PKA signaling drives mammary tumorigenesis through Src.

Protein kinase A (PKA) hyperactivation causes hereditary endocrine neoplasias; however, its role in sporadic epithelial cancers is unknown. Here, we show that heightened PKA activity in the mammary epithelium generates tumors. Mammary-restricted biallelic ablation of Prkar1a, which encodes for the critical type-I PKA regulatory subunit, induced spontaneous breast tumors characterized by enhanced type-II PKA activity. Downstream of this, Src phosphorylation occurs at residues serine-17 and tyrosine-416 and mammary cell transformation is driven through a mechanism involving Src signaling. The phenotypic consequences of these alterations consisted of increased cell proliferation and, accordingly, expansion of both luminal and basal epithelial cell populations. In human breast cancer, low PRKAR1A/high SRC expression defines basal-like and HER2 breast tumors associated with poor clinical outcome. Together, the results of this study define a novel molecular mechanism altered in breast carcinogenesis and highlight the potential strategy of inhibiting SRC signaling in treating this cancer subtype in humans.

In-depth mutational analysis of the promyelocytic leukemia zinc finger BTB/POZ domain reveals motifs and residues required for biological and transcriptional functions.

The promyelocytic leukemia zinc finger (PLZF) protein is a transcription factor disrupted in patients with t(11;17)(q23;q21)-associated acute promyelocytic leukemia. PLZF contains an N-terminal BTB/POZ domain which is required for dimerization, transcriptional repression, formation of high-molecular-weight DNA-protein complexes, nuclear sublocalization, and growth suppression. X-ray crystallographic data show that the PLZF BTB/POZ domain forms an obligate homodimer via an extensive interface. In addition, the dimer possesses several highly conserved features, including a charged pocket, a hydrophobic monomer core, an exposed hydrophobic surface on the floor of the dimer, and two negatively charged surface patches. To determine the role of these structures, mutational analysis of the BTB/POZ domain was performed. We found that point mutations in conserved residues that disrupt the dimer interface or the monomer core result in a misfolded nonfunctional protein. Mutation of key residues from the exposed hydrophobic surface suggests that these are also important for the stability of PLZF complexes. The integrity of the charged-pocket region was crucial for proper folding of the BTB/POZ domain. In addition, the pocket was critical for the ability of the BTB/POZ domain to repress transcription. Alteration of charged-pocket residue arginine 49 to a glutamine (mutant R49Q) yields a domain that can still dimerize but activates rather than represses transcription. In the context of full-length PLZF, a properly folded BTB/POZ domain was required for all PLZF functions. However, PLZF with the single pocket mutation R49Q repressed transcription, while the double mutant D35N/R49Q could not, despite its ability to dimerize. These results indicate that PLZF requires the BTB/POZ domain for dimerization and the charged pocket for transcriptional repression.

Toward the bilayer proteome, electrospray ionization-mass spectrometry of large, intact transmembrane proteins.

Genes encoding membrane proteins comprise a substantial proportion of genomes sequenced to date, but ability to perform structural studies on this portion of the proteome is limited. Electrospray ionization-MS (ESI-MS) of an intact protein generates a profile defining the native covalent state of the gene product and its heterogeneity. Here we apply ESI-MS technology with accuracy exceeding 0.01% to a hydrophobic membrane protein with 12-transmembrane alpha-helices, the full-length lactose permease from Escherichia coli. Furthermore, ESI-MS is used to titrate reactive thiols with N-ethylmaleimide. Treatment of the native protein solubilized in detergent micelles reveals only two reactive thiols, and both are protected by a substrate analog.

Packed protein bilayers in the 0.90 A resolution structure of a designed alpha helical bundle.

A 12-residue peptide designed to form an alpha-helix and self-associate into an antiparallel 4-alpha-helical bundle yields a 0.9 A crystal structure revealing unanticipated features. The structure was determined by direct phasing with the "Shake-and-Bake" program, and contains four crystallographically distinct 12-mer peptide molecules plus solvent for a total of 479 atoms. The crystal is formed from nearly ideal alpha-helices hydrogen bonded head-to-tail into columns, which in turn pack side-by-side into sheets spanning the width of the crystal. Within each sheet, the alpha-helices run antiparallel and are closely spaced (9-10 A center-to-center). The sheets are more loosely packed against each other (13-14 A between helix centers). Each sheet is amphiphilic: apolar leucine side chains project from one face, charged lysine and glutamate side chains from the other face. The sheets are stacked with two polar faces opposing and two apolar faces opposing. The result is a periodic biomaterial composed of packed protein bilayers, with alternating polar and apolar interfaces. All of the 30 water molecules in the unit cell lie in the polar interface or between the stacked termini of helices. A section through the sheet reveals that the helices packed at the apolar interface resemble the four-alpha-helical bundle of the design, but the helices overhang parts of the adjacent bundles, and the helix crossing angles are less steep than intended (7-11 degrees rather than 18 degrees).

