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K Ferrell

Publications and source records attributed to K Ferrell.

At least 19 recordsLinked to original sources
PubMed2000

Analysis of a gene encoding Rpn10 of the fission yeast proteasome reveals that the polyubiquitin-binding site of this subunit is essential when Rpn12/Mts3 activity is compromised.

Substrates are targeted for proteolysis by the ubiquitin pathway by the addition of a polyubiquitin chain before being degraded by the 26 S proteasome. Previously, a subunit of the proteasome, S5a, was identified that was able to bind to polyubiquitin in vitro and thus proposed to act as a substrate recognition component. Deletion of the corresponding Saccharomyces cerevisiae gene, MCB1/RPN10, rendered cells viable indicating that other proteasomal polyubiquitin receptors must exist. In this study, we describe pus1(+), the fission yeast homologue of RPN10. This gene is also not required for cell viability; however, the Deltapus1 mutant is synthetically lethal with mutations in other proteasomal component-encoding genes, namely mts3, pad1, and mts4 (RPN12, RPN11, and RPN1). Overexpression of pus1(+) is able to rescue mts3-1 at 32 degrees C but overexpression of a cDNA encoding a version of Pus1 that does not bind to polyubiquitin cannot and leads to greatly reduced viability when used to rescue the mts3-1Deltapus1 double mutant. The Mts3 protein was unable to bind to polyubiquitin in vitro, but the Pus1 and Mts3 proteins were found to bind to one another in vitro, which taken together with the genetic data suggests that they are also closely associated in vivo.

Amino Acid Sequence↗
PubMed2000

Regulatory subunit interactions of the 26S proteasome, a complex problem.

The 26S proteasome is the major non-lysosomal protease in eukaryotic cells. This multimeric enzyme is the integral component of the ubiquitin-mediated substrate degradation pathway. It consists of two subcomplexes, the 20S proteasome, which forms the proteolytic core, and the 19S regulator (or PA700), which confers ATP dependency and ubiquitinated substrate specificity on the enzyme. Recent biochemical and genetic studies have revealed many of the interactions between the 17 regulatory subunits, yielding an approximation of the 19S complex topology. Inspection of interactions of regulatory subunits with non-subunit proteins reveals patterns that suggest these interactions play a role in 26S proteasome regulation and localization.

Adenosine Triphosphatases↗
PubMed1999

COP9 signalosome-directed c-Jun activation/stabilization is independent of JNK.

The basic region-leucine zipper transcription factor c-Jun regulates gene expression and cell function. It participates in the formation of homo- or heterodimeric complexes that specifically bind to DNA sequences called activating protein 1 (AP-1) sites. The stability and activity of c-Jun is regulated by phosphorylation within the N-terminal activation domain. Mitogen- and stress-activated c-Jun N-terminal kinases (JNKs) were previously the only described enzymes phosphorylating c-Jun at the N terminus in vivo. In this report we demonstrate a JNK-independent activation of c-Jun in vivo directed by the constitutive photomorphogenesis (COP9) signalosome. The overexpression of signalosome subunit 2 (Sgn2), a subunit of the COP9 signalosome, leads to de novo assembly of the complex with a partial incorporation of the overexpressed subunit. The de novo formation of COP9 signalosome parallels an increase of c-Jun protein resulting in elevated AP-1 transcriptional activity. The c-Jun activation caused by Sgn2 overexpression is independent of JNK and mitogen-activated protein kinase kinase 4. The data demonstrate the existence of a novel COP9 signalosome-directed c-Jun activation pathway.

COP9 Signalosome Complex↗
PubMed1999

Hemizygosity for the COP9 signalosome subunit gene, SGN3, in the Smith-Magenis syndrome.

Smith-Magenis syndrome (SMS) is a multiple congenital anomaly/mental retardation syndrome associated with an interstitial deletion of chromosome band 17p11.2. The critical region is extremely gene-rich and spans approximately 1.5-2.0 Mb of DNA. Here we report the localization and partial characterization of the gene for subunit 3 of the COP9 signalosome, SGN3. SGN3 maps to the distal portion of the SMS critical interval, between SREBF1 and cCI17-638. We assessed the potential effect of haploinsufficiency of SGN3 in SMS patient lymphoblastoid cell lines through transfection studies and western analysis. Our results indicate that the COP9 signalosome assembles properly in these cells and appears to have normal expression and a kinase function intact. However, because the role of the COP9 signalosome in embryogenesis or differentiation is still uncertain, we cannot rule out the involvement of this gene in the Smith-Magenis syndrome.

Abnormalities, Multiple↗
PubMed1999

Comparison of human COP9 signalsome and 26S proteasome lid'.

