Biomedical subjects

Huilin Zhou

Publications and source records attributed to Huilin Zhou.

14 recordsLinked to original sources

PubMed2006

Checkpoint proteins control morphogenetic events during DNA replication stress in Saccharomyces cerevisiae.

In response to DNA replication stress in Saccharomyces cerevisiae, the DNA replication checkpoint maintains replication fork stability, prevents precocious chromosome segregation, and causes cells to arrest as large-budded cells. The checkpoint kinases Mec1 and Rad53 act in this checkpoint. Treatment of mec1 or rad53Delta mutants with replication inhibitors results in replication fork collapse and inappropriate partitioning of partially replicated chromosomes, leading to cell death. We describe a previously unappreciated function of various replication stress checkpoint proteins, including Rad53, in the control of cell morphology. Checkpoint mutants have aberrant cell morphology and cell walls, and show defective bud site selection. Rad53 shows genetic interactions with septin ring pathway components, and, along with other checkpoint proteins, controls the timely degradation of Swe1 during replication stress, thereby facilitating proper bud growth. Thus, checkpoint proteins play an important role in coordinating morphogenetic events with DNA replication during replication stress.

Actins↗

PubMed2006

An FHA domain-mediated protein interaction network of Rad53 reveals its role in polarized cell growth.

The DNA damage checkpoint kinase Rad53 is important for the survival of budding yeast under genotoxic stresses. We performed a biochemical screen to identify proteins with specific affinity for the two Forkhead associated (FHA) domains of Rad53. The N-terminal FHA1 domain was found to coordinate a complex protein interaction network, which includes nuclear proteins involved in DNA damage checkpoints and transcriptional regulation. Unexpectedly, cytosolic proteins involved in cytokinesis, including septins, were also found as FHA1 binding proteins. Consistent with this interaction, a Rad53 mutant defective in its nuclear localization was found to localize to the bud neck. Abnormal morphology was observed in cells overexpressing the FHA1 domain and in rad53Delta cells under DNA replication stress. Further, septin Shs1 appears to have an important role in the response to DNA replication stress. Collectively, the results suggest a novel function of Rad53 in the regulation of polarized cell growth in response to DNA replication stress.

Carrier Proteins↗

PubMed2006

Mechanism of Dun1 activation by Rad53 phosphorylation in Saccharomyces cerevisiae.

Despite extensive studies, the molecular mechanism of DNA damage checkpoint activation remains incompletely understood. To better dissect this mechanism, we developed an activity-based assay for Dun1, a downstream DNA damage check-point kinase in yeast, using its physiological substrate Sml1. Using this assay, we confirmed the genetic basis of Dun1 activation. Rad53 was found to be directly responsible for Dun1 activation. We reconstituted the activation of Dun1 by Rad53 and found that phosphorylation of Thr-380 in the activation loop of Dun1 by Rad53 is responsible for Dun1 activation. Interestingly, phosphorylation of the evolutionarily conserved Thr-354 in the activation loop of Rad53 is also important for the regulation of Rad53 activity. Thus, this conserved mode of activation loop phosphorylation appears to be a general mechanism for the activation of Chk2 family kinases.

Amino Acid Sequence↗

PubMed2006

Checkpoint functions are required for normal S-phase progression in Saccharomyces cerevisiae RCAF- and CAF-I-defective mutants.

The chromatin-assembly factor I (CAF-I) and the replication-coupling assembly factor (RCAF) complexes function in chromatin assembly during DNA replication and repair and play a role in the maintenance of genome stability. Here, we have investigated their role in checkpoints and S-phase progression. FACS analysis of mutants lacking Asf1 or Cac1 as well as various checkpoint proteins indicated that normal rates of S-phase progression in asf1 mutants have a strong requirement for replication checkpoint proteins, whereas normal S-phase progression in cac1 mutants has only a weak requirement for either replication or DNA-damage checkpoint proteins. Furthermore, asf1 mutants had high levels of Ddc2.GFP foci that were further increased in asf1 dun1 double mutants consistent with a requirement for checkpoint proteins in S-phase progression in asf1 mutants, whereas cac1 mutants had much lower levels of Ddc2.GFP foci that were not increased by a dun1 mutation. Our data suggest that RCAF defects lead to unstable replication forks that are then stabilized by replication checkpoint proteins, whereas CAF-I defects likely cause different types of DNA damage.