Two-dimensional crystallization of Escherichia coli lactose permease.

A chimeric protein consisting of lactose permease with cytochrome b562 in the middle cytoplasmic loop and six His residues at the C terminus (LacY/L6cytb562/417H6 or "red permease") was overexpressed in Escherichia coli and isolated by nickel affinity chromatography after solubilization with dodecyl-beta,d-maltopyranoside. Red permease was then reconstituted in the presence of phospholipids, yielding densely packed vesicles and well-ordered two-dimensional (2D) crystals as shown by electron microscopy of negatively stained specimens. Single-particle analysis of 16 383 protein particles in densely packed vesicles reveals a 5.4-nm-long trapeziform protein of 4.1 to 5.1 nm width, with a central stain-filled indentation. Depending on reconstitution conditions, trigonal and rectangular crystallographic packing arrangements of these elongated particles assembled into trimers are observed. The best ordered 2D crystals exhibit a rectangular unit cell, of dimensions a = 9.9 nm, b = 17.4 nm, that houses two trimeric complexes. Projection maps calculated to a resolution of 2 nm show that these crystals consist of two layers.

Crystal structure of the BTB domain from PLZF.

The BTB domain (also known as the POZ domain) is an evolutionarily conserved protein-protein interaction motif found at the N terminus of 5-10% of C2H2-type zinc-finger transcription factors, as well as in some actin-associated proteins bearing the kelch motif. Many BTB proteins are transcriptional regulators that mediate gene expression through the control of chromatin conformation. In the human promyelocytic leukemia zinc finger (PLZF) protein, the BTB domain has transcriptional repression activity, directs the protein to a nuclear punctate pattern, and interacts with components of the histone deacetylase complex. The association of the PLZF BTB domain with the histone deacetylase complex provides a mechanism of linking the transcription factor with enzymatic activities that regulate chromatin conformation. The crystal structure of the BTB domain of PLZF was determined at 1.9 A resolution and reveals a tightly intertwined dimer with an extensive hydrophobic interface. Approximately one-quarter of the monomer surface area is involved in the dimer intermolecular contact. These features are typical of obligate homodimers, and we expect the full-length PLZF protein to exist as a branched transcription factor with two C-terminal DNA-binding regions. A surface-exposed groove lined with conserved amino acids is formed at the dimer interface, suggestive of a peptide-binding site. This groove may represent the site of interaction of the PLZF BTB domain with nuclear corepressors or other nuclear proteins.

High-resolution structures of the ligand binding domain of the wild-type bacterial aspartate receptor.

The high-resolution structures of the wild-type periplasmic domain of the bacterial aspartate receptor have been determined in the absence and presence of bound aspartate to 1.85 and 2.2 A resolution, respectively. As we reported earlier, in the refined structure of the complexed form of the crosslinked cysteine mutant receptor, the binding of the aspartate at the first site was mediated through four bridging water molecules while the second site showed an occupant electron density that best fit a sulfate group, which was present in the crystallization solution at high concentration. In the wild-type periplasmic domain structure two aspartate residues are bound per dimer, but with different occupancies. There exists a "strong" aspartate-binding site whose binding is again mediated by four water molecules while the second site contains aspartate whose B-factor is about 10% higher, signifying weaker binding. The interaction between the second, "weaker" aspartate with the three ligand-binding arginine side-chains is slightly different from the first site. The major difference is that there are three water molecules mediating the binding of aspartate at the second site, whereas in the first site there are four bridging water molecules. The fact that aspartate-complexed crystals of the wild-type were grown with a large excess aspartate while the cross-linked crystals were grown with equal molar aspartate may explain the difference in the stoichiometry observed. The conservation of the four bridging water molecules in the strong aspartate site of both the cross-linked and wild-type periplasmic domain may reflect an important binding motif. The periplasmic domain in the apo form is a symmetrical dimer, in which each of the subunits is equivalent, and the two aspartate binding sites are identical. Upon the binding of aspartate, the subunits are no longer symmetrical. The main difference between the aspartate-bound and unbound forms is in a small, rigid-body rotation between the subunits within a dimer. The rotation is similar in both direction and magnitude in the crosslinked and wild-type periplasmic domains. The presence of the second aspartate in the wild-type structure does not make any additional rotation compared to the single-site binding. The conservation of the small angular change in vitro suggests that the inter-subunit rotation may have relevance to the understanding of the mechanism of transmembrane signal transduction in vivo.

