1. Volume 17, Issue 1, Pages 37-48 (January 2005)
MAPKAP Kinase-2 Is a Cell Cycle Checkpoint Kinase that Regulates the G2/M Transition and S Phase Progression in Response to UV Irradiation 
Isaac A. Manke, Anhco Nguyen, Daniel Lim, Mary Q. Stewart, Andrew E.H. Elia, Michael B. Yaffe 
Molecular Cell 
Volume 17, Issue 1, Pages (January 2005)
DOI: /j.molcel

2. Figure 1
Substrate Specificity and Kinetic Analysis of Substrate Phosphorylation by p38α SAPK
(A) p38 substrate specificity determined using oriented peptide library screening. Residues displaying the highest selectivity are shown; those with selection values >1.7 are in bold. MEF2A, myocyte enhancer factor 2; ATF2, activating transcription factor 2; 3PK1, MAP kinase-activated protein kinase-3.
(B) Kinetics of in vitro phosphorylation of an optimal p38 peptide (p38tide) and a peptide from p47phox (p47tide) by p38α kinase.
(C) Kinetics of in vitro phosphorylation of wild-type GST-p47phox, the Ser345→Ala mutant, and the Ser348→Ala mutant. Typical data from n = 3 experiments is shown.
(D) In vitro phosphorylation of full-length wild-type or mutant p47phox proteins. Samples were analyzed by SDS-PAGE/autoradiography.
(E) Table of kinetic parameters for the reactions in (C).
Molecular Cell  , 37-48DOI: ( /j.molcel )

3. Figure 2
Involvement of MAPKAP Kinase-2 in the Phosphorylation of Cdc25B and Cdc25C after DNA Damage
(A) Phosphorylation of full-length wild-type Cdc25B or a Ser323→Ala mutant by p38α SAPK or MAPKAP kinase-2. After phosphorylation, generation of a binding site on Cdc25B was determined by a MBP pull-down followed by SDS-PAGE/autoradiography.
(B) Kinetics of MAPKAP kinase-2 phosphorylation and generation of a binding site on Cdc25B were measured in U2OS cells following 20 J/m2 UV irradiation.
(C) Signaling events in the G2/M, G1, and S phase checkpoint response were analyzed in GFP siRNA- or MAPKAP kinase-2 siRNA-treated U2OS cells before and 2 hr after UV-induced DNA damage. Equal loading was determined by Western blotting for β-actin.
Molecular Cell  , 37-48DOI: ( /j.molcel )

4. Figure 3
Substrate Specificity and Kinetic Analysis of Substrate Phosphorylation by MAPKAP Kinase-2
(A) MAPKAP kinase-2 substrate specificity was determined by oriented peptide library screeing. HSP27, heat shock protein 27; 5-LO, 5-lipoxygenase; LSP1, lymphocyte-specific protein; SRF, serum response factor; GS, glycogen synthase; TH, tyrosine hydroxylase.
(B) Kinetics of in vitro phosphorylation of the optimal MAPKAP kinase-2 peptide (MK2tide) by MAPKAP kinase-2.
(C) Table of kinetic parameters for MAPKAP kinase-2 phosphorylation of wild-type and mutant MK2tides.
Molecular Cell  , 37-48DOI: ( /j.molcel )

5. Figure 4
MAPKAP Kinase-2 Is Required for G2/M Arrest following DNA Damage
(A) UV-C irradiation induces DNA damage as revealed by nuclear foci formation. U2OS cells were mock irradiated or exposed to 20 J/m2 of UV-C radiation and immunostained 2 hr later using an anti-phospho(Ser/Thr) ATM/ATR substrate antibody.
(B–I) GFP siRNA- or MAPKAP kinase-2 siRNA-treated U2OS cells were irradiated as in (A), then placed in 50 ng/ml nocodazole-containing media for an additional 16 hr. Cells were analyzed by FACS for DNA content by PI staining (B, D, F, and H) and for phospho-histone H3 staining as a marker of mitotic entry (C, E, G, and I).
(J–K) GFP siRNA- or MAPKAP kinase-2 siRNA-treated U2OS cells were either mock treated, exposed to 20 J/m2 of UV-C irradiation (J), or to 10 Gy of ionizing radiation (K) and analyzed as in (B)–(I). Representative results of each experiment are shown.
Molecular Cell  , 37-48DOI: ( /j.molcel )

