Biomedical subjects

Dale S Haines

Publications and source records attributed to Dale S Haines.

11 recordsLinked to original sources

PubMed2007

The proteomic reactor facilitates the analysis of affinity-purified proteins by mass spectrometry: application for identifying ubiquitinated proteins in human cells.

Mass spectrometry (MS) coupled to affinity purification is a powerful approach for identifying protein-protein interactions and for mapping post-translational modifications. Prior to MS analysis, affinity-purified proteins are typically separated by gel electrophoresis, visualized with a protein stain, excised, and subjected to in-gel digestion. An inherent limitation of this series of steps is the loss of protein sample that occurs during gel processing. Although methods employing in-solution digestion have been reported, they generally suffer from poor reaction kinetics. In the present study, we demonstrate an application of a microfluidic processing device, termed the Proteomic Reactor, for enzymatic digestion of affinity-purified proteins for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Use of the Proteomic Reactor enabled the identification of numerous ubiquitinated proteins in a human cell line expressing reduced amounts of the ubiquitin-dependent chaperone, valosin-containing protein (VCP). The Proteomic Reactor is a novel technology that facilitates the analysis of affinity-purified proteins and has the potential to aid future biological studies.

Adenosine Triphosphatases↗

PubMed2005

The p270 (ARID1A/SMARCF1) subunit of mammalian SWI/SNF-related complexes is essential for normal cell cycle arrest.

Mammalian SWI/SNF-related complexes are ATPase-powered nucleosome remodeling assemblies crucial for proper development and tissue-specific gene expression. The ATPase activity of the complexes is also critical for tumor suppression. The complexes contain seven or more noncatalytic subunits; only one of which, hSNF5/Ini1/BAF47, has been individually identified as a tumor suppressor thus far. The noncatalytic subunits include p270/ARID1A, which is of particular interest because tissue array analysis corroborated by screening of tumor cell lines indicates that p270 may be deficient in as many as 30% of renal carcinomas and 10% of breast carcinomas. The complexes can also include an alternative ARID1B subunit, which is closely related to p270, but the product of an independent gene. The respective importance of p270 and ARID1B in the control of cell proliferation was explored here using a short interfering RNA approach and a cell system that permits analysis of differentiation-associated cell cycle arrest. The p270-depleted cells fail to undergo normal cell cycle arrest on induction, as evidenced by continued synthesis of DNA. These lines fail to show other characteristics typical of arrested cells, including up-regulation of p21 and down-regulation of cyclins. The requirement for p270 is evident separately in both the up-regulation of p21 and the down-regulation of E2F-responsive products. In contrast, the ARID1B-depleted lines behaved like the parental cells in these assays. Thus, p270-containing complexes are functionally distinct from ARID1B-containing complexes. These results provide a direct biological basis to support the implication from tumor tissue screens that deficiency of p270 plays a causative role in carcinogenesis.

3T3 Cells↗

PubMed2004

A single PXY motif located within the carboxyl terminus of Spt23p and Mga2p mediates a physical and functional interaction with ubiquitin ligase Rsp5p.

Proteasome-dependent processing of the endoplasmic reticulum localized transcription factor Spt23p of Saccharomyces cerevisiae generates its transcriptionally competent form and requires the WW domain containing Rsp5p ubiquitin ligase. Although previous studies documented an Rsp5p-Spt23p association in cells, very little is known about the nature of this interaction. We report here the identification of an imperfect type I WW domain-binding site (LPKY) within the carboxyl-terminal region of Spt23p that is required for Rsp5p binding in vitro and in vivo. Deletion of this motif abrogates Rsp5p-induced ubiquitination of Spt23p in vitro and reduces ubiquitination of the Spt23p precursor in yeast. In addition, the Spt23pDeltaLPKY mutant is inefficiently processed and is defective at up-regulating target gene (OLE1) expression in cells. Deletion of the corresponding LPKY site within Mga2p, an Spt23p homologue, also abrogates Rsp5p binding and Rsp5p-dependent ubiquitination in vitro as well as Rsp5p binding and Mga2p polyubiquitination in cells. However, the Mga2pDeltaLPKY mutant undergoes efficient proteasome-dependent processing. These experiments indicate that the LPKY motif of Spt23p is required for Rsp5p binding, Rsp5-induced ubiquitination, proteasome-dependent processing, and its OLE1 inducing function. They also suggest that the LPKY motif of Mga2p is required for Rsp5p binding and ubiquitination, and Rsp5p regulates Mga2p function by a mechanism that is independent of providing the partial degradation signal.

