RAS interaction with effector target RIN1.
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Animals↗
Biomedical subjects
J Colicelli
Publications and source records attributed to J Colicelli.
BCR/ABL inhibition by an escort/phosphatase fusion protein.
Cellular transformation by the BCR/ABL oncogene depends on the ABL-encoded tyrosine kinase activity. To block BCR/ABL function, we created a unique tyrosine phosphatase by fusing the catalytic domain of SHP1 (SHP1c) to the ABL binding domain (ABD) of RIN1, an established binding partner and substrate for c-ABL and BCR/ABL. This fusion construct (ABD/SHP1c) binds to BCR/ABL in cells and functions as an active phosphatase. ABD/SHP1c effectively suppressed BCR/ABL function as judged by reductions in transformation of fibroblast cells, growth factor independence of hematopoietic cell lines, and proliferation of primary bone marrow cells. In addition, the leukemogenic properties of BCR/ABL in a murine model system were blocked by coexpression of ABD/SHP1c. Both the "escort" function provided by ABD and the inhibitor function provided by the phosphatase of SHP1c were necessary for effective BCR/ABL interference. Expression of ABD/SHP1c also reversed the transformed phenotype of K562, a human leukemia-derived cell line. These results have direct implications for leukemia therapeutics and suggest an approach to block aberrant signal transduction in other pathologies through the use of appropriately designed escort/inhibitors.
Animals↗
Human ERK1 induces filamentous growth and cell wall remodeling pathways in Saccharomyces cerevisiae.
Expression of an activated extracellular signal-regulated kinase 1 (ERK1) construct in yeast cells was used to examine the conservation of function among mitogen-activated protein (MAP) kinases. Sequence alignment of the human MAP kinase ERK1 with all Saccharomyces cerevisiae kinases reveals a particularly strong kinship with Kss1p (invasive growth promoting MAP kinase), Fus3p (pheromone response MAP/ERK kinase), and Mpk1p (cell wall remodeling MAP kinase). A fusion protein of constitutively active human MAP/ERK kinase 1 (MEK) and human ERK1 was introduced under regulated expression into yeast cells. The fusion protein (MEK/ERK) induced a filamentation response element promoter and led to a growth retardation effect concomitant with a morphological change resulting in elongated cells, bipolar budding, and multicell chains. Induction of filamentous growth was also observed for diploid cells following MEK/ERK expression in liquid culture. Neither haploids nor diploids, however, showed marked penetration of agar medium. These effects could be triggered by either moderate MEK/ERK expression at 37 degrees C or by high level MEK/ERK expression at 30 degrees C. The combination of high level MEK/ERK expression and 37 degrees C resulted in cell death. The deleterious effects of MEK/ERK expression and high temperature were significantly mitigated by 1 m sorbitol, which also enhanced the filamentous phenotype. MEK/ERK was able to constitutively activate a cell wall maintenance reporter gene, suggesting misregulation of this pathway. In contrast, MEK/ERK effectively blocked expression from a pheromone-responsive element promoter and inhibited mating. These results are consistent with MEK/ERK promoting filamentous growth and altering the cell wall through its ability to partially mimic Kss1p and stimulate a pathway normally controlled by Mpk1p, while appearing to inhibit the normal functioning of the structurally related yeast MAP kinase Fus3p.
Cell Division↗
Unified nomenclature for the COP9 signalosome and its subunits: an essential regulator of development.
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Animals↗
A consistent pattern of RIN1 rearrangements in oral squamous cell carcinoma cell lines supports a breakage-fusion-bridge cycle model for 11q13 amplification.
