crispr-cas9-gfp-par2 plasmid (espcas9-2a-gfp (px458) with cas9, gfp and f2rl1 sgrna  (GenScript corporation)

Journal: Biochemical Journal

Article Title: Unconventional binding of calmodulin to CHK2 kinase inhibits catalytic activity

doi: 10.1042/BCJ20253431

Figure Lengend Snippet: (A ) Schematic of the conventional mode of Ca 2+ -CaM activation in members of the Ca 2+ -CaM-dependent protein kinase (CaMK) family. Upon Ca 2+ influx, Ca 2+ -CaM binds a linear sequence (CaM-binding sequence; CaMBS; orange) that is proximal to the CaMK kinase domain (blue). This interaction restores activity by sequestering an autoinhibitory pseudosubstrate sequence, which formerly obstructed the active site. ( B ) Far Western blots show that recombinant full-length CHK2 and positive control kinase, CaMKK2, interact with His 6 -CaM in a Ca 2+ -dependent manner (Ca 2+ : 500 μM), as binding is abolished in the presence of the Ca 2+ -chelator, EGTA (10 mM). A negative control pseudokinase, MLKL, did not detectably bind CaM. CaM interaction was detected using an anti-His 6 HRP antibody. data are representative of two independent replicates. ( C ) CHK2 is inhibited by Ca 2+ -CaM. Full-length wildtype and K249A kinase-dead CHK2 were immunoprecipitated and activity was measured by radiometric assay containing 200 μM CHKtide, 200 μM [ 32 P]-γ-ATP and in the presence (red) or absence (white) of 200 μM CaCl 2 , 1 mM CaM for 10 min. Two-way ANOVA; **** P <0.0001; n.s = non-significant. ( D ) Double referenced SPR sensorgrams for immobilised full-length CHK2 binding to analyte, Ca 2+ -CaM. Shown is a representative sensorgram that is colour-coded based on CaM concentration. ( E ) Steady state analysis for the SPR experiment shown in ( D ), along with the dissociation constant. ( F ) Titration of CaM (range 0, 0.5, 1, 2, 5, 10, 20, 50, 100, 200 μM) using immunoprecipitated full-length CHK2 (~10 ng) in the presence of 1 mM CaCl 2 by radiometric assay. The half-maximal inhibitory concentration (IC 50 ) for CaM is 8.1 μM. Data represent mean ± SD; n = 3. ( G ) Plot of highest and lowest CHK2 kinase activity from CaM titration the presence of 1 mM CaCl 2 . Individual data points are shown as circles with accompanying mean ± SD; n = 3. Statistical analysis was performed by unpaired t-test; **** P <0.0001. ( H ) Titration of CaCl 2 (range 0, 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000 μM) using immunoprecipitated full-length CHK2 (~10 ng) in the presence of 200 μM CaM by radiometric assay. the IC 50 of CHK2 and CaM by Ca 2+ is 30.7 μM. Data represent mean ± SD; n = 3. ( I ) Plot of highest and lowest CHK2 kinase activity from CaCl 2 titration in the presence of 200 μM CaM. individual data points are shown as circles with accompanying mean ± SD; n = 3. Statistical analysis was performed by unpaired t-test; *** P <0.001. ( J ) Schematic of the amino acid residues of recombinant full-length CHK2 and CaM (red) chemically cross-linked by DMTMM (‘zero-length’) and ( K ) photoactivatable cross-linker, NHS-Diazarine (SDA; 3.9 Å spacer), following mass-spectrometry analysis. Cross-links (grey) are only observed within the CHK2 kinase domain (blue) for both chemical cross-linkers. ( L ) Six lysine residues in CHK2, identified by DMTMM chemical cross-linking are mapped to the domain architecture of CHK2. ( M ) Radiometric assay of immunoprecipitated CHK2 wildtype and select cross-linked lysine mutants in the presence (red) or absence (white) of 200 μM CaCl 2 , 1 mM CaM for 10 min. Note: wildtype and K249A kinase-dead data are reproduced from ( C ). Data represent mean ± SD; n = 3. Statistical analysis was performed by two-way ANOVA; * and **** P <0.1 and P <0.0001, respectively; n.s = non-significant. Assay data using recombinant CHK2 kinase domain with each cross-linked lysine mutant are shown in .

