cdk5 inhibitor r cr8  (Tocris)

Journal: Communications Biology

Article Title: Post-ischemic ubiquitination at the postsynaptic density reversibly influences the activity of ischemia-relevant kinases

doi: 10.1038/s42003-024-06009-8

Figure Lengend Snippet: a Proteins with increased and decreased post-ischemic ubiquitination were assessed for GO enrichment for biological process. Benjamini-corrected P values for the top four terms for the “Ub increased” dataset are depicted. b All proteins with increased ubiquitination after ischemia and enrichment in the four top categories for biological process were investigated for GO enrichment for molecular function and cellular components. Benjamini-corrected P values for the five highest-ranking terms are shown. c Post-ischemic ubiquitination of select PSD-associated kinases and phosphatases was confirmed by immunoprecipitation of proteins of interest from ipsi- and contralateral detergent-insoluble cortical lysates from MCAO/1 h reperfusion-treated mice and detection of ubiquitin by Western Blotting. IgG-isotype antibodies served as controls. Results from n = 3 mice/group were quantified. CaMKIIα: * P = 0.0009; CaMKIIβ: * P = 0.0040; PKCβ: * P = 0.0021; PKCγ: * P = 0.0099; Cdk5: * P = 0.0015; Pyk2: * P = 0.0016; CKβ: * P = 0.0002; Pten: * P = 0.0013. Two-tailed unpaired t -test. Data are expressed as mean ± s.e.m. d Domain structure of prominent PSD-associated kinases and phosphatases found ubiquitinated after ischemia. Numbers represent amino acid positions, and arrows indicate ubiquitinated residues identified by MS analysis. BP biological process, c contralateral, C carboxy-terminus, C1a, and C1b diacylglycerol-binding domain, C2 calcium-binding domain, CaM calmodulin, FAT focal adhesion kinase-targeting domain, FERM, 4.1 protein, Ezrin radixin, and moesin domain, GO gene ontology, i ipsilateral, LTP long-term potentiation, N amino-terminus, nc not called, P proline-rich region, PDZ-b PSD95, Dlg1 Zo-1-containing domain-binding domain, POI protein of interest, Ub ubiquitination.

Article Snippet: The specificity of substrate phosphorylation by Cdk5 was verified by adding 10 μM Cdk5 inhibitor ((R)-CR8, Tocris Bioscience, Bristol, UK) to companion reactions.

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Two Tailed Test, Binding Assay

Journal: Communications Biology

Article Title: Post-ischemic ubiquitination at the postsynaptic density reversibly influences the activity of ischemia-relevant kinases

doi: 10.1038/s42003-024-06009-8

Figure Lengend Snippet: a Phosphorylation levels of Cdk5 downstream targets were determined in PSD lysates of sham and MCAO-treated animals. Tau: * P = 0.0137 from s, # P = 0.0055 from s; Crmp2: * P = 0.0496 from s and P = 0.0430 from c; one-way ANOVA with Bonferroni test; n = 4–5 animals/group. b Cortical PSD-lysates from sham and MCAO-treated mice were assessed for Cdk5 activity (* P < 0.0001, two-tailed unpaired t -test; n = 6 mice/group). c Cdk5 activity was measured in post-ischemic PSD lysates untreated and treated with recombinant deubiquitinase USP2 (* P = 0.0057, two-tailed paired t -test; n = 6 animals). c contralateral, i ipsilateral, S serine, s sham. Data are expressed as mean ± s.e.m.

Article Snippet: The specificity of substrate phosphorylation by Cdk5 was verified by adding 10 μM Cdk5 inhibitor ((R)-CR8, Tocris Bioscience, Bristol, UK) to companion reactions.

Techniques: Phospho-proteomics, Activity Assay, Two Tailed Test, Recombinant

Journal: Communications Biology

Article Title: Post-ischemic ubiquitination at the postsynaptic density reversibly influences the activity of ischemia-relevant kinases

doi: 10.1038/s42003-024-06009-8

Figure Lengend Snippet: a Post-ischemic detergent-resistant ubiquitination is elevated in neurons, particularly at the postsynaptic density (PSD) of glutamatergic neurons, while it is reduced in all other brain cell types. EC endothelial cell, glut glutamatergic, Tx Triton X100, Ub ubiquitination. b Postsynaptic proteins with increased ubiquitination after ischemic stroke include receptors (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor (AMPAR), N-methyl-D-aspartate receptor (NMDAR), Densin-180, G protein-coupled receptor (GPCR), tropomyosin receptor kinase (TrkB)), scaffolding proteins and their adapters (brain-enriched guanylate kinase-associated protein (Begain), Disks large-associated protein 2–4 (Dlgap2-4), guanylate kinase-associated protein (GKAP), postsynaptic density protein 93/95 (PSD93/95), synapse-associated protein 97/102 (SAP97/102), SH3 and multiple ankyrin repeat domain (Shank)), G proteins (guanine nucleotide-binding protein G(i) subunit alpha (Gnai), guanine nucleotide-binding protein G(o) subunit alpha (Gnao), guanine nucleotide-binding protein G(s) subunit alpha isoforms short (Gnas), guanine nucleotide-binding protein G(t) subunit alpha (Gnat)), and signaling proteins (calcium-calmodulin-dependent protein kinase II (CaMKII), cyclin-dependent kinase 5 (Cdk5), creatine kinase b (CKb), Kalirin-7, protein kinase C (PKC), protein phosphatase 2 (PP2), phosphatase and TENsin homolog (Pten), proline-rich tyrosine kinase (Pyk2), synaptic Ras GTPase-activating protein 1 (SynGAP1)). c PSD-kinases, such as CaMKII, PKC, Cdk5, and Pyk2, are prominent post-ischemic ubiquitination targets. While CaMKII, PKC, and Cdk5 activities at the PSD are decreased after stroke, leading to reduced target phosphorylation, Pyk2 exhibits increased activity, thereby accelerating the phosphorylation of target proteins. In all cases, kinase activity regulation after stroke was dependent on ubiquitination, whose removal normalized activity. P phosphorylation. This figure was created with BioRender.com .

