Stephen J. Kron

Peptide chips for the quantitative evaluation of protein kinase activity.

Peptide chips are an emerging technology that could replace many of the bioanalytical methods currently used in drug discovery, diagnostics, and cell biology. Despite the promise of these chips, their development for quantitative assays has been limited by several factors, including a lack of well-defined surface chemistries to immobilize peptides, the heterogeneous presentation of immobilized ligands, and nonspecific adsorption of protein to the substrate. This paper describes a peptide chip that overcomes these limitations, and demonstrates its utility in activity assays of the nonreceptor tyrosine kinase c-Src. The chip was prepared by the Diels–Alder-mediated immobilization of the kinase substrate AcIYGEFKKKC-NH2 on a self-assembled monolayer of alkanethiolates on gold. Phosphorylation of the immobilized peptides was characterized by surface plasmon resonance, fluorescence, and phosphorimaging. Three inhibitors of the enzyme were quantitatively evaluated in an array format on a single, homogeneous substrate.

Assays for actin sliding movement over myosin-coated surfaces.

Publisher Summary One important result from in vitro studies of the interaction of the major proteins of muscle, actin and myosin, has been the growing recognition that nearly any aspect of muscle mechanics can be studied in a model system consisting of purified proteins. This chapter is a compilation of techniques for purified in vitro motility assays for actin sliding movement over myosin. Several forms of myosin, including filaments, monomers, and soluble proteolytic fragments, have been found to work well in aetin sliding movement assays. The focus is limited to studies using skeletal muscle proteins, but only slight modification of these protocols may be necessary for proteins derived from smooth muscle and nonmuscle sources. The properties of the protein preparations used are critical to reproducibility of actin sliding movement assays. The methods presented in the chapter are trustworthy preparations but are not singularly successful. However, in particular it should be noted that myosin subfragment preparations that work well in solution experiments might not be optimal for use in movement assays.

Myosin step size: Estimation from slow sliding movement of actin over low densities of heavy meromyosin☆

We have estimated the step size of the myosin cross-bridge (d, displacement of an actin filament per one ATP hydrolysis) in an in vitro motility assay system by measuring the velocity of slowly moving actin filaments over low densities of heavy meromyosin on a nitrocellulose surface. In previous studies, only filaments greater than a minimum length were observed to undergo continuous sliding movement. These filaments moved at the maximum speed (Vo), while shorter filaments dissociated from the surface. We have now modified the assay system by including 0.8% methylcellulose in the ATP solution. Under these conditions, filaments shorter than the previous minimum length move, but significantly slower than Vo, as they are propelled by a limited number of myosin heads. These data are consistent with a model that predicts that the sliding velocity (v) of slowly moving filaments is determined by the product of vo and the fraction of time when at least one myosin head is propelling the filament, that is, v = vo [1-(1-ts/tc)N], where ts is the time the head is strongly bound to actin, tc is the cycle time of ATP hydrolysis, and N is the average number of myosin heads that can interact with the filament. Using this equation, the optimum value of ts/tc to fit the measured relationship between v and N was calculated to be 0.050. Assuming d = vots, the step size was then calculated to be between 10nm and 28 nm per ATP hydrolyzed, the latter value representing the upper limit. This range is within that of geometric constraint for conformational change imposed by the size of the myosin head, and therefore is not inconsistent with the swinging cross-bridge model tightly coupled with ATP hydrolysis.

Histone H2AX Phosphorylation as a Predictor of Radiosensitivity and Target for Radiotherapy

Based on the role of phosphorylation of the histone H2A variant H2AX in recruitment of DNA repair and checkpoint proteins to the sites of DNA damage, we have investigated γH2AX as a reporter of tumor radiosensitivity and a potential target to enhance the effectiveness of radiation therapy. Clinically relevant ionizing radiation (IR) doses induced similar patterns of γH2AX focus formation or immunoreactivity in radiosensitive and radioresistant human tumor cell lines and xenografted tumors. However, radiosensitive tumor cells and xenografts retained γH2AX for a greater duration than radioresistant cells and tumors. These results suggest that persistence of γH2AX after IR may predict tumor response to radiotherapy. We synthesized peptide mimics of the H2AX carboxyl-terminal tail to test whether antagonizing H2AX function affects tumor cell survival following IR. The peptides did not alter the viability of unirradiated tumor cells, but both blocked induction of γH2AX foci by IR and enhanced cell death in irradiated radioresistant tumor cells. These results suggest that H2AX is a potential molecular target to enhance the effects of radiotherapy.