Cell Cycle Proteins↗

PubMed2006

Negative regulation of the E3 ubiquitin ligase itch via Fyn-mediated tyrosine phosphorylation.

Conjugation of ubiquitin (Ub) to a protein substrate targets the substrate for degradation or functional modification, which is tightly controlled by diverse mechanisms including phosphorylation of the substrate. An emerging mechanism involves regulation of the E3 Ub ligase, for example, the JNK-dependent phosphorylation and activation of Itch E3 ligase, which controls the turnover of Jun proteins and T cell differentiation. Here we show that Itch is also modulated by an Src kinase Fyn via tyrosine phosphorylation at the Tyr371 residue. Fyn associates with Itch, and loss of Fyn results in reduced Itch phosphorylation. Importantly, tyrosine phosphorylation of Itch appears to reduce its interaction with its substrate JunB. The turnover of JunB is accelerated in Fyn-deficient T cells, which is further reconstituted by Itch Tyr371 mutation. Thus, in contrast to the activation pathway mediated by serine/threonine phosphorylation, tyrosine phosphorylation of Itch plays a negative role in modulating Itch-promoted ubiquitination.

Amino Acid Sequence↗

PubMed2005

Mass spectrometric and kinetic analysis of ASF/SF2 phosphorylation by SRPK1 and Clk/Sty.

Assembly of the spliceosome requires the participation of SR proteins, a family of splicing factors rich in arginine-serine dipeptide repeats. The repeat regions (RS domains) are polyphosphorylated by the SRPK and Clk/Sty families of kinases. The two families of kinases have distinct enzymatic properties, raising the question of how they may work to regulate the function of SR proteins in RNA metabolism in mammalian cells. Here we report the first mass spectral analysis of the RS domain of ASF/SF2, a prototypical SR protein. We found that SRPK1 was responsible for efficient phosphorylation of a short stretch of amino acids in the N-terminal portion of the RS domain of ASF/SF2 while Clk/Sty was able to transfer phosphate to all available serine residues in the RS domain, indicating that SR proteins may be phosphorylated by different kinases in a stepwise manner. Both kinases bind with high affinity and use fully processive catalytic mechanisms to achieve either restrictive or complete RS domain phosphorylation. These findings have important implications on the regulation of SR proteins in vivo by the SRPK and Clk/Sty families of kinases.

Adenosine Triphosphate↗

PubMed2005

Tandem mass spectrometry identifies proteins phosphorylated by cyclic AMP-dependent protein kinase when sea urchin sperm undergo the acrosome reaction.

The exocytotic acrosome reaction (AR), which is required for fertilization, occurs when sea urchin sperm contact the egg jelly (EJ) layer. Among other physiological changes, increases in adenylyl cyclase activity, cAMP and cAMP-dependent protein kinase (PKA) activity occur coincident with the AR. By using inhibitors of PKA, a permeable analog of cAMP and the phosphodiesterase inhibitor IBMX, we show that PKA activity is required for AR induction by EJ. A minimum of six sperm proteins are phosphorylated by PKA upon exposure to EJ, as detected by a PKA substrate-specific antibody. The phosphorylation of these proteins and the percentage of acrosome reacted sperm can be regulated by PKA modulators. The fucose sulfate polymer (FSP), a major component of EJ, is the molecule that triggers sperm PKA activation. Extracellular Ca(2+) is required for PKA activation. Six sperm proteins phosphorylated by PKA were identified by tandem mass spectrometry (MS/MS) utilizing the emerging sea urchin genome. Based on their identities and localizations in sperm head and flagellum, the putative functions of these proteins in sperm physiology and AR induction are discussed.