Article Snippet: 1 × 10 6 cells were co-transfected with 5 μg of eSpCas9-hGeminin –CHK2 gRNA plasmid, 5 μg of ATP1A1_plasmid_donor_RD (Addgene plasmid, #86551), and 2.5 μl ssODN donor (100 μM).

Techniques: Activation Assay, Sequencing, Binding Assay, Activity Assay, Western Blot, Recombinant, Positive Control, Negative Control, Immunoprecipitation, Concentration Assay, Titration, Mass Spectrometry, Mutagenesis

Journal: Biochemical Journal

Article Title: Unconventional binding of calmodulin to CHK2 kinase inhibits catalytic activity

doi: 10.1042/BCJ20253431

Figure Lengend Snippet: (A ) Quantitative analysis of deuterium exchange differences of human CHK2 in the presence of Ca 2+ -CaM, following hydrogen-deuterium exchange mass spectrometry (HDX-MS). The graph shows the #D difference in deuterium incorporation for CHK2 over the entire time course. Each point represents an individual peptide, with those coloured in red showing a significant change (greater than 0.40 Da and 5% difference at any timepoint, with a two tailed t -test; P <0.01, n = 3), with error bars showing standard deviation. CHK2 domain architecture is annotated above. ( B ) Significant differences in deuterium exchange are mapped onto an AlphaFold3 model of CHK2 and are colour-coded according to the legend. Zoomed view of the CHK2 active site cleft is shown on the right. Key catalytic residues and cross-linked lysine residues (from Figure 1L ) are annotated. ( C ) Cross-linking mass spectrometry data are mapped onto an AlphaFold model of CaM:CHK2. ‘Zero-length’ DMTMM and SDA cross-links are shown in red and blue, respectively. Key catalytic residues of CHK2 are shown in yellow. ( D ) The electrostatic potential mapped onto the molecular surface of CHK2 reveals patches of positive charge that are proximal with residues in ( C ) and complement the negatively charged surface of CaM. ( E ) AlphaFold3 model of human CHK2 kinase domain (blue) in complex with CaM (red).

Article Snippet: 1 × 10 6 cells were co-transfected with 5 μg of eSpCas9-hGeminin –CHK2 gRNA plasmid, 5 μg of ATP1A1_plasmid_donor_RD (Addgene plasmid, #86551), and 2.5 μl ssODN donor (100 μM).

Techniques: Mass Spectrometry, Two Tailed Test, Standard Deviation, Structural Proteomics

Journal: Biochemical Journal

Article Title: Unconventional binding of calmodulin to CHK2 kinase inhibits catalytic activity

doi: 10.1042/BCJ20253431

Figure Lengend Snippet: (A ) Multiple sequence alignment of CHK2 orthologs, which highlights the conservation of CHK2 residues in animals and fungi (K255, K373 and K465; asterisk) implicated in interaction with Ca 2+ -CaM. Residues in the alignment are coloured according to the Clustal colouring scheme, which highlights chemical nature and patterns of conservation. ( B ) Chromatograms from Sanger sequencing confirm the successful integration of nucleotide changes for K373A in hTERT-immortalized RPE cells by CRISPR/Cas9. ( C ) Real-time proliferation analysis of wildtype and CRISPR-edited RPE cells using IncuCyte live-cell imaging, and ( D ) corresponding area under the curve. Data represent mean ± SD; n = 8. Statistical analysis was performed by paired t -test; **** P <0.0001. ( E ) Real-time proliferation analysis of wildtype and CRISPR-edited RPE cells cultured in the presence of DNA damaging agent, camptothecin (CPT), at 10 nM and 100 nM, and ( F ) corresponding area under the curve. Data represent mean ± SD; n = 8. Statistical analysis was performed by two-way ANOVA; **** P <0.0001. ( G ) Raw micrographs of cell confluence exported from the IncuCyte SX3 system after 72 h incubation; the scale bars (black) represent 100 µm. ( H ) Whole cell-lysates of hTERT-immortalised RPE cells (wildtype and CHK2 K373A ) were resolved by SDS-PAGE and probed by immunoblot for phospho-CHK2 (Thr383) and total CHK2 with anti-actin as a loading control. Immunoblots are representative of n = 2 independent experiments.