Gene amplification is a common feature of tumors. Overexpression of some amplified genes plays a role in tumor progression. Gene amplification can occur either extrachromosomally as double-minute chromosomes (dmin) or intrachromosomally in the form of homogeneously staining regions (hsrs). Approximately one-half of our oral squamous cell carcinomas (OSCCs) are characterized by amplification of band 11q13, usually as an hsr located entopically (occurring or situated at the normal chromosomal site, as opposed to ectopically). Using chromosomal fluorescence in situ hybridization (FISH), we confirmed the amplification of the cyclin D1 (CCND1/PRAD1) and fibroblast growth factor types 3 and 4 (FGF3/INT2 and FGF4/HSTF1) genes within the 11q13 amplicon in our series of primary OSCCs and derived cell lines. The human RIN1 gene was isolated as an RAS interaction/interference protein in a genetic selection in yeast and has been described as a putative effector of both the RAS and ABL oncogenes. We mapped RIN1 to 11q13.2. FISH analysis of 10 11q13-amplified OSCC cell lines revealed high-level RIN1 amplification in two cell lines. Three additional cell lines have what appear to be duplications and/or low-level amplification of RIN1, visible in both interphase and metaphase cells. The hybridization pattern of RIN1 on the metaphase chromosomes is particularly revealing; RIN1 signals flank the 11q13 hsr, possibly as a result of an inverted duplication. The gene amplification model of Coquelle et al. (1997) predicted that gene amplification occurs by breakage-fusion-bridge (BFB) cycles involving fragile sites. Our data suggest that the pattern of gene amplification at 11q13 in OSCC cell lines is consistent with a BFB model. RIN1 appears to be a valuable probe for investigating the process of gene amplification in general and, specifically, 11q13 amplification in oral cancer.
Adult↗
Identification of inhibitor specificity determinants in a mammalian phosphodiesterase.
Mammalian phosphodiesterase types 3 and 4 (PDE3 and PDE4) hydrolyze cAMP and are essential for the regulation of this intracellular second messenger in many cell types. Whereas these enzymes share structural and biochemical similarities, each can be distinguished by its sensitivity to isozyme-specific inhibitors. By using a series of chimeric enzymes, we have localized the region of PDE4 that confers sensitivity to selective inhibitors. This inhibitor specificity domain lies within a short sequence at the carboxyl terminus of the catalytic domain of the protein, consistent with the competitive nature of inhibition by these compounds. Surprisingly, the identified region also includes some of the most highly conserved residues among PDE isoforms. A yeast-based expression system was used for the isolation and characterization of mutations within this area that confer resistance to the PDE4-specific inhibitor rolipram. Analysis of these mutants indicated that both conserved and unique residues are required for isoform-specific inhibitor sensitivity. In some cases, combined point mutations contribute synergistically to the reduction of sensitivity (suppression of IC50). We also report that several mutations display differential sensitivity changes with respect to distinct structural classes of inhibitors.
3',5'-Cyclic-AMP Phosphodiesterases↗
Yeast model system for study of mammalian phosphodiesterases.
Facile manipulation and rapid regeneration have helped to establish the yeast Saccharomyces cerevisiae as a genetic workhorse. More recently, these simple eukaryotes have been used for the biochemical analysis of mammalian proteins. This article describes the use of a yeast expression system for both in vitro and in vivo assays of mammalian phosphodiesterase (PDE) activity using yeast cells devoid of endogenous PDEs. It also presents simple methodologies for the analysis of the pharmacological properties of mammalian PDEs and describes the use of a powerful genetic selection for mutant forms of PDEs with altered biochemical and pharmacological characteristics.
Animals↗
Protein binding and signaling properties of RIN1 suggest a unique effector function.
Human RIN1 was first characterized as a RAS binding protein based on the properties of its carboxyl-terminal domain. We now show that full-length RIN1 interacts with activated RAS in mammalian cells and defines a minimum region of 434 aa required for efficient RAS binding. RIN1 interacts with the "effector domain" of RAS and employs some RAS determinants that are common to, and others that are distinct from, those required for the binding of RAF1, a known RAS effector. The same domain of RIN1 that binds RAS also interacts with 14-3-3 proteins, extending the similarity between RIN1 and other RAS effectors. When expressed in mammalian cells, the RAS binding domain of RIN1 can act as a dominant negative signal transduction blocker. The amino-terminal domain of RIN1 contains a proline-rich sequence similar to consensus Src homology 3 (SH3) binding regions. This RIN1 sequence shows preferential binding to the ABL-SH3 domain in vitro. Moreover, the amino-terminal domain of RIN1 directly associates with, and is tyrosine phosphorylated by, c-ABL. In addition, RIN1 encodes a functional SH2 domain that has the potential to activate downstream signals. These data suggest that RIN1 is able to mediate multiple signals. A differential pattern of expression and alternate splicing indicate several levels of RIN1 regulation.
3T3 Cells↗
Regulation of the oncogenic activity of BCR-ABL by a tightly bound substrate protein RIN1.
