The C-terminal domain of p53 recognizes DNA damaged by ionizing radiation.

p53 accumulates after DNA damage and arrests cellular growth. These findings suggest a possible role for p53 in the cellular response to DNA damage. We have previously shown that the C terminus of p53 binds DNA nonspecifically and assembles stable tetramers. In this study, we have utilized purified segments of human and murine p53s to determine which p53 domains may participate in a DNA damage response pathway. We find that the C-terminal 75 amino acids of human or murine p53 are necessary and sufficient for the DNA annealing and strand-transfer activities of p53. In addition, both full-length wild-type p53 and the C-terminal 75 amino acids display an increased binding affinity for DNA damaged by restriction digestion, DNase I treatment, or ionizing radiation. In contrast, the central site-specific DNA-binding domain together with the tetramerization domain does not have these activities. We propose that interactions of the C terminus of p53 with damaged DNA may play a role in the activation of p53 in response to DNA damage.

An alternatively spliced form of the transcription factor Sp1 containing only a single glutamine-rich transactivation domain.

Protein-protein interactions involving specific transactivation domains play a central role in gene transcription and its regulation. The promoter-specific transcription factor Sp1 contains two glutamine-rich transcriptional activation domains (A and B) that mediate direct interactions with the transcription factor TFIID complex associated with RNA polymerase II and synergistic effects involving multiple Sp1 molecules. In the present study, we report the complementary DNA sequence for an alternatively spliced form of mouse Sp1 (mSp1-S) that lacks one of the two glutamine-rich activation regions present in the full-length protein. Corresponding transcripts were identified in mouse tissues and cell lines, and an Sp1-related protein identical in size to that predicted for mSp1-S was detected in mouse nuclear extracts. Cotransfection analysis revealed that mSp1-S lacks appreciable activity at promoters containing a single Sp1 response element but is active when multiple Sp1 sites are present, suggesting synergistic interactions between multiple mSp1-S molecules. The absence of a single glutamine-rich domain does not fully explain the properties of the smaller protein and indicates that additional structural features account for its unique transcriptional activity. The functional implications of this alternatively spliced form of Sp1 are discussed.

Mutational analysis of phytochrome B identifies a small COOH-terminal-domain region critical for regulatory activity.

Overexpression of phytochrome B (phyB) in transgenic Arabidopsis results in enhanced deetiolation in red light. To define domains of phyB functionally important for its regulatory activity, we performed chemical mutagenesis of a phyB-overexpressing line and screened for phenotypic revertants in red light. Four phyB-transgene-linked revertants that retain parental levels of full-length, dimeric, and spectrally normal overexpressed phyB were identified among 101 red-light-specific revertants. All carry single amino acid substitutions in the transgene-encoded phyB that reduce activity by 40- to 1000-fold compared to the nonmutagenized parent. The data indicate that the mutant molecules are fully active in photosignal perception but defective in the regulatory activity responsible for signal transfer to downstream components. All four mutations fall within a 62-residue region in the COOH-terminal domain of phyB, with two independent mutations occurring in a single amino acid, Gly-767. Accumulating evidence indicates that the identified region is a critical determinant in the regulatory function of both phyB and phyA.

Alpha 1(E)-catenin is an actin-binding and -bundling protein mediating the attachment of F-actin to the membrane adhesion complex.

Calcium-dependent homotypic cell-cell adhesion, mediated by molecules such as E-cadherin, guides the establishment of classical epithelial cell polarity and contributes to the control of migration, growth, and differentiation. These actions involve additional proteins, including alpha- and beta-catenin (or plakoglobin) and p120, as well as linkage to the cortical actin cytoskeleton. The molecular basis for these interactions and their hierarchy of interaction remain controversial. We demonstrate a direct interaction between F-actin and alpha (E)-catenin, an activity not shared by either the cytoplasmic domain of E-cadherin or beta-catenin. Sedimentation assays and direct visualization by transmission electron microscopy reveal that alpha 1(E)-catenin binds and bundles F-actin in vitro with micromolar affinity at a catenin/G-actin monomer ratio of approximately 1:7 (mol/mol). Recombinant human beta-catenin can simultaneously bind to the alpha-catenin/actin complex but does not bind actin directly. Recombinant fragments encompassing the amino-terminal 228 residues of alpha 1(E)-catenin or the carboxyl-terminal 447 residues individually bind actin in cosedimentation assays with reduced affinity compared with the full-length protein, and neither fragment bundles actin. Except for similarities to vinculin, neither region contains sequences homologous to established actin-binding proteins. Collectively these data indicate that alpha 1 (E)-catenin is a novel actin-binding and -bundling protein and support a model in which alpha 1(E)-catenin is responsible for organizing and tethering actin filaments at the zones of E-cadherin-mediated cell-cell contact.