Engineering the lac permease for purification and crystallization.

The lactose permease is being used as a model system for the rational redesign of a membrane protein with the goal of increasing the likelihood of crystallization. Various modifications to the protein have been added for the purposes of purification, stability, and potential for crystallization. The addition of six consecutive histidines at the C-terminus of the protein allows for rapid purification by nickel-chelate chromatography, and the insertion of an entire protein domain into one of the inner cytoplasmic loops of the permease gives the resulting protein a larger hydrophilic surface area. The increase in polar surface area makes the fusion protein easier to handle and more likely to crystallize. In particular, the introduction of cytochrome b562 of E. coli into the central hydrophilic domain the lac permease results in a fusion protein with the transport activity of the permease and the visible absorbance spectrum of the cytochrome. The "red permease" is very easy to monitor through the steps of expression, purification, concentration, and crystallization.

The lactose permease meets Frankenstein.

The lactose permease (lac) of Escherichia coli is a paradigm for membrane transport proteins. Encoded by the lacY gene, the permease has been solubilized, purified to homogeneity, reconstituted into phospholipid vesicles and shown to catalyse the coupled translocation of beta-galactosides and H+ with a stoichiometry of unity. Circular dichroism and other spectroscopic approaches demonstrate that the purified permease is about 80% helical. Based on hydropathy analysis of the primary amino-acid sequence, a secondary structure has been proposed in which the protein has 12 hydrophobic domains in alpha-helical conformation that traverse the membrane in zigzag fashion connected by hydrophilic loops. A variety of other approaches are consistent with the model and demonstrate that both the N and C termini are on the inner surface of the membrane, and studies on an extensive series of lac permease/alkaline phosphatase fusion proteins provide exclusive support for the topological predictions of the 12-helix motif. This presentation concentrates on the use of site-directed fluorescence spectroscopy to study structure-function relationships in the permease.

The BTB domain, found primarily in zinc finger proteins, defines an evolutionarily conserved family that includes several developmentally regulated genes in Drosophila.

The Drosophila bric à brac protein and the transcriptional regulators encoded by tramtrack and Broad-Complex contain a highly conserved domain of approximately 115 amino acids, which we have called the BTB domain. We have identified six additional Drosophila genes that encode this domain. Five of these genes are developmentally regulated, and one of them appears to be functionally related to bric à brac. The BTB domain defines a gene family with an estimated 40 members in Drosophila. This domain is found primarily at the N terminus of zinc finger proteins and is evolutionarily conserved from Drosophila to mammals.

Fusion proteins as tools for crystallization: the lactose permease from Escherichia coli.

A novel strategy is presented for the crystallization of membrane proteins or other proteins with low solubility and/or stability. The method is illustrated with the lactose permease from Escherichia coli, in which a fusion is constructed between the permease and a 'carrier' protein. The carrier is a soluble, stable protein with its C and N termini close together in space at the surface of the protein, so that the carrier can be introduced into an internal position of the target protein. The carrier is chosen with convenient spectral or enzymatic properties, making the fusion protein easier to handle than the native molecule. Data are presented for the successful construction, expression and purification of a fusion product between lactose permease and cytochrome b(562) from E. coli. The lactose transport activity of the fusion protein is similar to that of wild-type lactose permease, and the fusion product has an absorption spectrum in the visible range which is essentially identical to that of cytochrome b(562). The fusion protein has a higher proportional polar surface area than wild-type permease, and should have better possibilities of forming the strong directional intermolecular contacts required of a crystal lattice.

What's new with lactose permease.

The lactose permease of Escherichia coli is a paradigm for polytopic membrane transport proteins that transduce free energy stored in an electrochemical ion gradient into work in the form of a concentration gradient. Although the permease consists of 12 hydrophobic transmembrane domains in probable alpha-helical conformation that traverse the membrane in zigzag fashion connected by hydrophilic "loops", little information is available regarding the folded tertiary structure of the molecule. In a recent approach site-directed fluorescence labeling is being used to study proximity relationships in lactose permease. The experiments are based upon site-directed pyrene labeling of combinations of paired Cys replacements in a mutant devoid of Cys residues. Since pyrene exhibits excimer fluorescence if two molecules are within about 3.5A, the proximity between paired labeled residues can be determined. The results demonstrate that putative helices VIII and IX are close to helix X. Taken together with other findings indicating that helix VII is close to helices X and XI, the data lead to a model that describes the packing of helices VII to XI.