RIN1 was originally identified by its ability to physically bind to and interfere with activated Ras in yeast. Paradoxically, RIN1 potentiates the oncogenic activity of the BCR-ABL tyrosine kinase in hematopoietic cells and dramatically accelerates BCR-ABL-induced leukemias in mice. RIN1 rescues BCR-ABL mutants for transformation in a manner distinguishable from the cell cycle regulators c-Myc and cyclin D1 and the Ras connector Shc. These biological effects require tyrosine phosphorylation of RIN1 and binding of RIN1 to the Abl-SH2 and SH3 domains. RIN1 is tyrosine phosphorylated and is associated with BCR-ABL in human and murine leukemic cells. RIN1 exemplifies a new class of effector molecules dependent on the concerted action of the SH3, SH2, and catalytic domains of a cytoplasmic tyrosine kinase.
Adaptor Proteins, Signal Transducing↗
HIP-I: a huntingtin interacting protein isolated by the yeast two-hybrid system.
We report the discovery of the huntingtin interacting protein I (HIP-I) which binds specifically to the N-terminus of human huntingtin, both in the two-hybrid screen and in in vitro binding experiments. For the interaction in vivo, a protein region downstream of the polyglutamine stretch in huntingtin is essential. The HIP1 cDNA isolated by the two-hybrid screen encodes a 55 kDa fragment of a novel protein. Using an affinity-purified polyclonal antibody raised against recombinant HIP-I, a protein of 116 kDa was detected in brain extracts by Western blot analysis. The predicted amino acid sequence of the HIP-I fragment exhibits significant similarity to cytoskeleton proteins, suggesting that HIP-I and huntingtin play a functional role in the cell filament networks. The HIP1 gene is ubiquitously expressed in different brain regions at low level. HIP-I is enriched in human brain but can also be detected in other human tissues as well as in mouse brain. HIP-I and huntingtin behave almost identically during subcellular fractionation and both proteins are enriched in the membrane containing fractions.
Amino Acid Sequence↗
Two human cDNAs, including a homolog of Arabidopsis FUS6 (COP11), suppress G-protein- and mitogen-activated protein kinase-mediated signal transduction in yeast and mammalian cells.
We have isolated two novel human cDNAs, gps1-1 and gps2, that suppress lethal G-protein subunit-activating mutations in the pheromone response pathway of the yeast Saccharomyces cerevisiae. Suppression of other pathway-activating events was examined. In wild-type cells, expression of either gps1-1 or gps2 led to enhanced recovery from cell cycle arrest induced by pheromone. Sequence analysis indicated that gps1-1 contains only the carboxy-terminal half of the gps1 coding sequence. The predicted gene product of gps1 has striking similarity to the protein encoded by the Arabidopsis FUS6 (COP11) gene, a negative regulator of light-mediated signal transduction that is known to be essential for normal development. A chimeric construct containing gps1 and FUS6 sequences also suppressed the yeast pheromone pathway, indicating functional conservation between these human and plant genes. In addition, when overexpressed in mammalian cells, gps1 or gps2 potently suppressed a RAS- and mitogen-activated protein kinase-mediated signal and interfered with JNK activity, suggesting that signal repression is part of their normal function. For gps1, these results are consistent with the proposed function of FUS6 (COP11) as a signal transduction repressor in plants.
Amino Acid Sequence↗
Truncated forms of a novel yeast protein suppress the lethality of a G protein alpha subunit deficiency by interacting with the beta subunit.
