REAP: A two minute cell fractionation method

<p>Abstract</p> <p>Background</p> <p>The translocation or shuttling of proteins between the nucleus and cytoplasm (nucleocytoplasmic transport [NCPT]) is often a rapid event following stimulation with growth factors or in response to stress or other experimental manipulations. Commonly used methods to separate nuclei from cytoplasm employ lengthy steps such as density gradient centrifugation which exposes cells to non-physiological hyperosmotic conditions for extended time periods resulting in varying degrees of leakage between the nucleus and cytoplasm. To help maintain and quantify nuclear:cytoplasmic ratios of proteins, agents such as leptomycin B have been employed to be able to better analyze NCPT by inhibiting nuclear export. To track NCPT in the absence of these experimental manipulations that could introduce unknown artefacts, we have developed a rapid method that appears to produce pure nuclear and cytoplasmic fractions, suitable for obtaining accurate estimates of the nuclear:cytoplasmic ratios of proteins known to undergo NCPT.</p> <p>Findings</p> <p>We have developed a <b>R</b>apid, <b>E</b>fficient <b>A</b>nd <b>P</b>ractical (<b>REAP</b>) method for subcellular fractionation of primary and transformed human cells in culture. The REAP method is a two minute non-ionic detergent-based purification technique requiring only a table top centrifuge, micro-pipette and micro-centrifuge tubes. This inexpensive method has proven to efficiently separate nuclear from cytoplasmic proteins as estimated by no detectible cross-contamination of the nucleoporin and lamin A nuclear markers or the pyruvate kinase and tubulin cytoplasmic markers. REAP fractions also mirrored TNFα induced NF-κB NCPT observed in parallel by indirect immunofluorescence.</p> <p>Conclusions</p> <p>This method drastically reduces the time needed for subcellular fractionation, eliminates detectable protein degradation and maintains protein interactions. The simplicity, brevity and efficiency of this procedure allows for tracking ephemeral changes in subcellular relocalization of proteins while maintaining protein integrity and protein complex interactions.</p

Src Regulates the Activity of the ING1 Tumor Suppressor

<div><p>The INhibitor of Growth 1 (ING1) is stoichiometric member of histone deacetylase (HDAC) complexes and functions as an epigenetic regulator and a type II tumor suppressor. It impacts cell growth, aging, apoptosis, and DNA repair, by affecting chromatin conformation and gene expression. Down regulation and mislocalization of ING1 have been reported in diverse tumor types and Ser/Thr phosphorylation has been implicated in both of these processes. Here we demonstrate that both <i>in vitro</i> and <i>in vivo</i>, the tyrosine kinase Src is able to physically associate with, and phosphorylate ING1, which results in a nuclear to cytoplasmic relocalization of ING1 in cells and a decrease of ING1 stability. Functionally, Src antagonizes the ability of ING1 to induce apoptosis, most likely through relocalization of ING1 and down regulation of ING1 levels. These effects were due to both kinase-dependent and kinase-independent properties of Src, and were most apparent at elevated levels of Src expression. These findings suggest that Src may play a major role in regulating ING1 levels during tumorigenesis in those cancers in which high levels of Src expression or activity are present. These data represent the first report of tyrosine kinase-mediated regulation of ING1 levels and suggest that kinase activation can impact chromatin structure through the ING1 epigenetic regulator.</p> </div

HEK293 cells were transfected with constructs expressing the indicated proteins.

<p>Row 1: ING1b; Row2: Src wild type; Row3: Src wild type+ING1b; Row4: Src activated Y530F; Row5: Src activated Y530F +ING1b; Row6: Src kinase dead K298M; Row7: Src kinase dead K298M +ING1b. Analysis by fluorescence microscopy at 24 hours post transfection utilized a primary antibody hybridoma cocktail mix of Cab 2,4,5 and 9 for ING1, and Mab327 for Src. Secondary fluorescent antibodies were Alexa Fluor 488 and Alexa Fluor 568 (Invitrogen) for ING1 and Src respectively. Hoechst 33342 was used for nuclear staining.</p

Src-dependent decrease in ING1B stability.

<p>HEK293 cells were transfected overnight with plasmid constructs expressing ING1b and either control or Src-expressing plasmids. The following morning, cycloheximide (100 ug/ml) was added to each well and the cells were harvested at the indicated times following cycloheximide addition. Cell extracts were analyzed by western blotting with anti-ING1b antibody. The ING1b bands were quantitated by scanning and the data normalized to 100% at time 0 for each condition. The results are from triplicate wells of cells +/−1 S.E. (*p<0.01, **p<0.001).</p

Src phosphorylates ING1 <i>in vitro</i>.

<p>ING1, Src and ATP were added to lane 6 where phosphorylation of ING1 is seen when blotting with anti-phosphotyrosine. Lanes 1–5 serve as negative controls.</p

ING1 binds to Src and Src mutants.

<p>A) Wild-type Src and the mutants indicated were ectopically expressed in HEK293 cells. Expression levels of Src and ING1 are shown in immunoblots of whole cell extracts (upper two panels). Src immunoprecipitates from these cells were probed with antibodies against ING1 or Src (lower two panels). Bands at 55 and 23 kDa represent heavy and light chains of α−Src used in immunoprecipitation. The top two and bottom panels are data from the same experiment. The third panel showing ING1 co-immunoprecipitated with Src is from a separate experiment performed under the same conditions; however, a larger amount of total protein in the lysate was used for immunoprecipitation than in the bottom panel in order to aid in the visualization of endogenous ING1. Note: The level of activated Y530F Src (lanes 3 and 7, top and bottom panels) is less than that of wt or kinase-dead K298M Src because of its shorter half life. This effect is also seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060943#pone-0060943-g003" target="_blank">Fig. 3</a>. B) A431 cell extracts were immunoprecipitated using either antibody against Src (lane 1) or ING1 (lane 2), using control mouse IgG antibody (lane 3), or in the absence of antibody (lane 4), followed by immunoblotting of the immunoprecipitates for ING1. Untreated whole cell extract (approx. 8% of cell extract protein used in the immunoprecipitations for lanes 1–4) (lane 5) was also blotted for ING1.</p

Kinase active and kinase inactive forms of Src block the ability of ING1 to induce apoptosis in MDA-MB-468 cells.

<p>Exponentially growing cells were transfected with GFP expression construct in the absence or presence of the additional constructs indicated. ING1 expression increased the level of apoptotic cells from 20% to 80% within 24 hours as estimated by Annexin V staining in flow cytometry. Active forms of Src completely blocked ING1-induced apoptosis, and kinase-inactive Src was nearly as effective in preventing ING1-induced apoptois.</p

Subcellular localization of ING1 and Src.

<p><b>A)</b> Lamin A serves as a nuclear marker while tubulin is cytoplasmic. Src is found in both the nuclear (N) and cytoplasmic (C) fractions while ING1 is primarily nuclear. W indicates whole cell lysate. <b>B)</b> ING1 localization in response to Src. ING1 was coexpressed with the Src variants noted.</p

Phosphorylation of ING1 by Src <i>in vivo</i>.

<p>HEK293 cells were untransfected (lane 1), transfected with Src constructs alone (lanes 2–4) ING1 alone (lane 5) or cotransfected with ING1 plus wt Src (lane 6), activated Src (lane 7) or kinase dead Src (lane 8). Lysates (top panels) or ING1 immunoprecipitates (bottom panels) were blotted with the indicated antibodies.</p