Phage display selection of ligand residues important for Src homology 3 domain binding specificity.

The Src homology 3 (SH3) domain is a 50-aa modular unit present in many cellular proteins involved in intracellular signal transduction. It functions to direct protein-protein interactions through the recognition of proline-rich motifs on associated proteins. SH3 domains are important regulatory elements that have been demonstrated to specify distinct regulatory pathways important for cell growth, migration, differentiation, and responses to the external milieu. By the use of synthetic peptides, ligands have been shown to consist of a minimum core sequence and to bind to SH3 domains in one of two pseudosymmetrical orientations, class I and class II. The class I sites have the consensus sequence ZP(L/P)PP psi P whereas the class II consensus is PP psi PPZ (where psi is a hydrophobic residue and Z is a SH3 domain-specific residue). We previously showed by M13 phage display that the Src, Fyn, Lyn, and phosphatidylinositol 3-kinase (PI3K) SH3 domains preferred the same class I-type core binding sequence, RPLPP psi P. These results failed to explain the specificity for cellular proteins displayed by SH3 domains in cells. In the current study, class I and class II core ligand sequences were displayed on the surface of bacteriophage M13 with five random residues placed either N- or C-terminal of core ligand residues. These libraries were screened for binding to the Src, Fyn, Lyn, Yes, and PI3K SH3 domains. By this approach, additional ligand residue preferences were identified that can increase the affinity of SH3 peptide ligands at least 20-fold compared with core peptides. The amino acids selected in the flanking sequences were similar for Src, Fyn, and Yes SH3 domains; however, Lyn and PI3K SH3 domains showed distinct binding specificities. These results indicate that residues that flank the core binding sequences shared by many SH3 domains are important determinants of SH3 binding affinity and selectivity.

Genetic and comparative analyses reveal an alternative secondary structure in the region of nt 912 of Escherichia coli 16S rRNA.

Mutations at position 912 of Escherichia coli 16S rRNA result in two notable phenotypes. The C-->U transition confers resistance to streptomycin, a translational-error-inducing antibiotic, while a C-->G transversion causes marked retardation of cell growth rate. Starting with the slow-growing G912 mutant, random mutagenesis was used to isolate a second site mutation that restored growth nearly to the wild-type rate. The second site mutation was identified as a G-->C transversion at position 885 in 16S rRNA. Cells containing the G912 mutation had an increased doubling time, abnormal sucrose gradient ribosome/subunit profile, increased sensitivity to spectinomycin, dependence upon streptomycin for growth in the presence of spectinomycin, and slower translation rate, whereas cells with the G912/C885 double mutation were similar to wild type in these assays. Comparative analysis showed there was significant covariation between positions 912 and 885. Thus the second-site suppressor analysis, the functional assays, and the comparative data suggest that the interaction between nt 912 and nt 885 is conserved and necessary for normal ribosome function. Furthermore, the comparative data suggest that the interaction extends to include G885-G886-G887 pairing with C912-U911-C910. An alternative secondary structure element for the central domain of 16S rRNA is proposed.

Transcription factor TFIID is a direct functional target of the adenovirus E1A transcription-repression domain.

The 243-amino acid adenovirus E1A oncoprotein both positively and negatively modulates the expression of cellular genes involved in the regulation of cell growth. The E1A transcription repression function appears to be linked with its ability to induce cellular DNA synthesis, cell proliferation, and cell transformation, as well as to inhibit cell differentiation. The mechanism by which E1A represses the transcription of various promoters has proven enigmatic. Here we provide several lines of evidence that the "TATA-box" binding protein (TBP) component of transcription factor TFIID is a cellular target of the E1A repression function encoded within the E1A N-terminal 80 amino acids. (i) The E1A N-terminal 80 amino acids [E1A-(1-80)protein] efficiently represses basal transcription from TATA-containing core promoters in vitro. (ii) TBP reverses completely E1A repression in vitro. (iii) TBP restores transcriptional activity to E1A-(1-80) protein affinity-depleted nuclear extracts. (iv) The N-terminal repression domain of E1A interacts directly and specifically with TBP in vitro. These results may help explain how E1A represses a set of genes that lack common upstream promoter elements.