In Saccharomyces cerevisiae, the mating pheromone-initiated signal is transduced by a heterotrimeric G protein and normally results in transient cell cycle arrest and differentiation. A null allele of the G alpha (GPA1/SCG1) subunit results in cell death due to unchecked signaling from the G beta gamma (STE4, STE18, respectively) heterodimer. We have identified three high copy suppressors of gpa1 lethality. Two of these genes encode known transcription factors. Mat alpha 2p and Mcm1p. The third is a truncated form of a novel gene, SYG1. Overexpressed wild type SYG1 is a weak suppressor of gpa1. In contrast, the isolated mutant allele SYG1-1 is a strong suppressor that completely blocks the cell cycle arrest and differentiation phenotypes of gpa1 cells of both mating types. One deletion mutant (SYG1 delta 340) can suppress the cell cycle arrest associated with gpa1, but the cells retain a differentiated morphology. SYG1-1 can suppress the effects of overexpressed wild type G beta but is not able to suppress the lethality of an activated G beta mutant (STE4Hpl). Consistent with these genetic observations, the suppressing form of Syg1p can interact with the STE4 gene product, as determined by a two-hybrid assay. SYG1-1 is also capable of promoting pheromone recovery in wild type cells, as judged by halo assay. The sequence of SYG1 predicts eight membrane-spanning domains. Deletion mutants of SYG1 indicate that complete gpa1 suppression requires removal of all of these hydrophobic regions. Interestingly, this truncated protein localizes to the same plasma membrane-enriched subcellular fraction as does full-length Syg1p. Three hypothetical yeast proteins, identified by their similarity to the SYG1 primary sequence within the gpa1 suppression domain, also appear to have related structures. The properties of Syg1p are consistent with those of a transmembrane signaling component that can respond to, or transduce signals through, G beta or G beta gamma.
Amino Acid Sequence↗
A human protein selected for interference with Ras function interacts directly with Ras and competes with Raf1.
The overexpression of some human proteins can cause interference with the Ras signal transduction pathway in the yeast Saccharomyces cerevisiae. The functional block is located at the level of the effector itself, since these proteins do not suppress activating mutations further downstream in the same pathway. We now demonstrate, with in vivo and in vitro experiments, that the protein encoded by one human cDNA (clone 99) can interact directly with yeast Ras2p and with human H-Ras protein, and we have named this gene rin1 (Ras interaction/interference). The interaction between Ras and Rin1 is enhanced when Ras is bound to GTP. Rin1 is not able to interact with either an effector mutant or a dominant negative mutant of H-Ras. Thus, Rin1 displays a human H-Ras interaction profile that is the same as that seen for Raf1 and yeast adenylyl cyclase, two known effectors of Ras. Moreover, Raf1 directly competes with Rin1 for binding to H-Ras in vitro. Unlike Raf1, however, the Rin1 protein resides primarily at the plasma membrane, where H-Ras is localized. These data are consistent with Rin1 functioning in mammalian cells as an effector or regulator of H-Ras.
Amino Acid Sequence↗
Mutational mapping of kinetic and pharmacological properties of a human cardiac cAMP phosphodiesterase.
We have created a series of deletion mutants of a human cardiac cAMP phosphodiesterase in order to define sequences necessary for function and to identify residues required for inhibition by cGMP and by the drugs milrinone and trequinsin. These truncated constructs were expressed in yeast cells, and their biochemical properties were analyzed. The mutations define an amino acid sequence that is essential for function. Among the active constructs, there was considerable variability in the level of expression and in the stability of the proteins, with the full-length and near full-length constructs being the least stable. There were, however, no significant changes in Km values among the active enzymes. Cation studies confirmed that Mn2+ is a more efficient cofactor than Mg2+ or Co2+. Interestingly, Mn2+ acts as a more efficient cofactor for cGMP inhibition as well. Although IC50 values for the drugs trequinsin and milrinone were not significantly altered by deletions, there was a decrease in cGMP IC50 values for the smaller constructs, indicating a role for amino acid residues outside the catalytic region in cGMP inhibition. We also demonstrate in vivo inhibition of this enzyme in yeast cells grown in the presence of pharmacological inhibitors, allowing for the selection of drug-resistant mutants. Finally, we have constructed and analyzed chimeric genes in which portions of this phosphodiesterase are replaced with homologous sequences from a closely related phosphodiesterase isozyme that is expressed in brain. Our results demonstrate that sequence variations between related isozymes account for more than just pharmacological distinctions and may reflect significant structural differences.
3',5'-Cyclic-AMP Phosphodiesterases↗
Cloning of a rat cDNA encoding dihydroxypolyprenylbenzoate methyltransferase by functional complementation of a Saccharomyces cerevisiae mutant deficient in ubiquinone biosynthesis.
