Mutational activation of c-raf-1 and definition of the minimal transforming sequence.

A series of wild-type and mutant raf genes was transfected into NIH 3T3 cells and analyzed for transforming activity. Full-length wild-type c-raf did not show transforming activity. Two types of mutations resulted in oncogenic activity similar to that of v-raf: truncation of the amino-terminal half of the protein and fusion of the full-length molecule to gag sequences. A lower level of activation was observed for a mutant with a tetrapeptide insertion mapping to conserved region 2 (CR2), a serine- and threonine-rich domain located 100 residues amino-terminal of the kinase domain. To determine essential structural features of the transforming region of raf, we analyzed point and deletion mutants of v-raf. Substitutions of Lys-56 modulated the transforming activity, whereas mutation of Lys-53, a putative ATP binding residue, abolished it. Deletion analysis established that the minimal transforming sequence coincided precisely with CR3, the conserved Raf kinase domain. Thus, oncogenic activation of the Raf kinase can be achieved by removal of CR1 and CR2 or by steric distortion and requires retention of an active kinase domain. These findings are consistent with a protein structure model for the nonstimulated enzyme in which the active site is buried within the protein.

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The Ubiquitin-associated Domain of hPLIC-2 Interacts with the Proteasome

Molecular Biology of the Cell ◽

10.1091/mbc.e02-11-0766 ◽

2003 ◽

Vol 14 (9) ◽

pp. 3868-3875 ◽

Cited By ~ 72

Author(s):

Maurits F. Kleijnen ◽

Rodolfo M. Alarcón ◽

Peter M. Howley

Keyword(s):

Proteasomal Degradation ◽

Binding Domain ◽

Full Length ◽

Degradation Pathways ◽

Wild Type ◽

Amino Terminal ◽

Uba Domain ◽

Carboxyl Terminal ◽

Protein Ligases

The ubiquitin-like hPLIC proteins can associate with proteasomes, and hPLIC overexpression can specifically interfere with ubiquitin-mediated proteolysis ( Kleijnen et al., 2000 ). Because the hPLIC proteins can also interact with certain E3 ubiquitin protein ligases, they may provide a link between the ubiquitination and proteasomal degradation machineries. The amino-terminal ubiquitin-like (ubl) domain is a proteasome-binding domain. Herein, we report that there is a second proteasome-binding domain in hPLIC-2: the carboxyl-terminal ubiquitin-associated (uba) domain. Coimmunoprecipitation experiments of wild-type and mutant hPLIC proteins revealed that the ubl and uba domains each contribute independently to hPLIC-2–proteasome binding. There is specificity for the interaction of the hPLIC-2 uba domain with proteasomes, because uba domains from several other proteins failed to bind proteasomes. Furthermore, the binding of uba domains to polyubiquitinated proteins does not seem to be sufficient for the proteasome binding. Finally, the uba domain is necessary for the ability of full-length hPLIC-2 to interfere with the ubiquitin-mediated proteolysis of p53. The PLIC uba domain has been reported to bind and affect the functions of proteins such as GABAAreceptor and presenilins. It is possible that the function of these proteins may be regulated or mediated through proteasomal degradation pathways.

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The amino-terminal 14 amino acids of v-src can functionally replace the extracellular and transmembrane domains of v-erbB

Molecular and Cellular Biology ◽

10.1128/mcb.11.9.4760-4770.1991 ◽

1991 ◽

Vol 11 (9) ◽

pp. 4760-4770

Author(s):

M McMahon ◽

R C Schatzman ◽

J M Bishop

Keyword(s):