Role of Dot1-Dependent Histone H3 Methylation in G1 and S Phase DNA Damage Checkpoint Functions of Rad9

ABSTRACT We screened radiation-sensitive yeast mutants for DNA damage checkpoint defects and identified Dot1, the conserved histone H3 Lys 79 methyltransferase. DOT1 deletion mutants (dot1Δ) are G1 and intra-S phase checkpoint defective after ionizing radiation but remain competent for G2/M arrest. Mutations that affect Dot1 function such as Rad6-Bre1/Paf1 pathway gene deletions or mutation of H2B Lys 123 or H3 Lys 79 share dot1Δ checkpoint defects. Whereas dot1Δ alone confers minimal DNA damage sensitivity, combining dot1Δ with histone methyltransferase mutations set1Δ and set2Δ markedly enhances lethality. Interestingly, set1Δ and set2Δ mutants remain G1 checkpoint competent, but set1Δ displays a mild S phase checkpoint defect. In human cells, H3 Lys 79 methylation by hDOT1L likely mediates recruitment of the signaling protein 53BP1 via its paired tudor domains to double-strand breaks (DSBs). Consistent with this paradigm, loss of Dot1 prevents activation of the yeast 53BP1 ortholog Rad9 or Chk2 homolog Rad53 and decreases binding of Rad9 to DSBs after DNA damage. Mutation of Rad9 to alter tudor domain binding to methylated Lys 79 phenocopies the dot1Δ checkpoint defect and blocks Rad53 phosphorylation. These results indicate a key role for chromatin and methylation of histone H3 Lys 79 in yeast DNA damage signaling.

A Novel Mechanism of Ion Homeostasis and Salt Tolerance in Yeast: the Hal4 and Hal5 Protein Kinases Modulate the Trk1-Trk2 Potassium Transporter

ABSTRACT The regulation of intracellular ion concentrations is a fundamental property of living cells. Although many ion transporters have been identified, the systems that modulate their activity remain largely unknown. We have characterized two partially redundant genes fromSaccharomyces cerevisiae, HAL4/SAT4 andHAL5, that encode homologous protein kinases implicated in the regulation of cation uptake. Overexpression of these genes increases the tolerance of yeast cells to sodium and lithium, whereas gene disruptions result in greater cation sensitivity. These phenotypic effects of the mutations correlate with changes in cation uptake and are dependent on a functional Trk1-Trk2 potassium transport system. In addition, hal4 hal5 and trk1 trk2 mutants exhibit similar phenotypes: (i) they are deficient in potassium uptake; (ii) their growth is sensitive to a variety of toxic cations, including lithium, sodium, calcium, tetramethylammonium, hygromycin B, and low pH; and (iii) they exhibit increased uptake of methylammonium, an indicator of membrane potential. These results suggest that the Hal4 and Hal5 protein kinases activate the Trk1-Trk2 potassium transporter, increasing the influx of potassium and decreasing the membrane potential. The resulting loss in electrical driving force reduces the uptake of toxic cations and improves salt tolerance. Our data support a role for regulation of membrane potential in adaptation to salt stress that is mediated by the Hal4 and Hal5 kinases.

Regulation of cation transport in Saccharomyces cerevisiae by the salt tolerance gene HAL3.

Dynamic regulation of ion transport is essential for homeostasis as cells confront changes in their environment. The gene HAL3 encodes a novel component of this regulatory circuit in the yeast Saccharomyces cerevisiae. Overexpression of HAL3 improves growth of wild-type cells exposed to toxic concentrations of sodium and lithium and suppresses the salt sensitivity conferred by mutation of the calcium-dependent protein phosphatase calcineurin. Null mutants of HAL3 display salt sensitivity. The sequence of HAL3 gives little clue to its function. However, alterations in intracellular cation concentrations associated with changes in HAL3 expression suggest that HAL3 activity may directly increase cytoplasmic K+ and decrease Na+ and Li+. Cation efflux in S. cerevisiae is mediated by the P-type ATPase encoded by the ENA1/PMR24 gene, a putative plasma membrane Na+ pump whose expression is salt induced. Acting in concert with calcineurin, HAL3 is necessary for full activation of ENA1 expression. This functional complementarity is also reflected in the participation of both proteins in recovery from alpha-factor-induced growth arrest. Recently, HAL3 was isolated as a gene (named SIS2) which when overexpressed partially relieves loss of transcription of G1 cyclins in mutants lacking the protein phosphatase Sit4p. Therefore, HAL3 influences cell cycle control and ion homeostasis, acting in parallel to the protein phosphatases Sit4p and calcineurin.

Budding yeast morphogenesis: signalling, cytoskeleton and cell cycle

Yeast-like fungi such as Saccharomyces cerevisiae exhibit a range of cell types differing in cell shape, gene expression and growth pattern. Signal transduction pathways mediate transitions between different cell types. Nutritional signals induce rounded yeast-form cells either to enter invasive growth as elongated filamentous cells or to arrest to prepare for stationary phase, conjugation, or meiosis. An emerging theme is that development critically depends upon differential regulation of vegetative functions, including cytoskeletal organization and cell cycle progression, as much as on the expression of cell type specific gene products.