3,4-Dihydroxy-5-hexaprenylbenzoate methyltransferase (DHHB-MTase) is the product of the COQ3 gene in Saccharomyces cerevisiae and catalyses the fourth step in the biosynthesis of ubiquinone (coenzyme Q) from p-hydroxybenzoic acid. A full-length cDNA encoding a mammalian homologue of DHHB-MTase was isolated from a newly constructed rat testis cDNA library by functional complementation of a coq3 deletion mutant of S. cerevisiae. The complementing clone contained a 1.1-kb poly(A)(+)-tailed insert with a 858-bp open reading frame and presumably encodes 3,4-dihydroxy-5-polyprenylbenzoate-MTase. The deduced rat amino acid (aa) sequence has a 39% identity over 138 aa with the yeast DHHB-MTase and a 37% identity over this same region with an Escherichia coli protein encoded by the ubiG gene, a MTase that catalyses the terminal step of ubiquinone biosynthesis. The rescue of the yeast coq3 mutant by the rat homologue suggests that yeast and rat synthesize ubiquinone via the same early steps in this pathway.
Amino Acid Sequence↗
Use of a yeast expression system for the isolation and analysis of drug-resistant mutants of a mammalian phosphodiesterase.
Saccharomyces cerevisiae strain PP5 has a phosphodiesterase (PDE) deficiency that results in heat-shock sensitivity due to the intracellular accumulation of cAMP. This strain also carries the cam mutation, which confers permeability to cAMP and, as shown here, to other compounds. Expression of rat type IV PDE in these cells caused them to revert to heat-shock resistance. Treatment of the transformed PP5 cells with rolipram, an antidepressant in humans and a potent inhibitor of type IV PDEs, reinstated sensitivity to heat shock. The biochemical properties of deletion mutants of this PDE were determined, and an active enzyme of minimum length was created. Reversion to heat-shock resistance was then used to select for PDE mutants refractory to the inhibitory effects of rolipram. Four mutants (A1, A2, A3, and A5) were isolated. Each carries a single point mutation; two have mutations in the same codon. Each mutant showed distinct properties, based on analysis of their substrate kinetics and IC50 values for a variety of inhibitors. Mutant A5 had a reduced activity for substrate, mutants A1 and A3 showed no change in substrate kinetics, and mutant A2 displayed an increase in activity. For most mutants, the drug resistance was confined to the class of drug used in the selection. This study shows that it is possible to recreate in yeast cells the susceptibility of mammalian enzymes to pharmacological agents. Our study also demonstrates that such systems can be used to select rare mutants useful in the analysis of drug-protein interactions.
1-Methyl-3-isobutylxanthine↗
Expression of three mammalian cDNAs that interfere with RAS function in Saccharomyces cerevisiae.
Saccharomyces cerevisiae strains expressing the activated RAS2Val19 gene or lacking both cAMP phosphodiesterase genes, PDE1 and PDE2, have impaired growth control and display an acute sensitivity to heat shock. We have isolated two classes of mammalian cDNAs from yeast expression libraries that suppress the heat shock-sensitive phenotype of RAS2Val19 strain. Members of the first class of cDNAs also suppress the heat shock-sensitive phenotype of pde1- pde2- strains and encode cAMP phosphodiesterases. Members of the second class fail to suppress the phenotype of pde1- pde2- strains and therefore are candidate cDNAs encoding proteins that interact with RAS proteins. We report the nucleotide sequence of three members of this class. Two of these cDNAs share considerable sequence similarity, but none are clearly similar to previously isolated genes.
3',5'-Cyclic-AMP Phosphodiesterases↗
Analysis of mutations in the envelope gene of Moloney murine leukemia virus: separation of infectivity from superinfection resistance.
Six deletion mutations and an insertion were generated in the env gene of cloned copies of Moloney murine leukemia virus DNA. All seven mutants were replication-defective as tested by transformation of NIH/3T3 cells. The mutant DNAs were introduced into NIH/3T3 cells to generate stable producer lines; all released virion particles into the medium, suggesting that none of the mutations affected overall viral gene expression, gag and pol gene expression, gag and pol gene functions, or virion budding. Several of the mutations reduced the lifetime of the env protein or blocked its export to the cell surface. One mutation altering the membrane-spanning region and the cytoplasmic tail of the TM protein had no effect on export of the protein, proteolytic processing, or incorporation into virion particles, but still blocked the infectivity of the resulting virus. The results suggest that alterations in the transmembrane region can affect early steps of infection, such as the fusion of virion and host membranes. Cells expressing this mutant env protein were fully resistant to superinfection by wild-type virus. Thus, induction of virus resistance, presumably reflecting blocking the virus receptor, can be separated from virus infectivity.
Amino Acid Sequence↗