Amino Acid Motifs↗

PubMed2004

Ub on the move.

Ubiquitination is an increasingly common post-translation modification that controls both the expression and activity of numerous proteins in the eukaryotic cell. One frequent target of the ubiquitin (Ub) modification machinery is transcription factors. Although ubiquitination generally modulates their function by inducing proteasome-dependent degradation, past and recent studies indicate that ubiquitination also regulates nuclear-cytoplasmic trafficking of transcriptional regulators. Ubiquitination is known to modulate transcription factor localization by destroying sequestering proteins and by directly promoting nuclear import and export of modified substrates. This review discusses old and new paradigms relating Ub modification and the control of transcription factor shuttling in and out of the nucleus.

Humans↗

PubMed2003

Rsp5p is required for ER bound Mga2p120 polyubiquitination and release of the processed/tethered transactivator Mga2p90.

A number of eukaryotic transcription factors are held in a latent state by being embedded in, or tethered to, cellular membranes. Mga2p of Saccharomyces cerevisiae is an endoplasmic reticulum (ER)-localized transcription factor that plays an overlapping role with homologous Spt23p in upregulating expression of OLE1, a gene required for the synthesis of essential oleic acid. Previous studies have documented that proteasome-dependent processing of ER bound 120 kDa Mga2p and Spt23p proteins generates transcriptionally competent 90 kDa polypeptides. In the case of Spt23p90, it is held at the membrane prior to release via a self-interaction with the unprocessed Spt23p120 anchor. It is currently thought that the highly conserved Rsp5p ubiquitin ligase provides the signal for partial degradation of both proteins. Cells lacking Rsp5p function require oleic acid for growth, and Spt23p processing is suppressed in rsp5 Delta cells and in wild-type RSP5 cells upon expression of Rsp5p dominant-negative mutants. We report here that Rsp5p is dispensable for Mga2p90 generation but not for release of the processed product from the ER. In addition, we demonstrate that polyubiquitinated Mga2p120 accumulates in cells lacking Npl4p or proteasome function and Rsp5p is required for Mga2p120 polyubiquitination. Finally, we provide evidence that Mga2p90 and Mga2p120 dimerize and that Rsp5p binds heterodimeric Mga2p complexes both in vitro and in vivo. In light of these experiments, we propose that Rsp5p facilitates Mga2p90 release from the ER by promoting polyubiquitination and Npl4p-proteasome-mediated degradation of the interacting Mga2p120 ER bound anchor.

Blotting, Western↗

PubMed2003

Stimulation of human DNA polymerase epsilon by MDM2.

The human DNA polymerase epsilon catalytic subunit consists of a 140-kDa N-terminal domain that contains the catalytic activity and a 120-kDa C-terminal domain that binds to the other subunits and to exogenous peptides, including PCNA and MDM2. We report here that recombinant human MDM2 purified from insect cells or Escherichia coli stimulated the activity of DNA polymerase epsilon up to 10- and 40-fold, respectively, but not those of DNA polymerase beta or Klenow fragment of E.coli DNA polymerase I. Kinetic studies indicated that MDM2 increased the maximum velocity of the reaction, but did not change substrate affinities. The stimulation depended upon the interaction of the N-terminal 166 amino acid residues of MDM2 with the C-terminal domain of the full-length catalytic subunit, since the deletion of 166 amino acids from N-terminal of MDM2 or the removal of the C-terminal domain of DNA polymerase epsilon by trypsin digestion or competition for binding to it by the addition of excess C-terminal fragment eliminated the stimulation. Since DNA polymerase epsilon appears to be involved in DNA replication, recombination and repair synthesis, we suggest that MDM2 binding to DNA polymerase epsilon might be part of a reconfiguration process that allows DNA polymerase epsilon to associate with repair/recombination proteins in response to DNA damage.