The human core COP9 signalosome consists of eight subunits which have been identified, cloned and sequenced. The components of COP9 signalosome possess homologies with eight non-ATPase regulatory subunits of the 26S proteasome. These polypeptides of the 19S regulator form a reversibly binding subcomplex called the 'lid'. We isolated the 'lid' from human red blood cells and compared it with the COP9 signalosome complex. In addition to the non-ATPase regulatory polypeptides, we found a high molecular mass ATPase copurifying with the human 'lid'. The COP9 signalosome-associated kinase activity is either not at all or only weakly affected by common kinase inhibitors such as 1-(5-Isoquinolinesulfonyl)-2-methyl-piperazine (H7), 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB) or Wortmannin. Curcumin, a tumor suppressor and effector of AP-1 activation, is a potent inhibitor of the COP9 signalosome kinase activity with a Ki of about 10 microM. Since curcumin is known as an inhibitor of the c-Jun N-terminal kinase (JNK) signaling pathway acting upstream of the MAP kinase kinase kinase level, one site of action of the COP9 signalosome might be proximal to regulators on that level.

Animals↗
PubMed1998

The Pad1+ gene encodes a subunit of the 26 S proteasome in fission yeast.

We have isolated a fission yeast mutant, mts5-1, in a screen for mutations that confer both methyl 2-benzimidazolecarbamate resistance (MBCR) and temperature sensitivity (ts) on Schizosaccharomyces pombe. This screen has previously isolated mutations in the 26 S proteasome subunits Mts2, Mts3, and Mts4. We show that the mutation in the mts5-1 strain occurs in the pad1(+) gene. pad1(+) was originally isolated on a multicopy plasmid that was capable of conferring staurosporine resistance on a wild type strain. mts5-1/pad1-1 has a similar phenotype to 26 S proteasome mutants previously isolated in the same screen and we show that Pad1 interacts genetically with two of these subunits, Mts3 and Mts4. In this study we describe the identification of Pad1 as a subunit of the 26 S proteasome in fission yeast.

Amino Acid Sequence↗
PubMed1998

A novel protein complex involved in signal transduction possessing similarities to 26S proteasome subunits.

A novel protein complex has been identified in human cells that has a molecular mass of approximately 450 kDa. It consists of at least eight different subunits including JAB1, the Jun activation-domain binding protein 1, and Trip15, the thyroid hormone receptor-interacting protein 15. The purified complex contains COP9 and COP11 protein homologs and is very similar, if not identical, to the plant COP9 complex involved in light-mediated signal transduction. The isolated JAB1-containing particle has kinase activity that phosphorylates IkappaBalpha, the carboxy terminus of p105, and Ser63 and/or Ser73 of the amino-terminal activation domain of c-Jun. The phosphorylation of c-Jun requires the carboxy terminus of the protein containing the DNA binding and dimerization domains. Three subunits of the new complex--Sgn3, Sgn5/JAB1, and Sgn6--exhibit sequence similarities to regulatory components of the 26S proteasome, which could indicate the existence of common substrate binding sites. Immunofluorescence staining reveals that the new complex shows a subcellular distribution similar to that of the 26S proteasome. The functional relationship of the two particles in regulating transcriptional activity is discussed. Considering the putative role of the complex in signal transduction and its widespread occurrence, we suggest the name JAB1-containing signalosome.

Amino Acid Sequence↗
PubMed1997

HIV-1 tat inhibits the 20 S proteasome and its 11 S regulator-mediated activation.

The proteasomal system consists of a proteolytic core, the 20 S proteasome, which associates in an ATP-dependent reaction with the 19 S regulatory complex to form the functional 26 S proteasome. In the absence of ATP, the 20 S proteasome forms a complex with the gamma-interferon-inducible 11 S regulator. Both the 20 S proteasome and the 11 S regulator have been implied in the generation of antigenic peptides. The human immunodeficiency virus (HIV)-1 Tat protein causes a number of different effects during acquired immunodeficiency syndrome (AIDS). Here we show that HIV-1 Tat protein strongly inhibits the peptidase activity of the 20 S proteasome and that it interferes with formation of the 20 S proteasome-11 S regulator complex. In addition, it slightly increases the activity of purified 26 S proteasome. These results may explain the mechanism by which HIV-1-infected cells escape cytotoxic T lymphocyte response and at least in part immunodeficiency in AIDS patients.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1997

The 26S proteasome: a dynamic structure.