Use of site-directed fluorescence labeling to study proximity relationships in the lactose permease of Escherichia coli.

The lactose permease of Escherichia coli is a paradigm for polytopic membrane transport proteins that transduce free energy stored in an electrochemical ion gradient into work in the form of a concentration gradient. Although the permease consists of 12 hydrophobic transmembrane domains in probable alpha-helical conformation that traverse the membrane in zigzag fashion connected by hydrophilic "loops", little information is available regarding the folded tertiary structure of the molecule. In this paper, we describe an approach to studying proximity relationships in lactose permease that is based upon site-directed pyrene labeling of combinations of paired Cys replacements in a mutant devoid of Cys residues. Since pyrene exhibits excimer fluorescence if two molecules are within about 3.5 A, the proximity between paired labeled residues can be determined. The results demonstrate that putative helices VIII and IX are close to helix X. Taken together with other findings indicating that helix VII is close to helices X and XI, the data lead to a model that describes the packing of helices VII-XI.

Properties and purification of an active biotinylated lactose permease from Escherichia coli.

A simplified approach for purification of functional lactose permease from Escherichia coli is described that is based on the construction of chimeras between the permease and a 100-amino acid residue polypeptide containing the biotin acceptor domain from the oxaloacetate decarboxylase of Klebsiella pneumoniae [Cronan, J. E., Jr. (1990) J. Biol. Chem. 265, 10327-10333]. Chimeras were constructed with a factor Xa protease site and the biotin acceptor domain in the middle cytoplasmic loop (loop 6) or at the C terminus of the permease. Each construct catalyzes active lactose transport in cells and right-side-out membrane vesicles. Moreover, the constructs are biotinylated in vivo, and in both chimeras, the factor Xa protease site is accessible from the cytoplasmic surface of the membrane. Both biotinylated permeases bind selectively to immobilized monomeric avidin and are eluted with free biotin in a high state of purity, and the loop 6 chimera catalyzes active transport after reconstitution into proteoliposomes. The methodology described should be applicable to other membrane proteins.

Refined structures of the ligand-binding domain of the aspartate receptor from Salmonella typhimurium.

The aspartate receptor is a transmembrane-signalling protein that mediates chemotaxis behaviour in bacteria. Aspartate receptors in Salmonella typhimurium and Escherichia coli exist as dimers of two subunits in the presence as well as in the absence of aspartate. We have previously reported the three-dimensional structures of the external ligand-binding domain of the S. typhimurium aspartate receptor with and without bound aspartate. The external or periplasmic region of the aspartate receptor is a dimer of four-alpha-helical bundle subunits; a single aspartate molecule binds to one of two sites residing at the subunit interface, increasing the affinity of the subunits for one another. Here we report the results of a detailed analysis of the aspartate receptor ligand-binding domain structure (residues 25 to 188). The dimer interface between the twofold related subunits consists primarily of contacts mediated by the side-chains of the N-terminal helix of each four-alpha-helical bundle subunit. The N-terminal helices pack approximately 20 degrees from parallel as an approximate coiled-coil super-secondary structure. We have refined aspartate receptor ligand-binding domain structures in the presence and in the absence of a bound aromatic compound, 1,10-phenanthroline, to 2.2 A and 2.3 A resolution, respectively, as well as crystal structures in the presence of specifically bound Au(I), Hg(II) and Pt(IV) complex ions at 2.4 A, 3.0 A and 3.3 A resolution, respectively. The possible biological relevance of the aromatic ligand-binding site and the metal ion-binding sites is discussed. The dimer of four-alpha-helical bundle subunits composing the periplasmic region of the S. typhimurium aspartate receptor provides a basis for understanding the results of mutational analyses performed on related chemotaxis transmembrane receptors. The crystal structure analysis provides an explanation for the way in which mutations in the E. coli aspartate receptor affect its binding to the periplasmic maltose-binding protein and how mutations in the more distantly related E. coli Trg chemotaxis receptor affect its binding to the periplasmic ribose and glucose-galactose binding proteins.

A cDNA that suppresses MPP+ toxicity encodes a vesicular amine transporter.

