Quantification of Protein–Protein and Protein–DNA Interactions In Vivo, Using Fluorescence Recovery after Photobleaching

Carrero, Gustavo; Crawford, Ellen; Th'ng, John P.H.; Vries, Gerda de; Hendzel, Michael J.

doi:10.1016/s0076-6879(03)75026-5

Cited by 31 publications

(52 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Fluorescence Recovery after Photobleaching-The FRAP analysis was performed with the Zeiss LSM 510 META confocal laser-scanning microscope as described (30). Briefly, HeLa cells transfected with SPHK2-green fluorescent protein (GFP) were treated without or with 100 nM PMA for 30 min, and then each nuclear or cytoplasmic region of interest was photobleached by scanning for 8 s with an argon laser at the highest power.…”

Section: Methodsmentioning

confidence: 99%

Protein Kinase D-mediated Phosphorylation and Nuclear Export of Sphingosine Kinase 2

Ding¹,

Sonoda²,

et al. 2007

Journal of Biological Chemistry

135

120

View full text Add to dashboard Cite

Sphingosine kinase (SPHK) is a key enzyme producing important messenger sphingosine 1-phosphate and is implicated in cell proliferation and suppression of apoptosis. Because the extent of agonist-induced activation of SPHK is modest, signaling via SPHK may be regulated through its localization at specific intracellular sites. Although the SPHK1 isoform has been extensively studied and characterized, the regulation of expression and function of the other isoform, SPHK2, remain largely unexplored. Here we describe an important post-translational modification, namely, phosphorylation of SPHK2 catalyzed by protein kinase D (PKD), which regulates its localization. Upon stimulation of HeLa cells by tumor promoter phorbol 12-myristate 13-acetate, a serine residue in a novel and putative nuclear export signal, identified for the first time, in SPHK2 was phosphorylated followed by SPHK2 export from the nucleus. Constitutively active PKD phosphorylated this serine residue in the nuclear export signal both in vivo and in vitro. Moreover, downregulation of PKDs through RNA interference resulted in the attenuation of both basal and phorbol 12-myristate 13-acetateinduced phosphorylation, which was followed by the accumulation of SPHK2 in the nucleus in a manner rescued by PKD overexpression. These results indicate that PKD is a physiologically relevant enzyme for SPHK2 phosphorylation, which leads to its nuclear export for subsequent cellular signaling. Sphingosine kinases (SPHKs)3 catalyze the formation of sphingosine 1-phosphate, a bioactive lipid that regulates a diverse range of cellular processes, including cell growth, survival, differentiation, motility, and cytoskeletal organization (1, 2). Some of these cellular processes are mediated by five sphingosine 1-phosphatespecific G protein-coupled receptors, whereas others appear to be controlled by intracellular sphingosine 1-phosphate through as yet unidentified intracellular targets (2, 3).Two distinct SPHK isoforms, SPHK1 and SPHK2, have been cloned and characterized (4, 5). Diverse external stimuli, particularly growth and survival factors, stimulate SPHK1, and intracellularly generated sphingosine 1-phosphate has been implicated in their mitogenic and anti-apoptotic effects (6 -15). Expression of SPHK1 enhanced proliferation, promoted the G 1 /S transition, protected cells from apoptosis (6,8,16), and induced tumor formation in mice (8, 9).In contrast to SPHK1 much less is known about SPHK2. Although highly similar in amino acid sequence and possessing five evolutionarily conserved domains found in all SPHKs (17), SPHK2 diverges in its N terminus and central regions. These two isoforms have different kinetic properties and differ in developmental and tissue expression (5) implying that they may have distinct physiological functions. In fact, studies from our laboratory have demonstrated that, in contrast to cytosolic distribution of SPHK1, SPHK2 enters nuclei and inhibits DNA synthesis or induces apoptosis under stressful conditions such as serum deprivation (18,19)....

show abstract

Section: Methodsmentioning

confidence: 99%

Protein Kinase D-mediated Phosphorylation and Nuclear Export of Sphingosine Kinase 2

Ding¹,

Sonoda²,

et al. 2007

Journal of Biological Chemistry

135

120

View full text Add to dashboard Cite

show abstract

“…Following selection in G418, cells were plated onto number 1.5 glass coverslips, mounted in a small volume of tissue culture medium by creating a "well" with vacuum grease (Dow-Corning) on a glass slide. A 1.5-micrometer diameter region passing across the width of the cell nucleus was then photobleached, and recovery was monitored and quantified according to detailed procedures published elsewhere (37) …”

Section: Cloning Of Histone H1mentioning

confidence: 99%

“…This has been demonstrated both in vitro and in vivo (36). Using chimeric eGFP-histone H1 proteins stably expressed in human or mouse cell lines, it is possible to quantify this mobility inside the nuclei of live cells (37). Point mutations and deletion experiments using histone H1.1 showed that a single cyclindependent kinase phosphorylation site at specific regions within the C-terminal domain can control the association of the entire histone molecule (33).…”