Animals↗

PubMed2003

SKP2 associates with p130 and accelerates p130 ubiquitylation and degradation in human cells.

p130 is a member of the retinoblastoma family of pocket proteins, which includes pRB and p107. Unlike pRB and p107, p130 protein levels decrease dramatically following its hyperphosphorylation starting in the mid-G1 phase of the cell cycle. However, the mechanism leading to p130 downregulation is unknown. We have found that the proteasome inhibitor, lactacystin, inhibited p130 downregulation in T98G cells progressing through the G1/S transition and S phase and that p130 is multiubiquitylated in 293 cells. We have previously shown that ectopic expression of both cyclin D and E induces phosphorylation and downregulation of p130. Since the SKP1/Cul1/SKP2 E3 ubiquitin ligase complex mediates ubiquitylation of substrates previously phosphorylated by cyclin-dependent kinases, we investigated the potential role of this ubiquitin ligase in mediating p130 downregulation. We found that p130 interacts with SKP1, Cul-1 and SKP2 in human 293 cells. We also found that ectopic coexpression of SKP2 and p130 leads to dose-dependent downregulation of p130, reduces p130 protein half-life and induces p130 ubiquitylation in these cells. Moreover, adenoviral-mediated expression of SKP2 accelerates downregulation of endogenous hyperphosphorylated p130 in mitogen-stimulated T98G cells and primary WI38 fibroblasts. We conclude that p130 is a substrate of the SCF(SKP2) ubiquitin ligase and this E3 ligase regulates p130 abundance during the cell cycle.

Cell Cycle Proteins↗

PubMed2003

Functional analysis of the human orthologue of the RSP5-encoded ubiquitin protein ligase, hNedd4, in yeast.

hNedd4 and Rsp5p are orthologous ubiquitin ligases that contain a catalytic Hect domain, a C2 domain and multiple WW domains that mediate interactions with proteins. hNedd4 associates with the epithelial sodium channel and mutations disrupting this interaction lead to Liddle's syndrome, a heritable hypertension. Yeast Rsp5p ubiquitinates plasma membrane receptors and transporters and regulates their endocytosis. To determine whether the human enzyme has activity in yeast, hNEDD4 was expressed in yeast from the RSP5 or GAL1/10 promoters. Ectopic hNedd4 improved the growth and partially suppressed the endocytosis defect of rsp5 mutant cells, although it did not restore the viability of the rsp5-delta strain. Wild-type cells harboring hNedd4 grew better at elevated temperature and on media containing cycloheximide. In contrast, hNedd4 WW domain mutants inhibited the growth of yeast when expressed at high levels. Our results show that hNedd4 affects cell growth, endocytosis and cycloheximide tolerance of yeast cells.

Antifungal Agents↗

PubMed2003

CDK9 is constitutively expressed throughout the cell cycle, and its steady-state expression is independent of SKP2.

CDK9 is a CDC2-related kinase and the catalytic subunit of the positive-transcription elongation factor b and the Tat-activating kinase. It has recently been reported that CDK9 is a short-lived protein whose levels are regulated during the cell cycle by the SCF(SKP2) ubiquitin ligase complex (R. E. Kiernan et al., Mol. Cell. Biol. 21:7956-7970, 2001). The results presented here are in contrast to those observations. CDK9 protein levels remained unchanged in human cells entering and progressing through the cell cycle from G(0), despite dramatic changes in SKP2 expression. CDK9 levels also remained unchanged in cells exiting from mitosis and progressing through the next cell cycle. Similarly, the levels of CDK9 protein did not change as cells exited the cell cycle and differentiated along various lineages. In keeping with these observations, the kinase activity associated with CDK9 was found to not be regulated during the cell cycle. We have also found that endogenous CDK9 is a very stable protein with a half-life (t(1/2)) of 4 to 7 h, depending on the cell type. In contrast, when CDK9 is overexpressed, it is not stabilized and is rapidly degraded, with a t(1/2) of less than 1 h, depending on the level of expression. Treatment of cells with proteasome inhibitors blocked the degradation of short-lived proteins, such as p27, but did not affect the expression of endogenous CDK9. Ectopic overexpression of SKP2 led to reduction of p27 protein levels but had no effect on the expression of endogenous CDK9. Finally, downregulation of endogenous SKP2 gene expression by interfering RNA had no effect on CDK9 protein levels, whereas p27 protein levels increased dramatically. Therefore, the SCF(SKP2) ubiquitin ligase does not regulate CDK9 expression in a cell cycle-dependent manner.

