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First published online 1 March 2005
doi: 10.1242/jcs.01724

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p25/Cdk5-mediated retinoblastoma phosphorylation is an early event in neuronal cell death

Malika Hamdane¹, Alexis Bretteville^1,*, Anne-Véronique Sambo^1,*, Katharina Schindowski², Séverine Bégard¹, André Delacourte¹, Philippe Bertrand² and Luc Buée^1,

¹ INSERM U422, Institut de Médecine Prédictive et Recherche Thérapeutique, Université de Lille 2, Place de Verdun, 59045 Lille Cedex, France
² Aventis Pharma, CNS Research, 94400 Vitry Sur Seine, France

View larger version (13K): [in a new window]   Fig. 1. Representative western-blot analysis of caspase-3 cleavage in a time-course experiment of p25 expression. NGF-differentiated (7 days) mock and p25 cells were treated (+) or not (–) with tetracycline for 24 hours or 48 hours, and lysates were analysed using anti-caspase-3 antibody that recognizes the full-length protein (35 kDa, arrow) and the large fragments resulting from its cleavage (19/17 kDa, arrowheads). As positive control, lysates derived from cells treated (+) or not (–) with 1 µM staurosporine (SSP) were used.

View larger version (15K): [in a new window]   Fig. 2. (A) Representative immunoblot analyses of cell-cycle-regulatory proteins. NGF-differentiated mock and p25 cells were treated (+) or not (–) with tetracycline for 24 hours and lysates were analysed using antibodies against p21CIP1 (p21), p27KIP1 (p27), cyclin A, cyclin B1 and p34Cdc2. Equal amounts of proteins were loaded, and visualized with an antibody against neuron-specific -enolase (NSE). (B) Analysis of Rb phosphorylation in a time-course experiment of p25 expression. Lysates from NGF-differentiated mock and p25 cells treated (+) or not (–) with tetracycline for 6 hours, 12 hours and 24 hours were first immunolabelled with antibodies against Ser807/811-phosphorylated and Ser795-phosphorylated Rb. After stripping, phosphorylation-independent antibody (IF-8) was used to visualize the amount of loaded Rb (Rb). Concomitant with this, p25 expression was analysed during the kinetics by antibody against the p35 C-terminus.

View larger version (25K): [in a new window]   Fig. 3. (A) Analysis of p25-Cdk5 complex. Cell lysates from NGF-p25 and NGF-mock cells (used as negative control), treated for 6 hours with tetracycline, were subjected to immunoprecipitation (IP) by antibodies against either the p35 C-terminus or Cdk5. Immune complexes were then analysed by western blotting (WB) with antibodies against the p35 C-terminus and Cdk5. (B) Analysis of roscovitine's effect on earlier Rb phosphorylation. NGF-differentiated p25 cells were induced (+) or not (–) with tetracycline for 6 hours in the absence or the presence of 5 µM roscovitine (–/+). Lysates were immunoblotted with antibody against phospho-Ser807/811 Rb (P-Rb) followed, after stripping, with the phosphorylation-independent antibody (IF-8) against Rb protein (Rb).

View larger version (26K): [in a new window]   Fig. 4. Immunoblot analyses of cell-cycle-regulatory proteins at the earliest stage of p25 expression. NGF-differentiated p25 cells were treated (+) or not (–) with tetracycline for 6 hours and lysates were analysed using the indicated antibodies. Anti-NSE antibody was used after stripping the membranes to estimate the total amount of loaded proteins.

View larger version (25K): [in a new window]   Fig. 5. Cell fractionation analysis. Cytoplasmic and nuclear fractions were prepared from NGF-mock and NGF-p25 cells, both treated for 6 hours with tetracycline. For each cell line, 10 µg cytoplasmic fraction (Cy) and an equivalent volume of the nuclear fraction (Nu) were analysed by western blotting. Subcellular distribution of the specified proteins was visualized using specific antibodies. The arrowhead shows cyclin-A immunoreactivity (lower bands), as determined by comparison with the SDS-PAGE pattern of immunoprecipitated cyclin A (not shown). Stripped membranes were reprobed with antibodies against NSE and lamin B to evaluate the purity of the cytoplasmic and nuclear fractions, respectively.

View larger version (25K): [in a new window]   Fig. 6. Immunoprecipitation and in vitro kinase assays. Lysates from NGF-mock and NGF-p25 cells, both treated for 6 hours with tetracycline, were subjected to immunoprecipitation (IP) with the indicated antibodies. (A) Western-blot analyses. Following in vitro kinase assays, Rb-C fusion protein (68 kDa) phosphorylation was evaluated by immunoblotting using P-Rb Ser807/811 and P-Rb Ser795 antibodies that give similar results (representative immunoblot with P-Rb Ser807/811 is shown). To evaluate the amount of loaded Rb-C fusion protein, blots were stripped and reprobed with a phosphorylation-independent antibody (C-15) directed against the C terminus of Rb (Rb). The lower sections of blots were also probed with specific antibodies to check the presence of the following proteins: p25, Cdk5, Cdk2, Cdk4 and Cdk6. (B) In vitro kinase assays in the presence of (-32P)ATP. Arrowhead indicates Rb-C fusion protein with incorporated 32P, which is only seen in in vitro kinase assay with purified p25-Cdk5 complex.

View larger version (20K): [in a new window]   Fig. 7. Western blotting of in vitro kinase assays with crude cell extracts. Lysates from NGF/p25-expressing cells (6 hours of tetracycline treatment) were used for Rb-C fusion protein phosphorylation, alone and in the presence of the kinase inhibitors roscovitine, PD98059, SB203580 and SP600125. As negative control, an in vitro kinase assay was performed with lysate from NGF-mock cells treated for 6 hours with tetracycline. Phosphorylation of Rb-C fusion protein (P-Rb) was monitored by P-Rb Ser807/811 and Ser795 antibodies, which showed similar data. After stripping, membranes were probed with a phosphorylation-independent antibody against Rb (C-15) to evaluate the amount of loaded Rb-C fusion protein (Rb). The lower side of membrane was analysed by immunoblotting for the levels of p25 and Cdk5, which attests to the presence of equivalent amounts of crude lysate in each kinase reaction.

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