Classical neurotransmitters are transported into synaptic vesicles so that their release can be regulated by neural activity. In addition, the vesicular transport of biogenic amines modulates susceptibility to N-methyl-4-phenylpyridinium (MPP+), the active metabolite of the neurotoxin N-methyl-1,2,3,6-tetrahydropyridine that produces a model of Parkinson's disease. Taking advantage of selection in MPP+, we have used gene transfer followed by plasmid rescue to identify a cDNA clone that encodes a vesicular amine transporter. The sequence predicts a novel mammalian protein with 12 transmembrane domains and homology to a class of bacterial drug resistance transporters. We have detected messenger RNA transcripts for this transporter only in the adrenal gland. Monoamine cell populations in the brain stem express a distinct but highly related protein.

X-ray crystal structures of transforming p21 ras mutants suggest a transition-state stabilization mechanism for GTP hydrolysis.

RAS genes isolated from human tumors often have mutations at positions corresponding to amino acid 12 or 61 of the encoded protein (p21), while retroviral ras-encoded p21 contains substitutions at both positions 12 and 59. These mutant proteins are deficient in their GTP hydrolysis activity, and this loss of activity is linked to their transforming potential. The crystal structures of the mutant proteins are presented here as either GDP-bound or GTP-analogue-bound complexes. Based on these structures, a mechanism for the p21 GTPase reaction is proposed that is consistent with the observed structural and biochemical data. The central feature of this mechanism is a specific stabilization complex formed between the Gln-61 side-chain and the pentavalent gamma-phosphate of the GTP transition state. Amino acids other than glutamine at position 61 cannot stabilize the transition state, and amino acids larger than glycine at position 12 would interfere with the transition-state complex. Thr-59 disrupts the normal position of residue 61, thus preventing its participation in the transition-state complex.

Three-dimensional structures of the ligand-binding domain of the bacterial aspartate receptor with and without a ligand.

The three-dimensional structure of an active, disulfide cross-linked dimer of the ligand-binding domain of the Salmonella typhimurium aspartate receptor and that of an aspartate complex have been determined by x-ray crystallographic methods at 2.4 and 2.0 angstrom (A) resolution, respectively. A single subunit is a four-alpha-helix bundle with two long amino-terminal and carboxyl-terminal helices and two shorter helices that form a cylinder 20 A in diameter and more than 70 A long. The two subunits in the disulfide-bonded dimer are related by a crystallographic twofold axis in the apo structure, but by a noncrystallographic twofold axis in the aspartate complex structure. The latter structure reveals that the ligand binding site is located more than 60 A from the presumed membrane surface and is at the interface of the two subunits. Aspartate binds between two alpha helices from one subunit and one alpha helix from the other in a highly charged pocket formed by three arginines. The comparison of the apo and aspartate complex structures shows only small structural changes in the individual subunits, except for one loop region that is disordered, but the subunits appear to change orientation relative to each other. The structures of the two forms of this protein provide a step toward understanding the mechanisms of transmembrane signaling.

The structure of B-helical C-G-A-T-C-G-A-T-C-G and comparison with C-C-A-A-C-G-T-T-G-G. The effect of base pair reversals.

The crystal structure of the DNA decamer C-G-A-T-C-G-A-T-C-G has been solved to a resolution of 1.5 A, with a final R-factor of 16.1% for 5,107 two-sigma reflections. Crystals are orthorhombic space group P2(1)2(1)2(1), with cell dimensions a = 38.93 A, b = 39.63 A, c = 33.30 A, and 10 base pairs/asymmetric unit. The final structure contains 404 DNA atoms, 142 water molecules treated as oxygen atoms, and two Mg(H2O)6(2+) complexes. Decamers stack atop one another to simulate continuous helical columns through the crystal, as with three previously solved monoclinic decamers, but the lateral contacts between columns are quite different in the orthorhombic and monoclinic cells. Narrow and wide regions of the minor groove exhibit a single spine or two ribbons of hydration, respectively, and the minor groove is widest when BII phosphate conformations are opposed diagonally across the groove. Phosphate conformation, in turn, appears to have a base sequence dependence. Twist, rise, cup, and roll are linked as has been observed in the three monoclinic decamers and can be characterized by high or low twist profiles. In all five known decamer crystal structures and eight representative dodecamers, a high twist profile is observed with G-C and G-A steps whereas all other R-R steps are low twist profiles (R = purine). A-T and A-C steps are intermediate in character whereas C-A and C-G exhibit behavior that is strongly influenced by the profiles of the preceding and following steps. When sufficient data are in hand, sequence/structure relationships for all helix parameters probably should be considered in a 4-base pair context. At this stage of limited information the problem is compounded because there are 136 unique 4-base steps x-A-B-y in a double helix as compared with only 10 2-base steps A-B.