6. Figure 5
MAPKAP Kinase-2 Is Required for S Phase Arrest and Cell Survival following DNA Damage
(A) GFP siRNA- or MAPKAP kinase-2 siRNA-treated U2OS cells were mock treated or UV irradiated and allowed to recover for 30 min. BrdU was added and cells were fixed and analyzed by FACS for DNA content and BrdU incorporation 12 hr later.
(B) Percentage of cells in (A) showing BrdU incorporation at 2 and 12 hr following BrdU addition were measured.
(C) GFP siRNA- or MAPKAP kinase-2 siRNA-treated U2OS cells were either mock treated or UV irradiated, allowed to recover for 30 min, then pulse labeled with BrdU for 30 min. At the indicated times after irradiation the distribution of DNA content was analyzed in the BrdU-positive population.
(D) GFP siRNA- or MAPKAP kinase-2 siRNA-treated U2OS cells were either mock treated or irradiated at the indicated UV-C dose. Cells were stained with Crystal Violet 48 hr later and visualized. Insets show a magnified view.
(E) Quantitative colony forming assays were performed by plating cells at a density of ∼100 cells per 35 mm2 dish. Cells were treated as in (D), and assays were performed in triplicate for each condition.
Molecular Cell  , 37-48DOI: ( /j.molcel )

7. Figure 6
Comparison of Active Site Electrostatic Potentials and Hydrophobicity for the Substrate Binding Cleft of MAPKAP Kinase-2, Akt, and Chk1
(A) Optimal substrate phosphorylation motifs for Akt/PKB, Chk1, Chk2, and MAPKAP kinase-2.
(B) Ribbons representation of the MAPKAP kinase-2 kinase domain in a similar orientation as that shown in (C)–(E) (upper), and in an orthogonal orientation (lower) with stick representations of the substrate peptide in the active site cleft. Figure created using Molscript (Kraulis, 1991) and Raster3D (Merritt and Murphy, 1994).
(C) Molecular surface representations of the Akt/PKB active site (PDB code 1O6K) using GRASP (Nicholls et al., 1993). Electrostatic potentials (left) are colored red (negative) and blue (positive). Hydrophobicity (right) is shaded gray, yellow, and green for low, medium, and high hydrophobic potentials, respectively. The GSK3 substrate peptide Gly-Arg-Pro-Arg-Thr-Thr-Ser-Phe-Ala-Glu, with the phospho-acceptor indicated in bold and underlined, is shown in stick representation.
(D) Molecular surface representations of the MAPKAP kinase-2 active site (PDB code 1NY3). Electrostatic and hydrophobic potentials are colored as in (C). The optimal substrate peptide Leu-Gln-Arg-Gln-Leu-Ser-Ile-Ala is shown in stick representation.
(E) Molecular surface representations of the Chk1 active site (PDB code 1IA8). Electrostatic and hydrophobic potentials are colored as in (C). Stick representation of the modeled Cdc25C substrate peptide (Leu-Tyr-Arg-Ser-Pro-Ser-Met-Pro-Leu) is shown.
The region corresponding to the Ser−5, Ser−3, and Ser+1 positions of the substrate peptides in (C), (D), and (E) is indicated by dashed circles.
Molecular Cell  , 37-48DOI: ( /j.molcel )

8. Figure 7 A Unified Model of the Kinase-Dependent DNA Damage Checkpoint
Parallel pathways in the DNA damage checkpoint signal transduction network converge on common substrates by signaling to downstream kinases with similar phosphorylation motif specificities. φ indicates hydrophobic residues. The dashed line from Chk1 to Cdc25B/C indicates that this phosphorylation event remains controversial in response to ionizing radiation.
Molecular Cell  , 37-48DOI: ( /j.molcel )