Membrane Protein ◽

Tyrosine Kinase ◽

Protein Tyrosine Kinase ◽

Transmembrane Domain ◽

Transmembrane Protein ◽

Integral Membrane Protein ◽

Kinase Domain ◽

Protein Tyrosine ◽

Amino Terminal ◽

Transforming Activity

The retroviral oncogene v-erbB encodes a truncated form of the receptor for epidermal growth factor, an integral membrane protein-tyrosine kinase. By contrast, the oncogene v-src encodes a protein-tyrosine kinase that is a peripheral membrane protein. The morphologies and spectra of cells transformed by these two oncogenes differ. In an effort to identify the functional determinant(s) of these differences, we constructed and tested first deletion mutants of v-erbB and then chimeras between v-src and v-erbB. As reported previously, the absence of any membrane anchorage eliminated transformation by v-erbB. Anchorage of the cytoplasmic kinase domain of v-erbB to membranes with amino-terminal portions of the v-src protein permitted transformation. The phenotype and spectrum of transformation were those expected for v-erbB rather than for v-src. The transforming chimeras lost their biological activity if the signal for myristylation at the amino terminus of v-src was compromised by mutation. Biochemical fractionations revealed a correlation between transforming activity and the association of chimeric gene products with the membrane fraction of the cell. For reasons not yet apparent, the combined presence of membrane anchorage domains of v-src, and the transmembrane domain of v-erbB in the same chimera typically (but not inevitably) impeded transformation. Our results suggest that the specificity of transformation by v-erbB resides in the selection of substrates by the cytoplasmic domain of the gene product. The protein retains access to those substrates even when anchored to the membrane in the manner of a peripheral rather than a transmembrane protein.

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Mechanism of activation of the human trk oncogene

Molecular and Cellular Biology ◽

10.1128/mcb.9.1.15-23.1989 ◽

1989 ◽

Vol 9 (1) ◽

pp. 15-23

Author(s):

F Coulier ◽

D Martin-Zanca ◽

M Ernst ◽

M Barbacid

Keyword(s):

Tyrosine Kinase ◽

Deleterious Effect ◽

Kinase Domain ◽

Tyrosine Kinase Receptor ◽

Beta Globin ◽

Amino Terminal ◽

Tropomyosin Isoforms ◽

Beta Actin ◽

Transforming Activity

The human trk oncogene was generated by a genetic rearrangement that replaced the extracellular domain of the normal trk tyrosine kinase receptor by sequences coding for the 221 amino-terminal residues of a nonmuscle tropomyosin. Molecular dissection of a cDNA clone of the trk oncogene indicated that both the tropomyosin and tyrosine kinase domains were required for proper transforming activity. Replacement of nonmuscle tropomyosin sequences with those of other tropomyosin isoforms had no deleterious effect. However, when tropomyosin sequences were replaced with those of another cytoskeletal gene, such as beta-actin or beta-globin, transforming activity was completely abolished. These results illustrate the important role of tropomyosin sequences in endowing the trk kinase with transforming properties. Functionally unrelated subdomains of the tropomyosin molecule were equally efficient in activating the trk gene. Moreover, the transforming activity of the trk oncogene was not affected when its subcellular localization was drastically altered. Therefore, tropomyosin sequences are likely to contribute to the malignant activation of the trk oncogene not by facilitating its interaction with defined cytoskeletal structures as initially suspected, but by allowing its kinase domain to fold into a constitutively active configuration.

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Identification of primary structural features that define the differential actions of IL-3 and GM-CSF receptors

Blood ◽

10.1182/blood-2001-12-0235 ◽

2002 ◽

Vol 100 (9) ◽

pp. 3164-3174 ◽

Cited By ~ 17

Author(s):

Caroline A. Evans ◽

Shahrul Ariffin ◽

Andrew Pierce ◽

Anthony D. Whetton

Keyword(s):