Double-stranded-RNA-dependent protein kinase and TAR RNA-binding protein form homo- and heterodimers in vivo.

The yeast two-hybrid system and far-Western protein blot analysis were used to demonstrate dimerization of human double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in vivo and in vitro. A catalytically inactive mutant of PKR with a single amino acid substitution (K296R) was found to dimerize in vivo, and a mutant with a deletion of the catalytic domain of PKR retained the ability to dimerize. In contrast, deletion of the two dsRNA-binding motifs in the N-terminal regulatory domain of PKR abolished dimerization. In vitro dimerization of the dsRNA-binding domain required the presence of dsRNA. These results suggest that the binding of dsRNA by PKR is necessary for dimerization. The mammalian dsRNA-binding protein TRBP, originally identified on the basis of its ability to bind the transactivation region (TAR) of human immunodeficiency virus RNA, also dimerized with itself and with PKR in the yeast assay. Taken together, these results suggest that complexes consisting of different combinations of dsRNA-binding proteins may exist in vivo. Such complexes could mediate differential effects on gene expression and control of cell growth.

Tyrosine phosphorylation and activation of STAT5, STAT3, and Janus kinases by interleukins 2 and 15.

The cytokines interleukin 2 (IL-2) and IL-15 have similar biological effects on T cells and bind common hematopoietin receptor subunits. Pathways that involve Janus kinases (JAKs) and signal transducers and activators of transcription (STATs) have been shown to be important for hematopoietin receptor signaling. In this study we identify the STAT proteins activated by IL-2 and IL-15 in human T cells. IL-2 and IL-15 rapidly induced the tyrosine phosphorylation of STAT3 and STAT5, and DNA-binding complexes containing STAT3 and STAT5 were rapidly activated by these cytokines in T cells. IL-4 induced tyrosine phosphorylation and activation of STAT3 but not STAT5. JAK1 and JAK3 were tyrosine-phosphorylated in response to IL-2 and IL-15. Hence, the JAK and STAT molecules that are activated in response to IL-2 and IL-15 are similar but differ from those induced by IL-4. These observations identify the STAT proteins activated by IL-2 and IL-15 and therefore define signaling pathways by which these T-cell growth factors may regulate gene transcription.

Suppression of lung metastasis of B16 mouse melanoma by N-acetylglucosaminyltransferase III gene transfection.

The beta 1-6 structure of N-linked oligosaccharides, formed by beta-1,6-N-acetylglucosaminyltransferase (GnT-V), is associated with metastatic potential. We established a highly metastatic subclone, B16-hm, from low metastatic B16-F1 murine melanoma cells. The gene encoding beta-1,4-N-acetylglucosaminyltransferase (GnT-III) was introduced into the B16-hm cells, and three clones that stably expressed high GnT-III activity were obtained. In these transfectants, the affinity to leukoagglutinating phytohemagglutinin was reduced, whereas the binding to erythroagglutinating phytohemagglutinin was increased, indicating that the level of beta 1-6 structure was decreased due to competition for substrate between intrinsic GnT-V and ectopically expressed GnT-III. Lung metastasis after intravenous injection of the transfectants into syngeneic and nude mice was significantly suppressed, suggesting that the decrease in beta 1-6 structure suppressed metastasis via a mechanism independent of the murine system. These transfectants also displayed decreased invasiveness into Matrigel and inhibited cell attachment to collagen and laminin. Cell growth was not affected. Our results demonstrate a causative role for beta 1-6 branches in invasion and cell attachment in the extravasation stage of metastasis.

Apolipoprotein B mRNA-editing protein induces hepatocellular carcinoma and dysplasia in transgenic animals.

