-42 (42-kDa endochitinase-encoding) gene of T. harzianum. This gene was chosen as a model because its expression is triggered during mycoparasitic interaction [Carsolio, C., Gutierrez, A., Jimenez, B., van Montagu, M. & Herrera-Estrella, A. (1994) Proc. Natl. Acad. Sci. USA 91, 10903–10907]. All cell-free extracts formed high-molecular weight protein–DNA complexes, but those obtained from mycelia activated for mycoparasitic attack formed a complex with greater mobility. Competition experiments, using oligonucleotides containing functional and nonfunctional consensus sites for binding of the carbon catabolite repressor Cre1, provided evidence that the complex from nonmycoparasitic mycelia involves the binding of Cre1 to both fragments of the ech-42 promoter. The presence of two and three consensus sites for binding of Cre1 in the two ech-42 promoter fragments used is consistent with these findings. In contrast, the formation of the protein–DNA complex from mycoparasitic mycelia is unaffected by the addition of the competing oligonucleotides and hence does not involve Cre1. Addition of equal amounts of protein of cell-free extracts from nonmycoparasitic mycelia converted the mycoparasitic DNA–protein complex into the nonmycoparasitic complex. The addition of the purified Cre1::glutathione S-transferase protein to mycoparasitic cell-free extracts produced the same effect. These findings suggest that ech-42 expression in T. harzianum is regulated by (i) binding of Cre1 to two single sites in the ech-42 promoter, (ii) binding of a “mycoparasitic” protein–protein complex to the ech-42 promoter in vicinity of the Cre1 binding sites, and (iii) functional inactivation of Cre1 upon mycoparasitic interaction to enable the formation of the mycoparasitic protein–DNA complex.