Bioinformatic Analysis ◽

Structural Features ◽

Myeloid Differentiation ◽

Wild Type ◽

Α Subunit ◽

Gm Csf ◽

Rich Domain ◽

Cytokine Activation ◽

Cytosolic Domain ◽

Α Subunits

Abstract Activation of human interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors, ectopically expressed in FDCP-mix multipotent cells, stimulates self-renewal or myeloid differentiation, respectively. These receptors are composed of unique α subunits that interact with common βc subunits. A chimeric receptor (hGM/βc), comprising the extracellular domain of the hGM-CSF receptor α subunit (hGM Rα) fused to the intracellular domain of hβc, was generated to determine whether hβc activation is alone sufficient to promote differentiation. hGM-CSF activation of hGM/βc, expressed in the presence and absence of the hβc subunit, promoted maintenance of primitive phenotype. This indicates that the cytosolic domain of the hGM Rα chain is required for differentiation mediated by activation of the hGM Rα, βc receptor complex. We have previously demonstrated that the α cytosolic domain confers signal specificity for IL-3 and GM-CSF receptors. Bioinformatic analysis of the IL-3 Rα and GM Rα subunits identified a tripeptide sequence, adjacent to the conserved proline-rich domain, which was potentially a key difference between them. Cross-exchange of the equivalent tripeptides between the α subunits altered receptor function compared to the wild-type receptors. Both the mutant and the corresponding wild-type receptors promoted survival and proliferation in the short-term but had distinct effects on developmental outcome. The mutated hGM Rα promoted long-term proliferation and maintenance of primitive cell morphology, whereas cytokine activation of the corresponding hIL-3 Rα mutant promoted myeloid differentiation. We have thus identified a region of the α cytosolic domain that is of critical importance for defining receptor specificity.

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Functional dissection of Ctr4 and Ctr5 amino-terminal regions reveals motifs with redundant roles in copper transport

Microbiology ◽

10.1099/mic.0.28392-0 ◽

2006 ◽

Vol 152 (1) ◽

pp. 209-222 ◽

Cited By ~ 36

Author(s):

Jude Beaudoin ◽

Julie Laliberté ◽

Simon Labbé

Keyword(s):

Plasma Membrane ◽

Amino Acid ◽

Expression System ◽

Copper Transport ◽

Full Length ◽

Proper Function ◽

Copper Uptake ◽

Wild Type ◽

Amino Terminal ◽

Redundant Functions

Copper uptake in the fission yeast Schizosaccharomyces pombe is carried out by a heteromeric complex formed by two proteins, Ctr4 and Ctr5. In this study, a stable expression system using integrative plasmids was developed to investigate the respective roles of Ctr4 and Ctr5 in copper transport. It was shown that expression of full-length Ctr4 or truncated Ctr4 containing residues 106–289 was required for localization of Ctr5 to the plasma membrane. Likewise, when the full-length Ctr5 or truncated Ctr5 from residues 44–173 was co-expressed with Ctr4, this protein was visualized at the periphery of the cell. To determine the importance of the Mets motifs (consisting of five methionines arranged as Met-X2-Met-X-Met, where X is any amino acid) of Ctr4 and Ctr5 in the heteroprotein complex, we co-expressed Ctr5 lacking the Mets motif and Cys-X-Met-X-Met sequence with wild-type Ctr4 or its mutant derivatives. Conversely, Ctr4 lacking the Mets motif and Met122 was expressed with wild-type Ctr5 or its mutant derivatives. These experiments revealed that the five Mets motifs of Ctr4 and the Ctr4 residue Met122 have equally important roles in copper assimilation. Furthermore, the two partially overlapping Mets motifs and the Cys-X-Met-X-Met sequence in Ctr5 have redundant functions in copper transport, with the latter sequence making a greater contribution than the former. Together, the data reveal that co-expression of both Ctr4 and Ctr5 is necessary for the proper function and localization of the heteroprotein complex to the plasma membrane. Once on the cell surface, the N-terminal regions of Ctr4 and Ctr5 can function independently to transport copper; however, the greatest efficiency is achieved when both N termini are present.

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Definition of the human raf amino-terminal regulatory region by deletion mutagenesis.