Apolipoprotein (apo-) B mRNA editing is the deamination of cytidine that creates a new termination codon and produces a truncated version of apo-B (apo-B48). The cytidine deaminase catalytic subunit [apo-B mRNA-editing enzyme catalytic polypeptide 1 (APOBEC-1)] of the multiprotein editing complex has been identified. We generated transgenic rabbits and mice expressing rabbit APOBEC-1 in their livers to determine whether hepatic expression would lower low density lipoprotein cholesterol concentrations. The apo-B mRNA from the livers of the transgenic mice and rabbit was extensively edited, and the transgenic animals had reduced concentrations of apo-B100 and low density lipoproteins compared with control animals. Unexpectedly, all of the transgenic mice and a transgenic rabbit had liver dysplasia, and many transgenic mice developed hepatocellular carcinomas. Many of the mouse livers were hyperplastic and filled with lipid. Other hepatic mRNAs with sequence motifs similar to apo-B mRNA were examined for this type of editing (i.e., cytidine deamination). One of these, tyrosine kinase, was edited in livers of transgenic mice but not of controls. This result demonstrates that other mRNAs can be edited by the overexpressed editing enzyme and suggests that aberrant editing of hepatic mRNAs involved in cell growth and regulation is the cause of the tumorigenesis. Finally, these findings compromise the potential use of APOBEC-1 for gene therapy to lower plasma levels of low density lipoproteins.

A synthetic inhibitor of the mitogen-activated protein kinase cascade.

Treatment of cells with a variety of growth factors triggers a phosphorylation cascade that leads to activation of mitogen-activated protein kinases (MAPKs, also called extracellular signal-regulated kinases, or ERKs). We have identified a synthetic inhibitor of the MAPK pathway. PD 098059 [2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one] selectively inhibited the MAPK-activating enzyme, MAPK/ERK kinase (MEK), without significant inhibitory activity of MAPK itself. Inhibition of MEK by PD 098059 prevented activation of MAPK and subsequent phosphorylation of MAPK substrates both in vitro and in intact cells. Moreover, PD 098059 inhibited stimulation of cell growth and reversed the phenotype of ras-transformed BALB 3T3 mouse fibroblasts and rat kidney cells. These results indicate that the MAPK pathway is essential for growth and maintenance of the ras-transformed phenotype. Further, PD 098059 is an invaluable tool that will help elucidate the role of the MAPK cascade in a variety of biological settings.

Adenoviral E1B-55kDa protein inhibits yeast mRNA export and perturbs nuclear structure.

The mechanisms of export of RNA from the nucleus are poorly understood; however, several viral proteins modulate nucleocytoplasmic transport of mRNA. Among these are the adenoviral proteins E1B-55kDa and E4-34kDa. Late in infection, these proteins inhibit export of host transcripts and promote export of viral mRNA. To investigate the mechanism by which these proteins act, we have expressed them in Saccharomyces cerevisiae. Overexpression of either or both proteins has no obvious effect on cell growth. By contrast, overexpression of E1B-55kDa bearing a nuclear localization signal (NLS) dramatically inhibits cell growth. In this situation, the NLS-E1B-55kDa protein is localized to the nuclear periphery, fibrous material is seen in the nucleoplasm, and poly(A)+ RNA accumulates in the nucleus. Simultaneous overexpression of E4-34kDa bearing or lacking an NLS does not modify these effects. We discuss the mechanisms of selective mRNA transport.

De novo decorin gene expression suppresses the malignant phenotype in human colon cancer cells.

The rapid progress in the cloning of proteoglycan genes has enabled investigators to examine in depth the functional roles these polyhedric molecules play in the control of cell proliferation. Decorin, a leucine-rich proteoglycan expressed by most connective tissues, is a prototype molecule that regulates cellular growth via two mechanisms: modulation of growth factor activity and matrix assembly. We now provide direct evidence that human colon cancer cells stably transfected with decorin cDNA exhibit a marked suppression of the transformed phenotype: the cells have a reduced growth rate in vitro, form small colonies in soft agar, and do not generate tumors in scid/scid mice. Several independent clones are arrested in the G1 phase of the cell cycle, and their growth suppression can be restored by treatment with decorin antisense oligodeoxynucleotides. These effects are independent of growth factors and are not due to either clonal selection or integration site of the decorin gene. These findings correlate well with the observation that decorin gene expression is markedly up-regulated during quiescence. Decorin thus appears to be one component of a negative loop that controls cell growth.