The proteasomal system consists of a proteolytic core, the 20S proteasome, which associates in ATP-dependent and independent reactions with endogenous regulators providing specific substrate binding sites, chaperone function and regulation of activity to the protease. The best known regulators of the 20S proteasome are the 11S and the 19S complexes. Three subunits of the 20S proteasome and the two subunits of the 11S regulator are induced by gamma-Interferon. However, there are no indications for an influence of gamma-interferon on the subunit composition of the 19S regulator and only a few data exist about the dynamics of this complex. The analysis of 19S regulator subunits from yeast mutants reveals that the ATPases appear to be stringently organized in the 26S complex, while peripheral non-ATPases, such as S5a, might serve as subunits which shuttle substrates to the enzyme. A novel non-ATPase has been cloned, sequenced and identified in a complex besides the 19S regulator, the function of which is presently unknown. The dynamic structure of the 26S proteasome is also characterized by transient associations with components such as the modulator and isopeptidases. Certain viral proteins can also be associated with components of the proteasomal system and alter enzymatic activities.

Humans↗
PubMed1996

Characteristics of 26 S proteases from fission yeast mutants, which arrest in mitosis.

We have isolated the 26 S protease from the fission yeast Schizosaccharomyces pombe. The affinity-purified enzyme contains the two regulatory ATPases mts2+, a homolog of human S4, and CIM5, a homolog of human MSS1 = S7. We show that mts3+, a homolog of the budding yeast NIN1 protein and human S14, is a true component of the 19 S regulatory complex from the fission yeast. The 26 S proteases purified from two thermosensitive mutants, mts2-1 and mts3-1, which arrest in cell cycle at the restrictive temperature (37 degrees C), have been compared with the wild-type enzyme after growing cells at permissive (25 degrees C) and non-permissive temperatures. We demonstrate that mutated mts2 protein is integrated into the protease complex prepared from mts2 cells, whereas mutated mts3 is not present in the 19 S regulatory complex from mts3 cells. The two mutant 26 S proteases isolated after growing cells at 37 degrees C remain stable for two hours at 37 degrees C as measured by ATP-dependent cleavage of the fluorogenic peptide sucLLVY-MCA. At the restrictive temperature, the mutant 26 S proteases do not degrade ubiquitin-[125I]lysozyme conjugates in an ATP-dependent manner, indicating that mts2+ and mts3+ are essential for ubiquitin conjugate degradation. This explains the conditional lethality of the mutants and the cell-cycle arrest in metaphase to anaphase transition. In addition, our data demonstrate that the ATPases of the 26 S enzyme are not redundant.

Adenosine Triphosphate↗
PubMed1996

Molecular cloning and expression of a multiubiquitin chain binding subunit of the human 26S protease.

S5a is a subunit of the 26S protease that binds and presumably selects multiubiquitinated proteins for destruction. We recently identified an Arabidopsis protein, MBP1, that is physically, immunologically and biochemically similar to S5a from the human erythrocyte 26S protease. Based upon the MBP1 cDNA sequence we have now isolated a HeLa cell cDNA coding for human S5a. The HeLa cDNA sequence is highly similar to MBP1 and it encodes peptides obtained directly from human erythrocyte S5a. Moreover, expression of the isolated cDNA in E. coli results in a recombinant protein with an apparent molecular mass and multiubiquitin binding properties that match those of human S5a obtained from the purified 26S enzyme.

Amino Acid Sequence↗
PubMed1995

Molecular cloning and expression of subunit 12: a non-MCP and non-ATPase subunit of the 26 S protease.

A cDNA encoding subunit 12 (S12) of human erythrocyte 26 S protease has been isolated, sequenced and expressed. The cDNA contains an open reading frame that encodes a 36.6 kDA protein 96% identical to mouse Mov-34 and 67% identical to its Drosophila melanogaster homolog. Based on the high degree of sequence identity between human S12, mouse and Drosophila Mov-34 proteins, we conclude that the Mov-34 gene product is a component of the 26 S protease. Antibodies produced against two S12 fragments, Met1-Tyr95 (S12f95) and Met1-Leu205 (S12f205), react with S12 transferred to nitrocellulose from SDS-PAGE. In contrast, after transfer from native gels, the epitope(s) recognized by anti-S12f205 is exposed in the regulatory complex but appears to be masked when the regulatory complex associates with the multicatalytic protease.

Amino Acid Sequence↗
PubMed1994

Molecular cloning and expression of a gamma-interferon-inducible activator of the multicatalytic protease.

