Distinct and Site-Specific Phosphorylation of the Retinoblastoma Protein at Serine 612 in Differentiated Cells

<div><p>The retinoblastoma susceptibility protein (pRB) is a phosphoprotein that regulates cell cycle progression at the G1/S transition. In quiescent and early G1 cells, pRB predominantly exists in the active hypophosphorylated form. The cyclin/cyclin-dependent protein kinase complexes phosphorylate pRB at the late G1 phase to inactivate pRB. This event leads to the dissociation and activation of E2F family transcriptional factors. At least 12 serine/threonine residues in pRB are phosphorylated <i>in vivo</i>. Although there have been many reports describing bulk phosphorylation of pRB, detail research describing the function of each phosphorylation site remains unknown. Besides its G1/S inhibitory function, pRB is involved in differentiation, prevention of cell death and control of tissue fate. To uncover the function of phosphorylation of pRB in various cellular conditions, we have been investigating phosphorylation of each serine/threonine residue in pRB with site-specific phospho-serine/threonine antibodies. Here we demonstrate that pRB is specifically phosphorylated at Ser612 in differentiated cells in a known kinase-independent manner. We also found that pRB phosphorylated at Ser612 still associates with E2F-1 and tightly binds to nuclear structures including chromatin. Moreover, expression of the Ser612Ala mutant pRB failed to induce differentiation. The findings suggest that phosphorylation of Ser612 provides a distinct function that differs from the function of phosphorylation of other serine/threonine residues in pRB.</p></div

Phosphorylation of pRB at Ser612 is mediated by an unknown kinase in particular contexts.

<p>(A) U-937 cells were pretreated with 10 µM of the Chk2 inhibitor II for 24 h, and the cells were then treated with 20 nM TPA. After the indicated periods, the cells were harvested for immunoblotting with the indicated antibodies. (B) MOLT-4 cells were treated with 0.1% DMSO (lane 1), 1 µM GW8510 (lane 2), 10 µM Chk2 inhibitor II (lane 3), 1 µM SB218078 (lane 4) or 50 µM PD98059 (lane 5) for 6 h, then the cells were harvested for immunoblotting with the indicated antibodies. All compounds were dissolved in DMSO as a 1000 times stock solution. (C) SAOS-2 cells were transfected with empty (mock), wild-type RB (WT), or Ser612Ala RB (S612A) expression vectors. Forty-eight hours after transfection, the cells were harvested for immunoblotting with the indicated antibodies. A cell extract prepared from MOLT-4 cells was also loaded as a control for the antibodies.</p

pRB is phosphorylated at Ser612 in differentiated cells.

<p>(A) U-937 cells were treated with 20 nM TPA for the indicated periods and then the cells were harvested for immunoblotting with the indicated antibodies. (B and C) K562 cells were treated with 30 µM hemin (B) or 10 nM TPA (C) for the indicated periods and then the cells were harvested for immunoblotting with the indicated antibodies. (D) SH-SY5Y cells were treated with 10 µM ATRA for the indicated periods and then the cells were harvested for immunoprecipitation and immunoblotting with the indicated antibodies. The asterisks denote non-specific bands.</p

pRB phosphorylated at Ser612 tightly associates with chromatin and the nuclear structure in differentiated cells.

<p>U-937 cells were treated with 20 nM TPA for the indicated periods and the cells were harvested. The cells were fractionated as described in the Materials and Methods section. The extracts were analyzed by immunoblotting with the indicated antibodies.</p

pRB phosphorylated at Ser612 does not release E2F-1.

<p>U-937 cells were treated with 20 nM TPA for the indicated periods and the cells were harvested. Cell extracts were prepared as described in the Materials and Methods section. Immunoprecipitation and immunoblotting was performed with the indicated antibodies.</p

Anti-phospho-Ser612 antibodies recognize pRB only when it is phosphorylated at Ser612.

<p>Recombinant pRB was synthesized as described in the Materials and Methods section. pRB was incubated with or without λ phosphatase (PPase) in the absence or presence of a phosphatase inhibitor cocktail. The reaction was stopped by adding the Laemmli sample buffer and then the sample was analyzed by immunoblotting with the indicated antibodies.</p

Expression of the pRB Ser612Ala mutant impairs the ability of the differentiation in ATRA-treated SH-SY5Y cells.

<p>(A) SH-SY5Y cells were transfected with empty (mock), wild-type RB (WT), or Ser612Ala RB (S612A) expression vectors. After 24 h, the cells were cultured in the presence or absence of 10 µM ATRA in 200 µg/ml hygromycin B containing medium for 14 d. Bar: 100 µm. (B) Neurite bearing cells were counted and the percentage of differentiated cells per field was calculated. Values are the means ± standard deviation of 3 independent fields. An asterisk represents a statistically significant difference from the values at ATRA-treated mock cells and ATRA-treated WT cells (<i>p</i><0.001, <i>t</i>-test).</p

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Functional analysis of DS1.

<p>(<i>A</i>) Isogenic yeast strain ∆lpp1∆dpp1∆pah1 containing empty pDO105 plasmid (+pDO105) or pDO105 containing DS1 (+pDO105: DS1) were cultured on agar YPD plates and incubated at 30°C or 37°C. (<i>B</i>) PAP activity of DS1 and dehydrofolate reductase (DHFR; negative control) was determined in the presence or absence of Mg²⁺ as described in Materials and Methods. Values are means and SD from triplicate experiments. Asterisks denote values significantly different from those of control (*; <i>P</i> < 0.05). (<i>C</i>) PAP activity in control (white bar) and DS1 plants (black bar). Crude protein fractions were isolated from control and DS1 plants 0 and 24 h after inoculation with <i>R</i>. <i>solanacearum</i>. PAP activity was determined without Mg²⁺ as described in Materials and Methods. Values are means and SD from triplicate experiments. Asterisks denote values significantly different from those of control (*; <i>P</i> < 0.05). (<i>D</i>) Phosphatidic acid contents in control and DS1 plants. Total lipid fraction was extracted from control and DS1 plants 0 to 24 h after inoculation with <i>R</i>. <i>solanacearum</i>. PA was separated by ethyl acetate TLC as described in Materials and Methods.</p

Role of jasmonic acid pathway in DS1 phenotype.

<p>Control, DS1, and DS1:Coi1 double-knockdown plant leaves were infiltrated with <i>R</i>. <i>solanacearum</i>. (<i>A</i>) Total RNA was isolated from control (Control), DS1 (DS1), and DS1:NbCoi1 (DS1:Coi1) double-knockdown plant 24 h after inoculation with <i>R</i>. <i>solanacearum</i>. Expression values of <i>PR-4</i> are expressed as [Qty] after normalization to actin. Values represent means and SD from triplicate experiments. Different letters show significant differences among control, DS1, and double-knockdown plants (ANOVA test). (<i>B</i>) Bacterial population was determined by plating at specified time points. Values are means of four replicate experiments with SD. Different letters show significant differences among controls, DS1, and double-knockdown plants (<i>p</i><0.05 ANOVA). (<i>C</i>) Disease development of bacterial wilt was rated daily on a 0–4 disease index in control (open circles), DS1 (solid circles), or DS1:Coi1 (closed squares) plants. Values are means of four replicate experiments with SD. Asterisks denote values significantly different from those of empty PVX control (*; <i>P</i> < 0.05, <i>t</i>-test). (<i>D</i>) Characteristic symptoms in control, DS1, and DS1:Coi1 plants. Photograph was taken 12 days after inoculation with <i>R</i>. <i>solanacearum</i>.</p