Journal: FEBS letters

Article Title: A novel site of AKT-mediated phosphorylation in the human MDM2 onco-protein.

doi: 10.1016/j.febslet.2004.09.081

Figure Lengend Snippet: Fig. 1. Cell extracts contain protein kinase activities that phosphorylate the region of human MDM2 comprising amino acids 108–200. (a) MCF-7 cell extracts were fractionated on a HiTrap Q column and eluted with a gradient of NaCl. Kinase activities were measured using the MP2 mini- protein. (b) Western blot showing the presence of the AKT in the fractions. (c) MDM2 kinase activity following immuno-depletion of AKT with anti-AKT and anti-Ran (control) antibodies. (d) Schematic representation showing the MDM2 protein and the four GST-MDM2 fusion proteins (MP1-4) used as substrates. (e) Phosphorylation of equal amounts of each of the four mini-proteins, MP1-MP4, by AKT. The positions of the mini- proteins are indicated.

Article Snippet: The following antibodies were used: MDM2 (D12 and SMP-14, both from Santa Cruz Biotechnology Inc.); AKT (polyclonal antibody from Prof. D. Alessi, University of Dundee), Ran (from Dr. B. McStay, University of Dundee), and Myc 9E10 (CRUK).

Techniques: Western Blot, Activity Assay, Immunodepletion, Control, Phospho-proteomics

Journal: FEBS letters

Article Title: A novel site of AKT-mediated phosphorylation in the human MDM2 onco-protein.

doi: 10.1016/j.febslet.2004.09.081

Figure Lengend Snippet: Fig. 2. AKT phosphorylates Ser-166 and Ser-188 in MDM2. (a) Phosphorylation of GST-MDM2 fusion protein MP2, or a series of Ser-Ala substitution mutants, in vitro by recombinant AKT. Ap- proximately equal amounts of substrate proteins were used in each assay (lower panel). (b) Kinase activity of HiTrap Q column fractions towards WT GST-MDM2 (MP2) and a S188A mutant. (c) Western blotting of in vitro-phosphorylated MP2, or alanine substitution mu- tants, using phospho-specific antibodies. Each of the small left hand panels shows positive (MP2 phosphorylated by the Chk1 kinase) and negative (unphosphorylated MP2) controls for each phospho-specific antibody. Approximately equal amounts of substrate proteins were used in each assay (lower panel).

Article Snippet: The following antibodies were used: MDM2 (D12 and SMP-14, both from Santa Cruz Biotechnology Inc.); AKT (polyclonal antibody from Prof. D. Alessi, University of Dundee), Ran (from Dr. B. McStay, University of Dundee), and Myc 9E10 (CRUK).

Techniques: Phospho-proteomics, In Vitro, Recombinant, Activity Assay, Mutagenesis, Western Blot

Journal: FEBS letters

Article Title: A novel site of AKT-mediated phosphorylation in the human MDM2 onco-protein.

doi: 10.1016/j.febslet.2004.09.081

Figure Lengend Snippet: Fig. 3. AKT phosphorylates full length MDM2. Tryptic phosphopeptide mapping of MP2 (a), GST-tagged MDM2 (FL WT (b)), and GST-tagged MDM2 in which Ser-188 is substituted by alanine (FL 188, panel (d)), phosphorylated in vitro by AKT. In panel (c), equal amounts of radioactive phosphopeptides from the MP2 and the full length MDM2 phosphorylation reactions were mixed prior to loading onto the thin layer plate (FLwt/ MP2 mix, panel (c)). The arrow in panel (d) indicates the absence of the major phosphopeptide in the map obtained from the FL188 mutant protein.

Article Snippet: The following antibodies were used: MDM2 (D12 and SMP-14, both from Santa Cruz Biotechnology Inc.); AKT (polyclonal antibody from Prof. D. Alessi, University of Dundee), Ran (from Dr. B. McStay, University of Dundee), and Myc 9E10 (CRUK).

Techniques: Phospho-proteomics, In Vitro, Mutagenesis

Journal: FEBS letters

Article Title: A novel site of AKT-mediated phosphorylation in the human MDM2 onco-protein.

doi: 10.1016/j.febslet.2004.09.081

Figure Lengend Snippet: Fig. 4. MDM2 is phosphorylated at Ser-166 in response to serum stimulation. U2OS cells were maintained in 10% FBS or in the absence of serum for 24 h. Cells deprived of serum were stimulated in the presence of 20% FBS for up to 8 h. (a, b) Cell extracts were prepared and MDM2 phos- phorylation reactions were carried out in vitro on GST-MDM2. (a) Detection by autoradiography when [c-32P]ATP was used as the phosphate donor. (b) Western blotting was performed using the anti-phospho-serine 166, or anti-GST antibodies. (c) Phosphorylation of endogenous MDM2 in response to serum and in the presence or absence of the PI3-kinase inhibitor, LY294002. MDM2 was immunoprecipitated using the monoclonal antibodies SMP14 and D12. Immunoprecipitated proteins were Western blotted for the presence of phospho-serine 166 and MDM2 (SMP14/D12).

Article Snippet: The following antibodies were used: MDM2 (D12 and SMP-14, both from Santa Cruz Biotechnology Inc.); AKT (polyclonal antibody from Prof. D. Alessi, University of Dundee), Ran (from Dr. B. McStay, University of Dundee), and Myc 9E10 (CRUK).

Techniques: In Vitro, Autoradiography, Western Blot, Phospho-proteomics, Immunoprecipitation, Bioprocessing

Journal: FEBS letters

Article Title: A novel site of AKT-mediated phosphorylation in the human MDM2 onco-protein.

doi: 10.1016/j.febslet.2004.09.081

Figure Lengend Snippet: Fig. 5. MDM2 is phosphorylated physiologically at Ser-188. (a) Tryptic phosphopeptide mapping of MCF-7 cells transfected with plasmids encoding Myc-tagged WT or S188A MDM2 and pulse-labeled with 32P-orthophosphate. Arrowheads mark the origins. Black arrows show the Ser-188 phosphopeptides. The sequence shown at the base of the plates represents amino acids 185-204 of MDM2 and indicates the position of phospho- serine 188. The vertical arrows indicate the sites of trypsin digestion. (b) SJSA cells deprived of serum were stimulated in the presence of 20% FBS for up to 2 h. Cell extracts were prepared and MDM2 phosphorylation reactions were carried out in vitro on GST-MDM2. Western blotting was performed using the anti-phospho-serine 188, or anti-GST antibody. (c) Phosphorylation of endogenous MDM2 in response to serum and in the presence or absence of the PI3-kinase inhibitor, LY294002. Immunoprecipitated MDM2 proteins were Western blotted for the presence of phospho- serine 188 and MDM2 (SMP14/D12).

Article Snippet: The following antibodies were used: MDM2 (D12 and SMP-14, both from Santa Cruz Biotechnology Inc.); AKT (polyclonal antibody from Prof. D. Alessi, University of Dundee), Ran (from Dr. B. McStay, University of Dundee), and Myc 9E10 (CRUK).

Techniques: Phospho-proteomics, Transfection, Labeling, Sequencing, In Vitro, Western Blot, Immunoprecipitation