Caged phosphopeptides reveal a temporal role for 14-3-3 in G1 arrest and S-phase checkpoint function

Nguyen, Anhco; Rothman, Deborah; Stehn, Justine R.; Imperiali, Barbara; Yaffe, Michael B.

doi:10.1038/nbt997

Cited by 87 publications

(60 citation statements)

References 47 publications

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“…The mechanisms of loading are far from being completely understood; endocytosis and simple plasma membrane permeation are two possible routes into cells, and it is sometimes thought that both mechanisms occur in parallel. This technique has much potential, but has only been used in one experiment with caged compounds so far 53 .…”

Section: Cell Loading and Extracellular Applicationmentioning

confidence: 99%

Caged compounds: photorelease technology for control of cellular chemistry and physiology

2007

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Caged compounds are light-sensitive probes that functionally encapsulate biomolecules in an inactive form. Irradiation liberates the trapped molecule, permitting targeted perturbation of a biological process. Uncaging technology and fluorescence microscopy are 'optically orthogonal': the former allows control, and the latter, observation of cellular function. Used in conjunction with other technologies (for example, patch clamp and/or genetics), the light beam becomes a uniquely powerful tool to stimulate a selected biological target in space or time. Here I describe important examples of widely used caged compounds, their design features and synthesis, as well as practical details of how to use them with living cells.The idea behind the caging technique is that a molecule of interest can be rendered biologically inert (or caged) by chemical modification with a photoremovable protecting group (Fig. 1). Illumination results in a concentration jump of the biologically active molecule that can bind to its cellular receptor, switching on (or off) the targeted process. Virtually every kind of signaling molecule or second messenger, of every size| [mdash]|from protons to proteins| [mdash]|has been caged 1 .Why are caged compounds so useful? A single component of cellular chemistry can control the function of a cell, and such cellular regulation can be temporally or spatially defined, intracellular or extracellular, and amplitude-or frequency-modulated 2 . Photomanipulation of cellular chemistry using caged compounds provides a uniquely powerful means to interact with such cellular dynamics, as it can touch upon any one of the above dimensions. Thus, since light passes through cell membranes, uncaging can rapidly release a biomolecule in an intracellular compartment. This space is not readily accessible to many second messengers (for example, inositol-1,4,5-trisphosphate (IP 3 ), ATP, Ca 2+ , cAMP, cGMP) when they are applied to cells externally, as their charge makes them impermeable to the plasma membrane. Furthermore, uniform illumination results in release throughout the cytosol, or the release can be localized by focusing the uncaging beam on one part of a cell. Likewise, extracellular uncaging of neurotransmitters and hormones is tunable, allowing stimulation of many neurons simultaneously or of single synapses| [mdash]|by global or focused illumination, respectively. Light cannot only be directed, but also modulated in time andCorrespondence should be addressed to Graham C R Ellis-Davies (cagedca@hotmail.com). (Fig. 2) has been widely used to study many Ca 2+ -controlled processes. In particular, secretory processes in neuronal and non-neuronal cells have been extensively studied with caged calcium 22 . Rapid uncaging of glutamate using two-photon photolysis is particularly useful for the rational stimulation of visually identified synapses in complex tissue preparations such as acutely isolated brain slices from the hippocampus 23 (Fig. 2b). Finally, uncaging of mRNA in vivo in zebrafish is an excellen...

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Section: Cell Loading and Extracellular Applicationmentioning

confidence: 99%

Caged compounds: photorelease technology for control of cellular chemistry and physiology

2007

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“…We have been particularly interested in understanding the roles of different 14-3-3 proteins in cell proliferation, cell cycle control, and human tumorigenesis (5,13,14). In epithelial cells, one particular 14-3-3 isoform, 14-3-3, appears to play a particularly important role in this regard.…”