Role of Oxidative Phosphorylation in Bax Toxicity

ABSTRACT The Bcl-2-related protein Bax is toxic when expressed either in yeast or in mammalian cells. Although the mechanism of this toxicity is unknown, it appears to be similar in both cell types and dependent on the localization of Bax to the outer mitochondrial membrane. To investigate the role of mitochondrial respiration in Bax-mediated toxicity, a series of yeast mutant strains was created, each carrying a disruption in either a component of the mitochondrial electron transport chain, a component of the mitochondrial ATP synthesis machinery, or a protein involved in mitochondrial adenine nucleotide exchange. Bax toxicity was reduced in strains lacking the ability to perform oxidative phosphorylation. In contrast, a respiratory-competent strain that lacked the outer mitochondrial membrane Por1 protein showed increased sensitivity to Bax expression. Deficiencies in other mitochondrial proteins did not affect Bax toxicity as long as the ability to perform oxidative phosphorylation was maintained. Characterization of Bax-induced toxicity in wild-type yeast demonstrated a growth inhibition that preceded cell death. This growth inhibition was associated with a decreased ability to carry out oxidative phosphorylation following Bax induction. Furthermore, cells recovered following Bax-induced growth arrest were enriched for a petite phenotype and were no longer able to grow on a nonfermentable carbon source. These results suggest that Bax expression leads to an impairment of mitochondrial respiration, inducing toxicity in cells dependent on oxidative phosphorylation for survival. Furthermore, Bax toxicity is enhanced in yeast deficient in the ability to exchange metabolites across the outer mitochondrial membrane.

Sensing, signalling and integrating physical processes during Saccharomyces cerevisiae invasive and filamentous growth.

Simple fungi have evolved sophisticated mechanisms to sense and respond to environmental cues by activating developmental switches that result in coordinated changes in cell physiology, morphology and cell adherence. Critical depletion of nutrients often induces growth arrest to form spores capable of tolerating a wide range of environmental stresses. However, an alternative response is the dimorphic switch to filamentous growth, characterized by branching networks of chains of cells or hyphae to form a mycelium (Gancedo, 2001; Kron & Gow, 1995). Filamentous growth is considered an important adaptive response that functions analogously to cell motility in allowing a starving fungal colony to forage for nutrients (Lee & Elion, 1999). Branching filaments permit wider exploration of the environment at a lower biomass (energy) cost than nonfilamentous growth. Cell–cell adherence and highly polarized growth promote invasion of the substrate (Gancedo, 2001). Further, the high surface-to-volume ratio of filaments may facilitate transport of nutrients. The dimorphic switch is essentially modular and may be activated by a wide range of stimuli appropriate to the lifestyle of the fungus. Typically, the dimorphic switch in pathogenic fungi is tuned such that cells elongate or increase cell–cell adherence when exposed to their host (San-Blas et al., 2000; Sanchez-Martinez & Perez-Martin, 2001). Such regulated dimorphism has been established as an important virulence factor, determining invasion and colonization by pathogenic organisms such as Candida albicans, Magnaporthe grisea (rice blast) and Ustilago maydis (corn smut) (Lengeler et al., 2000; Sanchez-Martinez & PerezMartin, 2001). Thus the dimorphic switch may prove to be an attractive target for chemical intervention in the prophylaxis and treatment of fungal infections in medicine and agriculture. In turn, many industrial biochemical processes employ dimorphic yeasts and filamentous fungi to drive production of desired chemical compounds. Fermentation of carbohydrates in fruits, grains and other biomass to ethanol by Saccharomyces cerevisiae is the critical process for a wide range of products from fine wines to gasoline additives (Bothast et al., 1999; Pretorius, 2000). S. cerevisiae and other simple fungi are also widely utilized in industrial production of small-molecule metabolites such as citrate and amino acids, bioremediation of toxic contaminants, fermentation of foodstuffs, production of complex organic compounds such as antibiotics, growth of starting material for protein purifications, and expression of heterologous proteins for vaccines, drugs and enzymes (Bennett, 1998; Chartrain et al., 2000; Ostergaard et al., 2000). The manner of organism proliferation, whether in a dispersed or aggregated form, is potentially a limiting factor in production of the desired metabolite(s), utilization of the growth medium and}or separation of cell mass from the soluble product. The classic example of self-clearing of beers at the end of the fermentation by the flocculation and settling of ale yeast underlines that control over cell morphology and}or cell–cell adhesion is a highly desirable characteristic of an industrial micro-organism (Hammond, 1995; Straver et al., 1993). A detailed understanding of how metabolite sensing and signalling leads to changes in physical properties of cells will be critical for cellular engineering approaches to control dimorphism. For example, maintaining cell dispersion is desired during a fermentation to maximize mass transport and reaction rates but inducing flocculation can facilitate separation of the cells from fermentation products. In turn, it should be kept inmind that adhesion and morphology are not the only characteristics that change during the dimorphic transition. Many fungi also switch their patterns of metabolic conversions, gene expression and protein secretion (Viard & Kuriyama,