Article Snippet: 1 × 10 6 cells were co-transfected with 5 μg of eSpCas9-hGeminin –CHK2 gRNA plasmid, 5 μg of ATP1A1_plasmid_donor_RD (Addgene plasmid, #86551), and 2.5 μl ssODN donor (100 μM).

Techniques: Sequencing, CRISPR, Live Cell Imaging, Cell Culture, Incubation, SDS Page, Western Blot, Control

Journal: Biochemical Journal

Article Title: Unconventional binding of calmodulin to CHK2 kinase inhibits catalytic activity

doi: 10.1042/BCJ20253431

Figure Lengend Snippet: Following DNA damage, the upstream kinase, ATM (green), phosphorylates Thr68 within the CHK2 SCD, which leads to the formation of a transient homodimer, mediated by the CHK2 FHA domains binding to pThr68. This dimerization results in CHK2 activation loop phosphorylation on T383 in trans and increased catalytic activity. Following activation, CHK2 phosphorylates multiple substrates, including CDC25C, BRCA1, HDMX, PLK1 and TP53, to promote cell cycle arrest and impede DNA repair. Upon sensing increased Ca 2+ levels, CaM can directly interact with the CHK2 kinase domain to inhibit catalytic activity and enable cell cycle progression and DNA repair.

Article Snippet: 1 × 10 6 cells were co-transfected with 5 μg of eSpCas9-hGeminin –CHK2 gRNA plasmid, 5 μg of ATP1A1_plasmid_donor_RD (Addgene plasmid, #86551), and 2.5 μl ssODN donor (100 μM).

Techniques: Binding Assay, Activation Assay, Phospho-proteomics, Activity Assay

Journal: Biochemical Journal

Article Title: Unconventional binding of calmodulin to CHK2 kinase inhibits catalytic activity

doi: 10.1042/BCJ20253431

Figure Lengend Snippet: (A ) Ca 2+ -CaM-dependent protein kinase II (PDB 2WEL) ; ( B ) Death-associated protein kinases (DAPK)-1 (PDB 2X0G) ; and ( C ) DAPK2 (PDB 6PAW) harbor a conventional CaM-binding sequence C-terminal to the protein kinase domain. ( D ) GPCR kinase 5 (GRK5; PDB 6PJX) interacts with Ca 2+ -CaM via two helical regions N- and C-terminal to the protein kinase domain. ( E ) Ca 2+ -CaM directly interacts with the protein kinase domain of CHK2 (AlphaFold model; from this study). Each mode of Ca 2+ -CaM regulation is illustrated in a Richardson (ribbons) diagram (top panel) and schematic representation (lower panel). The protein kinase domain is coloured blue, while other domains are grey. CaM and CaM-binding sequences (CaMBS) are coloured red and orange, respectively. Ca 2+ ions are coloured yellow.

Article Snippet: 1 × 10 6 cells were co-transfected with 5 μg of eSpCas9-hGeminin –CHK2 gRNA plasmid, 5 μg of ATP1A1_plasmid_donor_RD (Addgene plasmid, #86551), and 2.5 μl ssODN donor (100 μM).

Techniques: Binding Assay, Sequencing