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confidence: 99%

A Structural Basis for 14-3-3σ Functional Specificity*♦

Wilker

Grant

Artim

et al. 2005

Journal of Biological Chemistry

233

239

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The 14-3-3 family of proteins includes seven isotypes in mammalian cells that play numerous diverse roles in intracellular signaling. Most 14-3-3 proteins form homodimers and mixed heterodimers between different isotypes, with overlapping roles in ligand binding. In contrast, one mammalian isoform, 14-3-3, expressed primarily in epithelial cells, appears to play a unique role in the cellular response to DNA damage and in human oncogenesis. The biological and structural basis for these 14-3-3-specific functions is unknown. We demonstrate that endogenous 14-3-3 preferentially forms homodimers in cells. We have solved the x-ray crystal structure of 14-3-3 bound to an optimal phosphopeptide ligand at 2.4 Å resolution. The structure reveals the presence of stabilizing ring-ring and salt bridge interactions unique to the 14-3-3 homodimer structure and potentially destabilizing electrostatic interactions between subunits in 14-3-3-containing heterodimers, rationalizing preferential homodimerization of 14-3-3 in vivo. The interaction of the phosphopeptide with 14-3-3 reveals a conserved mechanism for phospho-dependent ligand binding, implying that the phosphopeptide binding cleft is not the critical determinant of the unique biological properties of 14-3-3. Instead, the structure suggests a second ligand binding site involved in 14-3-3-specific ligand discrimination. We have confirmed this by site-directed mutagenesis of three -specific residues that uniquely define this site. Mutation of these residues to the alternative sequence that is absolutely conserved in all other 14-

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“…These studies provide the first evidence of regulation of the ING family of class II tumor suppressors by the 14-3-3 family which has been shown to affect cell cycle checkpoints (35).…”

Section: Waf1mentioning

confidence: 99%

“…14-3-3 proteins mainly reside in the cytoplasmic compartment of the cell, forming homodimers or heterodimers with other 14-3-3 family members (1,12). Most reports indicate that many of the 14-3-3 members are interchangeable with respect to protein binding, and emerging evidence suggests that 14-3-3 proteins are central regulators of the cell cycle, especially at cell cycle checkpoints, where they might function as regulators of DNA damage checkpoints (25,30,35). In fission yeast, the 14-3-3 proteins Rad24 and Rad25 are required for checkpoint responses and are essential for cell survival (11).…”

mentioning

confidence: 99%

Subcellular Targeting of p33^ING1b by Phosphorylation-Dependent 14-3-3 Binding Regulates p21^WAF1 Expression

Gong

Russell

Suzuki

et al. 2006

Molecular and Cellular Biology

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ING1 is a type II tumor suppressor that affects cell growth, stress signaling, apoptosis, and DNA repair by altering chromatin structure and regulating transcription. Decreased ING1 expression is seen in several human cancers, and mislocalization has been noted in diverse types of cancer cells. Aberrant targeting may, therefore, functionally inactivate ING1. Bioinformatics analysis identified a sequence between the nuclear localization sequence and plant homeodomain domains of ING1 that closely matched the binding motif of 14-3-3 proteins that target cargo proteins to specific subcellular locales. We find that the widely expressed p33ING1b splicing isoform of ING1 interacts with members of the 14-3-3 family of proteins and that this interaction is regulated by the phosphorylation status of ING1. 14-3-3 binding resulted in significant amounts of p33ING1b protein being tethered in the cytoplasm. As shown previously, ectopic expression of p33 ING1bincreased levels of the p21 Waf1 cyclin-dependent kinase inhibitor upon UV-induced DNA damage. Overexpression of 14-3-3 inhibited the up-regulation of p21Waf1 by p33 ING1b , consistent with the idea that mislocalization blocks at least one of ING1's biological activities. These data support the idea that the 14-3-3 proteins play a crucial role in regulating the activity of p33ING1b by directing its subcellular localization.

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Caged phosphopeptides reveal a temporal role for 14-3-3 in G1 arrest and S-phase checkpoint function

Cited by 87 publications

References 47 publications

Caged compounds: photorelease technology for control of cellular chemistry and physiology

Caged compounds: photorelease technology for control of cellular chemistry and physiology

A Structural Basis for 14-3-3σ Functional Specificity*♦

Subcellular Targeting of p33^ING1b by Phosphorylation-Dependent 14-3-3 Binding Regulates p21^WAF1 Expression

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Caged phosphopeptides reveal a temporal role for 14-3-3 in G1 arrest and S-phase checkpoint function

Cited by 87 publications

References 47 publications

Caged compounds: photorelease technology for control of cellular chemistry and physiology

Caged compounds: photorelease technology for control of cellular chemistry and physiology

A Structural Basis for 14-3-3σ Functional Specificity*♦

Subcellular Targeting of p33ING1b by Phosphorylation-Dependent 14-3-3 Binding Regulates p21WAF1 Expression

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Subcellular Targeting of p33^ING1b by Phosphorylation-Dependent 14-3-3 Binding Regulates p21^WAF1 Expression