mentioning

confidence: 99%

H1 Family Histones in the Nucleus

Th'ng

Sung

et al. 2005

Journal of Biological Chemistry

Self Cite

177

106

View full text Add to dashboard Cite

H1 histones bind to DNA as they enter and exit the nucleosome. H1 histones have a tripartite structure consisting of a short N-terminal domain, a highly conserved central globular domain, and a lysine-and arginine-rich C-terminal domain. The C-terminal domain comprises approximately half of the total amino acid content of the protein, is essential for the formation of compact chromatin structures, and contains the majority of the amino acid variations that define the individual histone H1 family members. This region contains several cell cycle-regulated phosphorylation sites and is thought to function through a charge-neutralization process, neutralizing the DNA phosphate backbone to allow chromatin compaction. In this study, we use fluorescence microscopy and fluorescence recovery after photobleaching to define the behavior of the individual histone H1 subtypes in vivo. We find that there are dramatic differences in the binding affinity of the individual histone H1 subtypes in vivo and differences in their preference for euchromatin and heterochromatin. Further, we show that subtype-specific properties originate with the C terminus and that the differences in histone H1 binding are not consistent with the relatively small changes in the net charge of the C-terminal domains.The H1 or "linker" histones are a family of very lysine-rich proteins that associate with the stretch of DNA that enters and exits the nucleosome. In the absence of histone H1, the nucleosome comprises ϳ146 bp of DNA that wraps 1.75 turns around the outer surface of the histone octamer (1-3). In the presence of histone H1, the resulting chromatosome contains two complete turns of 168-bp DNA. Histone H1, which is in an ϳ1:1 ratio with the number of nucleosomes in the cell, bends and alters the path of the DNA entering and exiting the nucleosome such that the nucleosome adopts a lollipop-like conformation (4, 5). This change in the trajectory of the linker DNA defines the first step in folding the polynucleosome chain into interphase chromosomes.The discovery of the H1 histones and the identification of its seven variants in the mid 1970s suggested possible roles in development (6). An additional variant, referred to as H1oo, was recently isolated from the oocytes of mice (7). These studies revealed associations between histone H1 subtypes in cell growth and differentiation (8 -12) and in the development of higher eukaryotes (9 -15). In simplistic terms, variants that have been associated with cellular differentiation are the histone H5 and histone H1.0 subtypes, and they are closer in amino acid sequences and most divergent from the other somatic histone H1 variants. Histone H5 is restricted to amphibian and reptile species, where it is found in high abundance in the nucleated but transcriptionally inert erythrocytes (16,17). The nuclei of these mature erythrocytes are almost entirely heterochromatic, allowing them to maintain a very small total volume. In this case, histone H5 expression correlates with the cessation of the RNA polymerase . I...

show abstract

“…Mathematical models have been successful in the quantitative analysis of FRAP curves involving protein binding reactions (Carrero et al, 2004a;Carrero et al, 2004b;McDonald et al, 2006). These models share one important assumption: the fluorescent proteins have spatially homogeneous distributions.…”

Section: Discussionmentioning

confidence: 99%

“…This technique has been used widely to determine the diffusion constant of biomolecules in membranes or various intracellular compartments (Chen et al, 2006;Carrero et al, 2003). Recent FRAP experiments have investigated direct protein activities, such as interaction with binding partners, with the fluorescence recovery curve analyzed to differentiate fast and slow components that correspond to diffusion and binding events, or different binding states (Tardy et al, 1995;Kimura et al, 2002;Dundr et al, 2002;Carrero et al, 2003;Carrero et al, 2004a;Carrero et al, 2004b;Sprague et al, 2004;McDonald et al, 2006). One recent study combined the FRAP technique with model convolution methods and measured a gradient in microtubule dynamics in yeast spindles at ~65-nm spatial intervals (Pearson et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

In vivo assay of presynaptic microtubule cytoskeleton dynamics in Drosophila

Yan

Broadie

2007

Journal of Neuroscience Methods

View full text Add to dashboard Cite

Disrupted microtubule dynamics in neuronal synapses has been suggested as an underlying cause for several devastating neurological diseases, including Hereditary Spastic Paraplegia (HSP) and Fragile X Syndrome (FXS). However, previous studies have been restricted to indirect assays of synaptic microtubules, i.e. immunocytochemistry of microtubule-associated proteins and posttranslationally modified tubulins characteristic of microtubules with different stabilities. Very little is known about synaptic microtubule dynamics in vivo, or how microtubule dynamics may be disrupted in disease states. In this study, we develop methods to analyze microtubule dynamics directly in living synaptic boutons in situ. We use fluorescence recovery after photobleaching (FRAP) of transgenic green fluorescent protein (GFP) tagged tubulin at the well-characterized Drosophila neuromuscular junction (NMJ) synapse. FRAP measurements of tubulin-GFP demonstrate biphasic recovery kinetics. Treatment with taxol to stabilize microtubules and promote microtubule assembly reduces both recovery phases. Treatment with vinblastine to disassemble microtubules increases the fast recovery phase and decreases the slow recovery phase. These data indicate that the fast recovery phase is generated by rapid diffusion of tubulin subunits and the slow phase is generated by the relatively slow turnover of microtubules. This study demonstrates that tubulin-GFP fluorescence recovery after photobleaching can be used to assay microtubule dynamics directly in living synapses.

show abstract

Quantification of Protein–Protein and Protein–DNA Interactions In Vivo, Using Fluorescence Recovery after Photobleaching

Cited by 31 publications

References 46 publications

Protein Kinase D-mediated Phosphorylation and Nuclear Export of Sphingosine Kinase 2

Protein Kinase D-mediated Phosphorylation and Nuclear Export of Sphingosine Kinase 2

H1 Family Histones in the Nucleus

In vivo assay of presynaptic microtubule cytoskeleton dynamics in Drosophila

Contact Info

Product

Resources

About