Acrosome Reaction↗

PubMed2005

Cell junction-associated proteins IQGAP1, MAGI-2, CASK, spectrins, and alpha-actinin are components of the nephrin multiprotein complex.

Nephrin is a cell surface receptor of the Ig superfamily that localizes to slit diaphragms, the specialized junctions between the interdigitating foot processes of the glomerular epithelium (podocytes) in the kidney. Mutations in the NPHS1 gene encoding nephrin lead to proteinuria and congenital nephrotic syndrome, indicating that nephrin is essential for normal glomerular development and function. To identify nephrin-binding proteins, we performed mass spectrometry on proteins obtained from pull-down assays with GST-nephrin cytoplasmic domain. Nephrin specifically pulled down six proteins from glomerular lysates, MAGI-2/S-SCAM (membrane-associated guanylate kinase inverted 2/synaptic scaffolding molecule), IQGAP1 (IQ motif-containingGTPase-activatingprotein1),CASK(calcium/calmodulin-dependent serine protein kinase), alpha-actinin, alphaII spectrin, and betaII spectrin. All of these scaffolding proteins are often associated with cell junctions. By immunofluorescence these proteins are expressed in glomerular epithelial cells, where they colocalize with nephrin in the foot processes. During glomerular development, IQGAP1 is expressed in the junctional complexes between the earliest identifiable podocytes, MAGI-2/S-SCAM is first detected in junctional complexes in podocytes after their migration to the base of the cells. Thus, the nephrin-slit diaphragm protein complex contains a group of scaffolding proteins that function to connect junctional membrane proteins to the actin cytoskeleton and signaling cascades. Despite their special morphology and function, there is considerable compositional similarity between the podocyte slit diaphragm and typical junctional complexes of other epithelial cells.

Actinin↗

PubMed2005

Dynamic changes in protein-protein interaction and protein phosphorylation probed with amine-reactive isotope tag.

We present an approach for quantitative analysis of changes in the composition and phosphorylation of protein complexes by MS. It is based on a new class of stable isotope-labeling reagent, the amine-reactive isotope tag (N-isotag), for specific and quantitative labeling of peptides following proteolytic digestion of proteins. Application of the N-isotag method to the analysis of Rad53, a DNA damage checkpoint kinase in Saccharomyces cerevisiae, led to the identification of dynamic associations between Rad53 and the nuclear transport machinery, histones, and chromatin assembly proteins in response to DNA damage. Over 30 phosphorylation sites of Rad53 and its associated proteins were identified and quantified, and they showed different changes in phosphorylation in response to DNA damage. Interestingly, Ser789 of Rad53 was found to be a major initial phosphorylation site, and its phosphorylation regulates the Rad53 abundance in response to DNA damage. Collectively, these results demonstrate that N-isotag-based quantitative MS is generally applicable to study dynamic changes in the composition of protein complexes and their phosphorylation patterns in a site-specific manner in response to different cell stimuli.

Alanine↗

PubMed2004

Genome-wide lethality screen identifies new PI4,5P2 effectors that regulate the actin cytoskeleton.

To further understand the roles played by the essential phosphoinositide PI4,5P(2), we have used a synthetic lethal analysis, which systematically combined the mss4(ts) mutation, partially defective in PI4P 5-kinase activity, with each of approximately 4700 deletion mutations. This genomic screening technique uncovered numerous new candidate effectors and regulators of PI4,5P(2) in yeast. In particular, we identified Slm1 (Yil105c), a previously uncharacterized PI4,5P(2) binding protein. Like Mss4, Slm1 and its homolog Slm2 (Ynl047c) were required for actin cytoskeleton polarization and viability. Co-immunoprecipitation experiments revealed that Slm1 interacts with a component of TORC2, a Tor2 kinase-containing complex, which also regulates the actin cytoskeleton. Consistent with these findings, phosphorylation of Slm1 and Slm2 was dependent on TORC2 protein kinase activity, both in vivo and in vitro, and Slm1 localization required both PI4,5P(2) and functional TORC2. Together, these data suggest that Slm1 and Slm2 function downstream of PI4,5P(2) and the TORC2 kinase pathway to control actin cytoskeleton organization.