The multicatalytic protease (MCP) can be activated by two distinct multisubunit complexes. One is the regulatory component of the 26 S protease, which contains at least 15 distinct subunits. The other is a hexameric activator composed of 31- and 29-kDa subunits. A cDNA for the smaller subunit has been cloned and sequenced. The cDNA encodes a protein of 249 amino acids. Embedded between sequences typical of globular protein domains is a stretch of 28 "alternating" lysine and glutamic acid residues. Similar regions, which we call KEKE motifs, are also found in two MCP subunits, in subunit 12 of the 26 S protease and in a variety of chaperonins including hsp90, hsp70, and calnexin. Expression of the activator cDNA in Escherichia coli produced a functional protein virtually indistinguishable from MCP activator purified directly from red blood cells. The recombinant protein formed three isoelectric species on two-dimensional polyacrylamide gel electrophoresis, and it reacted with antibodies to red blood cell activator. Recombinant activator also bound the multicatalytic protease and stimulated cleavage at the carboxyl terminus of hydrophobic or charged residues. Synthesis of the activator subunit was induced by gamma interferon treatment of HeLa cells. These last two findings have implications for antigen presentation by class I major histocompatibility receptors.

Amino Acid Sequence↗
PubMed1994

Tat-binding protein 7 is a subunit of the 26S protease.

Subunit 6 (S6), an integral component of the 26S protease from human erythrocytes, has been studied by SDS-PAGE, peptide mapping and sequence analysis. S6 was cleaved with CNBr and three internal peptides were sequenced. A comparison with known proteins in Genbank revealed that all three S6 peptides match the predicted sequence of TBP7, Tat-binding protein 7. Based on peptide matches covering more than 10% of the TBP7 sequence, and the fact that the migration of S6 on SDS-PAGE is consistent with the estimated molecular mass for TBP7, we conclude that subunit 6 of the 26S protease is TBP7.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1993

Peptide sequencing identifies MSS1, a modulator of HIV Tat-mediated transactivation, as subunit 7 of the 26 S protease.

Subunit 7 is an integral component of the human erythrocyte 26 S protease. Peptide sequence analysis reveals that 22 amino acids from the N-terminus of subunit 7 correspond exactly to the N-terminus of MSS1, a modulator of HIV gene expression. Additional internal peptides from subunit 7 obtained by CNBr cleavage also match 100% with the deduced amino acid sequence of MSS1. Based on the fact that directly sequenced peptides from subunit 7 are identical to more than 12% of the hypothetical translation product of MSS1, and the fact that the molecular weight of subunit 7 (49 kDa) corresponds to the predicted molecular weight of MSS1 (48,633 Da), we conclude that subunit 7 is MSS1.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1992

Subunit 4 of the 26 S protease is a member of a novel eukaryotic ATPase family.

Ubiquitinated proteins are degraded by a 26 S ATP-dependent protease. SDS-polyacrylamide gel electrophoresis analysis of the purified 26 S enzyme reveals more than 20 polypeptides ranging in apparent molecular masses from 20 to 110 kDa. Although many of the subunits smaller than 30 kDa are members of the multicatalytic protease family, the identity and function of the larger polypeptides have remained unknown. We report here the cDNA sequence for subunit 4, a 51-kDa chain of the 26 S protease. Subunit 4 belongs to a recently identified eukaryotic ATPase family, which includes proteins involved in peroxisome formation, secretion, and human immunodeficiency virus gene expression. Subunit 4 also shows weak similarity to ClpA, the ATP-binding subunit of the Escherichia coli protease, Clp.

Adenosine Triphosphatases↗
PubMed1992

Purification of an 11 S regulator of the multicatalytic protease.

We have identified and purified a protein complex from human red blood cells that activates the multicatalytic protease (MCP). The complex, which we call the regulator, sediments at 11 S and is composed of 30-kDa subunits. The regulator does not hydrolyze fluorogenic peptides, but when multicatalytic protease and regulator are combined, MCP cleaves succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin and Leu-Leu-Glu-p-nitroanilide as much as 60-fold faster. Hydrolysis of several other fluorogenic peptides is stimulated to a lesser extent, and activated MCP does not degrade ubiquitin-lysozyme conjugates, bovine serum albumin, or lysozyme. Latent and activated forms of MCP display similar sensitivity to protease inhibitors, suggesting that activation does not generate new kinds of catalytic sites. In addition, ATP suppresses peptide hydrolysis by activated and latent MCPs to the same extent. Activation involves binding of regulator to MCP, and activated MCP migrates slower on native acrylamide gels. Dissociation of the MCP regulator complex during prolonged sedimentation on glycerol gradients releases active regulator and MCP molecules capable of being reactivated. Moreover, two-dimensional electrophoresis does not reveal changes in MCP or regulator subunits following activation. Thus, activation appears to result from reversible association of regulator subunits with MCP.

Adenosine Triphosphate↗