The ethylene hormone response in Arabidopsis: a eukaryotic two-component signaling system.

The simple gas ethylene affects numerous physiological processes in the growth and development of higher plants. With the use of molecular genetic approaches, we are beginning to learn how plants perceive ethylene and how this signal is transduced. Components of ethylene signal transduction are defined by ethylene response mutants in Arabidopsis thaliana. The genes corresponding to two of these mutants, etr1 and etr1, have been cloned. The ETR1 gene encodes a homolog of two-component regulators that are known almost exclusively in prokaryotes. The two-component regulators in prokaryotes are involved in the perception and transduction of a wide range of environmental signals leading to adaptive responses. The CTR1 gene encodes a homolog of the Raf family of serine/threonine protein kinases. Raf is part of a mitogen-activated protein kinase cascade known to regulate cell growth and development in mammals, worms, and flies. The ethylene response pathway may, therefore, exemplify a conserved protein kinase cascade regulated by a two-component system. The dominance of all known mutant alleles of ETR1 may be due to either constitutive activation of the ETR1 protein or dominant interference of wild-type activity. The discovery of Arabidopsis genes encoding proteins related to ETR1 suggests that the failure to recover recessive etr1 mutant alleles may be due to the presence of redundant genes.

FtsH is required for proteolytic elimination of uncomplexed forms of SecY, an essential protein translocase subunit.

When secY is overexpressed over secE or secE is underexpressed, a fraction of SecY protein is rapidly degraded in vivo. This proteolysis was unaffected in previously described protease-defective mutants examined. We found, however, that some mutations in ftsH, encoding a membrane protein that belongs to the AAA (ATPase associated with a variety of cellular activities) family, stabilized oversynthesized SecY. This stabilization was due to a loss of FtsH function, and overproduction of the wild-type FtsH protein accelerated the degradation. The ftsH mutations also suppressed, by alleviating proteolysis of an altered form of SecY, the temperature sensitivity of the secY24 mutation, which alters SecY such that its interaction with SecE is weakened and it is destabilized at 42 degrees C. We were able to isolate a number of additional mutants with decreased ftsH expression or with an altered form of FtsH using selection/screening based on suppression of secY24 and stabilization of oversynthesized SecY. These results indicate that FtsH is required for degradation of SecY. Overproduction of SecY in the ftsH mutant cells proved to deleteriously affect cell growth and protein export, suggesting that elimination of uncomplexed SecY is important for optimum protein translocation and for the integrity of the membrane. The primary role of FtsH is discussed in light of the quite pleiotropic mutational effects, which now include stabilization of uncomplexed SecY.

L-arginine may mediate the therapeutic effects of low protein diets.

We have previously shown beneficial effects of dietary protein restriction on transforming growth factor beta (TGF-beta) expression and glomerular matrix accumulation in experimental glomerulonephritis. We hypothesized that these effects result from restriction of dietary L-arginine intake. Arginine is a precursor for three pathways, the products of which are involved in tissue injury and repair: nitric oxide, an effector molecule in inflammatory and immunological tissue injury; polyamines, which are required for DNA synthesis and cell growth; and proline, which is required for collagen production. Rats were fed six isocaloric diets differing in L-arginine and/or total protein content, starting immediately after induction of glomerulonephritis by injection of an antibody reactive to glomerular mesangial cells. Mesangial cell lysis and monocyte/macrophage infiltration did not differ with diet. However, restriction of dietary L-arginine intake, even when total protein intake was normal, resulted in decreased proteinuria, decreased expression of TGF-beta 1 mRNA and TGF-beta 1 protein, and decreased production and deposition of matrix components. L-Arginine, but not D-arginine, supplementation to low protein diets reversed these effects. These results implicate arginine as a key component in the beneficial effects of low protein diet.