Actins↗

PubMed2004

Global analyses of sumoylated proteins in Saccharomyces cerevisiae. Induction of protein sumoylation by cellular stresses.

We have undertaken a global analysis of sumoylated proteins in Saccharomyces cerevisiae by tandem mass spectrometry. Exposure of cells to oxidative and ethanol stresses caused large increases in protein sumoylation. A large number of new sumoylated proteins were identified in untreated, hydrogen peroxide-treated, and ethanol-treated cells. These proteins are known to be involved in diverse cellular processes, including gene transcription, protein translation, DNA replication, chromosome segregation, metabolic processes, and stress responses. Additionally, the known enzymes, including E1, E2, and E3 of the sumoylation cascade were found to be auto-sumoylated. Taken together, these results show that protein sumoylation is broadly involved in many cellular functions and this mass spectrometry-based proteomic approach is useful in studying the regulation of protein sumoylation in the cells.

Blotting, Western↗

PubMed2004

Quantitative protein analysis by solid phase isotope tagging and mass spectrometry.

Here we describe a method for stable isotope labeling and solid-phase capture of cysteinyl peptides from complex protein mixtures. Site-specific, quantitative labeling of cysteine residues with tags that differ in isotopic content enables quantification of relative peptide abundance between samples. Labeling on a solid phase provides for simultaneous simplification of a complex peptide mixture by isolating cysteinyl, and subsequently tagged, peptides. Peptides from proteolytic digests of protein samples are labeled in preparation for analysis by microcapillary liquid chromatography and tandem mass spectrometry (microLC-MS/MS) to determine their sequences and relative abundance between samples. This approach enables rapid identification and accurate quantification of relative abundance of individual proteins from different biological contexts.

Amino Acid Sequence↗

PubMed2002

Quantitative proteome analysis by solid-phase isotope tagging and mass spectrometry.

The adaptation of sequences of chemical reactions to a solid-phase format has been essential to the automation, reproducibility, and efficiency of a number of biotechnological processes including peptide and oligonucleotide synthesis and sequencing. Here we describe a method for the site-specific, stable isotopic labeling of cysteinyl peptides in complex peptide mixtures through a solid-phase capture and release process, and the concomitant isolation of the labeled peptides. The recovered peptides were analyzed by microcapillary liquid chromatography and tandem mass spectrometry (microLC-MS/MS) to determine their sequences and relative quantities. The method was used to detect galactose-induced changes in protein abundance in the yeast Saccharomyces cerevisiae. A side-by-side comparison with the isotope-coded affinity tag (ICAT) method demonstrated that the solid-phase method for stable isotope tagging of peptides is comparatively simpler, more efficient, and more sensitive.

Chromatography, Liquid↗

PubMed2002

Quantitative protein profiling using two-dimensional gel electrophoresis, isotope-coded affinity tag labeling, and mass spectrometry.

Quantitative protein profiling is an essential part of proteomics and requires new technologies that accurately, reproducibly, and comprehensively identify and quantify the proteins contained in biological samples. We describe a new strategy for quantitative protein profiling that is based on the separation of proteins labeled with isotope-coded affinity tag reagents by two-dimensional gel electrophoresis and their identification and quantification by mass spectrometry. The method is based on the observation that proteins labeled with isotopically different isotope-coded affinity tag reagents precisely co-migrate during two-dimensional gel electrophoresis and that therefore two or more isotopically encoded samples can be separated concurrently in the same gel. By analyzing changes in the proteome of yeast (Saccharomyces cerevisiae) induced by a metabolic shift we show that this simple method accurately quantifies changes in protein abundance even in cases in which multiple proteins migrate to the same gel coordinates. The method is particularly useful for the quantitative analysis and structural characterization of differentially processed or post-translationally modified forms of a protein and is therefore expected to find wide application in proteomics research.

Affinity Labels↗