Acetylcysteine↗

PubMed2002

Substrate proteolysis is inhibited by dominant-negative Nedd4 and Rsp5 mutants harboring alterations in WW domain 1.

Mammalian Nedd4 and its budding yeast orthologue Rsp5 are members of a large family of HECT-domain-containing ubiquitin ligases. Besides possessing a Ca(2+)/lipid-binding domain, both ligases have multiple protein-interacting modules termed WW domains. The C-terminal WW domains mediate interactions with substrates, but the function of the first WW domain remains unclear. We found that expression of a WW domain 1 Nedd4 mutant inhibits the growth of budding yeast by affecting the rsp5-ole1 pathway. The WW domain 1 mutant-induced phenotype is suppressed by ole1 cDNA overexpression or oleic acid supplementation of growth media and ole1 RNA levels are reduced in cells expressing this Nedd4 mutant. Also, the WW domain 1 Nedd4 mutant associates via WW domains 2 and 3 with Spt23, a Rsp5 target and ole1 transactivator. The dominant-negative activity of this mutant is associated with promoting accumulation of unprocessed Spt23 and inhibiting generation of processed and presumably active protein. Also, Spt23 processing is inhibited by a Nedd4 mutant that lacks ubiquitin ligase activity and Spt23-binding-competent Rsp5 mutants harboring WW domain 1 or ligase domain mutations. Interestingly, in mammalian cells, wild-type Nedd4 promotes proteasome-mediated degradation of the precursor form of Spt23. WW domain 1 and ligase domain Nedd4 mutants block its degradation. These results indicate that WW domain 1 of these ligases interacts with cofactors that are required for ubiquitin/proteasome-dependent proteolysis of bound substrates.

Calcium-Binding Proteins↗

PubMed2002

Diminished expression of the type II receptor for TGFbeta (TGFbetaRII) in T lymphocytes from patients with Sezary syndrome is not due to mutations in the receptor's poly-A tract: limitations of the standard RT-PCR in cDNA sequence analysis of homopolymeric base stretches.

Peripheral blood lymphocytes from patients with Sezary syndrome (SzS) frequently demonstrate decreased surface expression of transforming growth factor beta receptor II (TGFbetaRII). The mechanism of this low TGFbetaRII expression remains unknown. Because mutations within the poly-A tract of the TGFbetaRII sequence (nucleotides 709-718) were shown to result in diminished TGFbetaRII expression in other types of malignant tumors, we examined the sequence of the TGFbetaRII poly-A tract in two SzS-derived cell lines and in peripheral blood SzS cells from 17 SzS patients and 4 control, healthy individuals using DNA sequencing and single-stranded conformation polymorphism (SSCP) analysis. A standard bidirectional, automated sequence analysis of the RT-PCR-generated cDNA TGFbetaRII fragment showed a heterogenous population of the normal length, 10-, with admixed, shortened, 9-base poly-A stretches. Surprisingly, this mixture was present not only in the cells from 5 SzS patients and 2 SzS cell lines, but also in cells from 2 healthy control individuals. Importantly, the proportion of the shortened, 9-base fragments was markedly reduced or practically eliminated when the procedure was modified by usage of high-fidelity DNA polymerase, labeled primers and/or cloned RT-PCR products, which indicates that the presence of the shortened, 9-base fragments represented a procedural phenomenon rather than a true deletional mutation within an allele of the TGFbetaRII gene. Accordingly, SSCP analysis of genomic DNA did not reveal any mutations within the poly-A tract-containing region. These results indicate that a mechanism different from mutations in the polyadenine tract underlies the diminished TGFbetaRII expression in SzS cells and that the results of an unmodified, direct sequence analysis of homopolymeric base streaches in RT-PCR-derived cDNA should be interpreted with caution.

Base Sequence↗