Molecular and Cellular Biology ◽

10.1128/mcb.9.2.639 ◽

1989 ◽

Vol 9 (2) ◽

pp. 639-647 ◽

Cited By ~ 142

Author(s):

V P Stanton ◽

D W Nichols ◽

A P Laudano ◽

G M Cooper

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Regulatory Region ◽

Kinase Domain ◽

Coding Sequences ◽

Amino Terminal ◽

Murine Sarcoma Virus ◽

Transforming Activity ◽

Definition Of ◽

Initiation Sequence

Activation of transforming potential of the cellular raf gene has uniformly been associated with the deletion of amino-terminal coding sequences. In order to determine whether 5' truncation alone could activate cellular raf, we constructed 21 human c-raf-1 cDNAs with variable BAL 31-generated deletions distal to a Moloney murine sarcoma virus long terminal repeat and a consensus translation initiation sequence. The deletions ranged from 136 to 1,399 nucleotides of coding sequence and shortened the 648-amino-acid raf protein by 44 to 465 amino acids. The full-length c-raf-1 cDNA was nontransforming upon transfection of NIH 3T3 cells, as were four mutants with deletions of 142 or fewer amino acids. Seven of nine mutants with deletions of 154 to 273 amino acids induced transformation with efficiencies ranging from 0.25 to 70 foci per micrograms of DNA. Mutants with deletions of 303 to 324 amino acids displayed high transforming activities (comparable with that of v-raf), with a peak activity of 2,400 foci per microgram of DNA when 305 amino acids were deleted. Deletions of greater than 383 amino acids, extending into the raf kinase domain, lacked transforming activity. Northern (RNA) blotting and immunoprecipitation assays indicated that transfected NIH cells expressed raf RNAs and proteins of the expected sizes. Thus, 5' truncation alone can activate raf transforming potential, with a sharp peak of activation around amino acid 300. Analysis of three raf genes previously detected by transfection of tumor DNAs indicated that these genes were activated by recombination in raf intron 7 and encoded fusion proteins containing amino-terminal non-raf sequences. The extend of deletion of raf sequences in these recombinant genes corresponded to BAL 31 mutants which did not display high transforming activity, suggesting that the fused non-raf coding sequences may also contribute to biological activity.

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γ-Catenin is regulated by the APC tumor suppressor and its oncogenic activity is distinct from that of β-catenin

Genes & Development ◽

10.1101/gad.14.11.1319 ◽

2000 ◽

Vol 14 (11) ◽

pp. 1319-1331 ◽

Cited By ~ 2

Author(s):

Frank T. Kolligs ◽

Barbara Kolligs ◽

Karen M. Hajra ◽

Gang Hu ◽

Masachika Tani ◽

...

Keyword(s):

Tumor Suppressor ◽

Wnt Signaling ◽

Target Genes ◽

Wnt Signaling Pathway ◽

Tumor Suppressor Protein ◽

Wild Type ◽

Downstream Target ◽

Amino Terminal ◽

Transforming Activity ◽

Altered Regulation

β-Catenin and γ-catenin (plakoglobin), vertebrate homologs of Drosophila armadillo, function in cell adhesion and the Wnt signaling pathway. In colon and other cancers, mutations in the APC tumor suppressor protein orβ-catenin's amino terminus stabilizeβ-catenin, enhancing its ability to activate transcription of Tcf/Lef target genes. Thoughβ- and γ-catenin have analogous structures and functions and like binding to APC, evidence that γ-catenin has an important role in cancer has been lacking. We report here that APC regulates bothβ- and γ-catenin andγ-catenin functions as an oncogene. In contrast to β-catenin, for which only amino-terminal mutated forms transform RK3E epithelial cells, wild-type and several amino-terminal mutated forms of γ-catenin had similar transforming activity. γ-Catenin's transforming activity, like β-catenin's, was dependent on Tcf/Lef function. However, in contrast toβ-catenin, γ-catenin strongly activated c-Myc expression and c-Myc function was crucial for γ-catenin transformation. Our findings suggest APC mutations alter regulation of bothβ- and γ-catenin, perhaps explaining why the frequency of APC mutations in colon cancer far exceeds that of β-catenin mutations. Elevated c-Myc expression in cancers with APC defects may be due to altered regulation of both β- andγ-catenin. Furthermore, the data implyβ- and γ-catenin may have distinct roles in Wnt signaling and cancer via differential effects on downstream target genes.

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The Srp54 GTPase is essential for protein export in the fission yeast Schizosaccharomyces pombe.