Catalytic domain of human immunodeficiency virus type 1 integrase: identification of a soluble mutant by systematic replacement of hydrophobic residues.

The integrase protein of human immunodeficiency virus type 1 is necessary for the stable integration of the viral genome into host DNA. Integrase catalyzes the 3' processing of the linear viral DNA and the subsequent DNA strand transfer reaction that inserts the viral DNA ends into host DNA. Although full-length integrase is required for 3' processing and DNA strand transfer activities in vitro, the central core domain of integrase is sufficient to catalyze an apparent reversal of the DNA strand transfer reaction, termed disintegration. This catalytic core domain, as well as the full-length integrase, has been refractory to structural studies by x-ray crystallography or NMR because of its low solubility and propensity to aggregate. In an attempt to improve protein solubility, we used site-directed mutagenesis to replace hydrophobic residues within the core domain with either alanine or lysine. The single substitution of lysine for phenylalanine at position 185 resulted in a core domain that was highly soluble, monodisperse in solution, and retained catalytic activity. This amino acid change has enabled the catalytic domain of integrase to be crystallized and the structure has been solved to 2.5-A resolution [Dyda, F., Hickman, A. B., Jenkins, T. M., Engelman, A., Craigie, R. & Davies, D. R. (1994) Science 266, 1981-1986]. Systematic replacement of hydrophobic residues may be a useful strategy to improve the solubility of other proteins to facilitate structural and biochemical studies.

Effective ribozyme delivery in plant cells.

Hammerhead ribozyme sequences were incorporated into a tyrosine tRNA (tRNA(Tyr)) and compared with nonembedded molecules. To increase the levels of ribozyme and control antisense in vivo, sequences were expressed from an autonomously replicating vector derived from African cassava mosaic geminivirus. In vitro, the nonembedded ribozyme cleaved more target RNA, encoding chloramphenicol acetyltransferase (CAT), than the tRNA(Tyr) ribozyme. In contrast, the tRNA(Tyr) ribozyme was considerably more effective in vivo than either the nonembedded ribozyme or antisense sequences, reducing CAT activity to < 20% of the control level. A target sequence (CM2), mutated to be noncleavable, showed no reduction in CAT activity in the presence of the tRNA(Tyr) ribozyme beyond that for the antisense construct. The reduction in full-length CAT mRNA and the presence of specific cleavage products demonstrated in vivo cleavage of the target mRNA by the tRNA(Tyr) ribozyme. The high titer of tRNA(Tyr) ribozyme was a result of transcription from the RNA polymerase III promoter and led to the high ribozyme/substrate ratio essential for ribozyme efficiency.

Phosphatidylinositol 3-kinase signals activation of p70 S6 kinase in situ through site-specific p70 phosphorylation.

The p70 S6 kinase is activated by insulin and mitogens through multisite phosphorylation of the enzyme. One set of activating phosphorylations occurs in a putative autoinhibitory domain in the noncatalytic carboxyl-terminal tail. Deletion of this tail yields a variant (p70 delta CT104) that nevertheless continues to be mitogen regulated. Coexpression with a recombinant constitutively active phosphatidylinositol (PI) 3-kinase (EC 2.7.1.137) gives substantial activation of both full-length p70 and p70 delta CT104 but not Rsk. Activation of p70 delta CT104 by PI 3-kinase and inhibition by wortmannin are each accompanied by parallel and selective changes in the phosphorylation of p70 Thr-252. A Thr or Ser at this site, in subdomain VIII of the catalytic domain just amino-terminal to the APE motif, is necessary for p70 40S kinase activity. The inactive ATP-binding site mutant K123M p70 delta CT104 undergoes phosphorylation of Thr-252 in situ but does not undergo direct phosphorylation by the active PI 3-kinase in vitro. PI 3-kinase provides a signal necessary for the mitogen activation of the p70 S6 kinase, which directs the site-specific phosphorylation of Thr-252 in the p70 catalytic domain, through a distinctive signal transduction pathway.

Mouse mammary tumor viruses with functional superantigen genes are selected during in vivo infection.

Mouse mammary tumor virus (MMTV) encodes a superantigen that is important for viral infectivity in vivo. To determine whether superantigen function was required for infection by milk-borne MMTV, we created HYB PRO/Cla transgenic mice. These mice produced a full-length, packaged viral RNA with a frameshift mutation that caused premature termination of the superantigen protein. Young HYB PRO/Cla mice showed no deletion of their cognate V beta 14+ T cells, although they shed virus in their milk. The nontransgenic offspring of the HYB PRO/Cla mice were infected with this virus, since transgene-specific viral transcripts were detected in their mammary glands. Surprisingly, these offspring demonstrated the progressive deletion of V beta 14+ T cells characteristic of exogenous MMTV (C3H) infection. Sequence analysis demonstrated that these newly acquired viruses had reconstituted superantigen open reading frames resulting from recombination between the HYB PRO/Cla and endogenous Mtv-1 proviral RNAs. Thus, there is selection during the infection process for MMTVs with functional superantigen genes.

Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue.

Complexed with its intracellular receptor, FKBP12, the natural product rapamycin inhibits G1 progression of the cell cycle in a variety of mammalian cell lines and in the yeast Saccharomyces cerevisae. Previously, a mammalian protein that directly associates with FKBP12-rapamycin has been identified and its encoding gene has been cloned from both human (designated FRAP) [Brown, E.J., Albers, M.W., Shin, T.B., Ichikawa, K., Keith, C.T., Lane, W.S. & Schreiber, S.L. (1994) Nature (London) 369, 756-758] and rat (designated RAFT) [Sabatini, D.M., Erdjument-Bromage, H., Lui, M., Tempst, P. & Snyder, S.H. (1994) Cell 78, 35-43]. The full-length FRAP is a 289-kDa protein containing a putative phosphatidylinositol kinase domain. Using an in vitro transcription/translation assay method coupled with proteolysis studies, we have identified an 11-kDa FKBP12-rapamycin-binding domain within FRAP. This minimal binding domain lies N-terminal to the kinase domain and spans residues 2025-2114. In addition, we have carried out mutagenesis studies to investigate the role of Ser2035, a potential phosphorylation site for protein kinase C within this domain. We now show that the FRAP Ser2035-->Ala mutant displays similar binding affinity when compared with the wild-type protein, whereas all other mutations at this site, including mimics of phosphoserine, abolish binding, presumably due to either unfavorable steric interactions or induced conformational changes.

Recombinant vesicular stomatitis viruses from DNA.

We assembled a DNA clone containing the 11,161-nt sequence of the prototype rhabdovirus, vesicular stomatitis virus (VSV), such that it could be transcribed by the bacteriophage T7 RNA polymerase to yield a full-length positive-strand RNA complementary to the VSV genome. Expression of this RNA in cells also expressing the VSV nucleocapsid protein and the two VSV polymerase subunits resulted in production of VSV with the growth characteristics of wild-type VSV. Recovery of virus from DNA was verified by (i) the presence of two genetic tags generating restriction sites in DNA derived from the genome, (ii) direct sequencing of the genomic RNA of the recovered virus, and (iii) production of a VSV recombinant in which the glycoprotein was derived from a second serotype. The ability to generate VSV from DNA opens numerous possibilities for the genetic analysis of VSV replication. In addition, because VSV can be grown to very high titers and in large quantities with relative ease, it may be possible to genetically engineer recombinant VSVs displaying foreign antigens. Such modified viruses could be useful as vaccines conferring protection against other viruses.

Folding of a nascent polypeptide chain in vitro: cooperative formation of structure in a protein module.

We have prepared a family of peptide fragments of the 64-residue chymotrypsin inhibitor 2, corresponding to its progressive elongation from the N terminus. The growing polypeptide chain has little tendency to form stable structure until it is largely synthesized, and what structures are formed are nonnative and lack, in particular, the native secondary structural elements of alpha-helix and beta-sheet. These elements then develop as sufficient tertiary interactions are made in the nearly full-length chain. The growth of structure in the small module is highly cooperative and does not result from the hierarchical accretion of substructures.

Analysis of the mouse Splotch-delayed mutation indicates that the Pax-3 paired domain can influence homeodomain DNA-binding activity.

The murine Pax-3 protein contains two DNA-binding domains, a paired domain and a homeodomain, and alterations in the Pax-3 gene are responsible for the neural tube defects observed in the Splotch (Sp) mouse mutant. Of five Sp alleles, Splotch-delayed (Spd) is the only one that encodes a full-length Pax-3 protein, containing a single glycine-to-arginine substitution within the paired domain. To better understand the consequence of this mutation on Pax-3 function, we have analyzed the DNA-binding properties of wild-type and Spd Pax-3, using oligonucleotides that bind primarily to the paired domain (e5) or exclusively to the homeodomain (P2). Wild-type Pax-3 was found to bind e5 in a specific manner. In contrast, the Spd mutation reduced binding of Pax-3 to e5 17-fold, revealing a defect in DNA binding by the paired domain. Surprisingly, the Spd mutation also drastically reduced the homeodomain-specific binding to P2 by 21-fold when compared with the wild-type protein. Interestingly, a deletion which removes the Spd mutation was found to restore P2-binding activity, suggesting that within the full-length Pax-3 protein, the paired domain and homeodomain may interact. We conclude, therefore, that the Spd mutation is phenotyically expressed in vitro by a defect in the DNA-binding properties of Pax-3. Furthermore, it is apparent that the paired domain and homeodomain of Pax-3 do not function as independent domains, since a mutation in the former impairs the DNA-binding activity of the latter.