Molecular and Cellular Biology ◽

10.1128/mcb.14.12.7839 ◽

1994 ◽

Vol 14 (12) ◽

pp. 7839-7854 ◽

Cited By ~ 20

Author(s):

S M Althoff ◽

S W Stevens ◽

J A Wise

Keyword(s):

Schizosaccharomyces Pombe ◽

Protein Targeting ◽

Signal Recognition Particle ◽

Secretory Protein ◽

Wild Type ◽

Amino Terminal ◽

Rich Domain ◽

Reduced Rate

Signal recognition particle (SRP) is a cytoplasmic ribonucleoprotein required for targeting a subset of presecretory proteins to the endoplasmic reticulum (ER) membrane. Here we report the results of a series of experiments to define the function of the Schizosaccharomyces pombe homolog of the 54-kDa subunit of mammalian SRP. One-step gene disruption reveals that the Srp54 protein, like SRP RNA, is essential for viability in S. pombe. Precursor to the secretory protein acid phosphatase accumulates in cells in which Srp54 synthesis has been repressed under the control of a regulated promoter, indicating that S. pombe SRP functions in protein targeting. In common with other Srp54 homologs, the S. pombe protein has a modular structure consisting of an amino-terminal G (GTPase) domain and a carboxyl-terminal M (methionine-rich) domain. We have analyzed the effects of 17 site-specific mutations designed to alter the function of each of the four GTPase consensus motifs individually. Several alleles, including some with relatively conservative amino acid substitutions, confer lethal or conditional phenotypes, indicating that GTP binding and hydrolysis are critical to the in vivo role of the protein. Two mutations (R to L at position 194 [R194L] and R194H) which were designed, by analogy to oncogenic mutations in rats, to dramatically decrease the catalytic rate and one (T248N) predicted to alter nucleotide binding specificity produce proteins that are unable to support growth at 18 degrees C. Consistent with its design, the R194L mutant hydrolyzes GTP at a reduced rate relative to wild-type Srp54 in enzymatic assays on immunoprecipitated proteins. In strains that also contain wild-type srp54, this mutant protein, as well as others designed to be locked in a GTP-bound conformation, exhibits temperature-dependent dominant inhibitory effects on growth, while a mutant predicted to be GDP locked does not interfere with the function of the wild-type protein. These results form the basis of a simple model for the role of GTP hydrolysis by Srp54 during the SRP cycle.

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Mechanism of activation of the human trk oncogene.

Molecular and Cellular Biology ◽

10.1128/mcb.9.1.15 ◽

1989 ◽

Vol 9 (1) ◽

pp. 15-23 ◽

Cited By ~ 27

Author(s):

F Coulier ◽

D Martin-Zanca ◽

M Ernst ◽

M Barbacid

Keyword(s):

Tyrosine Kinase ◽

Deleterious Effect ◽

Kinase Domain ◽

Tyrosine Kinase Receptor ◽

Beta Globin ◽

Amino Terminal ◽

Tropomyosin Isoforms ◽

Beta Actin ◽

Transforming Activity

The human trk oncogene was generated by a genetic rearrangement that replaced the extracellular domain of the normal trk tyrosine kinase receptor by sequences coding for the 221 amino-terminal residues of a nonmuscle tropomyosin. Molecular dissection of a cDNA clone of the trk oncogene indicated that both the tropomyosin and tyrosine kinase domains were required for proper transforming activity. Replacement of nonmuscle tropomyosin sequences with those of other tropomyosin isoforms had no deleterious effect. However, when tropomyosin sequences were replaced with those of another cytoskeletal gene, such as beta-actin or beta-globin, transforming activity was completely abolished. These results illustrate the important role of tropomyosin sequences in endowing the trk kinase with transforming properties. Functionally unrelated subdomains of the tropomyosin molecule were equally efficient in activating the trk gene. Moreover, the transforming activity of the trk oncogene was not affected when its subcellular localization was drastically altered. Therefore, tropomyosin sequences are likely to contribute to the malignant activation of the trk oncogene not by facilitating its interaction with defined cytoskeletal structures as initially suspected, but by allowing its kinase domain to fold into a constitutively active configuration.

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