Article Snippet: The specificity of substrate phosphorylation by Cdk5 was verified by adding 10 μM Cdk5 inhibitor ((R)-CR8, Tocris Bioscience, Bristol, UK) to companion reactions.

Techniques: Ubiquitin Proteomics, Scaffolding, Binding Assay, Phospho-proteomics, Activity Assay

Journal: Cell Death & Disease

Article Title: CDK5-induced p-PPAR γ (Ser 112) downregulates GFAP via PPREs in developing rat brain: effect of metal mixture and troglitazone in astrocytes

doi: 10.1038/cddis.2013.514

Figure Lengend Snippet: MM and TZ activate CDK5 and induce binding of CDK5 with PPAR γ , resulting in increased p-PPAR γ (S112) and reduced GFAP in rat brain astrocytes. Five- μ m-thick cryostat sections of cerebral cortex (coronal section) from vehicle (V)- and MM-treated rats were co-labeled for p-CDK5, GFAP and nuclear Hoechst. ( a ) Representative photomicrograph ( × 20 magnification) of p-CDK5 (red fluorescence), GFAP (green fluorescence) and nucleus (blue fluorescence), and the three merged in the same field for MM (LHS). The p-CDK5/GFAP ratio normalized with nuclear Hoechst, for MM (RHS). Sections are representatives of four rats from four different litters, and bar diagrams represent mean±S.E. *** P <0.001 (compared with V). ( b ) Representative photomicrograph ( × 20 magnification) of p-CDK5 (green fluorescence), GFAP (red fluorescence) and nucleus (blue fluorescence), and the three merged in the same field for TZ (LHS). The p-CDK5/GFAP ratio normalized with nuclear Hoechst for TZ (RHS). Sections are representatives of four rats from four different litters, and bar diagrams represent mean±S.E. *** P <0.001 (compared with V). ( c ) Rat primary astrocytes were treated with MM or TZ for 15 min. The cell lysates were collected and subjected to immune precipitation with PPAR γ antibody. Immunoprecipitated (IP) complexes along with input samples were resolved on SDS-PAGE and immunoblotted (IB) with CDK5 or PPAR γ antibodies. Representative blots of CDK5 (LHS) and PPAR γ (RHS) IP by PPAR γ . Results are representative of three independent experiments. Rat primary astrocytes were pre-incubated in reduced serum medium, or R-CR8 in reduced serum medium, and then co-treated with MM or TZ for the indicated time points. ( d ) Representative western blot (upper panel) and densitometric analysis (lower panel) of p-PPAR γ (S112) relative to PPAR γ for MM or R-CR8 with MM. Data represent mean±S.E. of four independent experiments in triplicate. *** P <0.001 and ** P <0.01 (compared with 0 min). ( e ) Representative western blot (upper panel) and densitometric analysis (lower panel) of p-PPAR γ (S112) relative to PPAR γ for TZ or R-CR8 with TZ. Data represent mean±S.E. of four independent experiments in triplicate. *** P <0.001, ** P <0.01 and * P <0.05 (compared with 0 min). ( f ) Representative western blot (upper panel) and densitometric analysis (lower panel) of GFAP relative to β -actin for MM or R-CR8 with MM. Data represent mean±S.E. of four independent experiments in triplicate. *** P <0.001 and ** P <0.01 (compared with 0 h). ( g ) Representative western blot (upper panel) and densitometric analysis (lower panel) of GFAP relative to β -actin for TZ or R-CR8 with TZ. Data represent mean±S.E. of four independent experiments in triplicate. *** P <0.001 and ** P <0.01 (compared with 0 h)

Article Snippet: PPAR- γ agonist TZ, rosiglitazone and pioglitazone, PPAR- γ antagonist T0070907 and CDK5 inhibitors (R)-CR8 were purchased from Tocris Biosciences (Bristol, UK).

Techniques: Binding Assay, Labeling, Fluorescence, Immunoprecipitation, SDS Page, Incubation, Western Blot

Journal: Cell Death & Disease

Article Title: CDK5-induced p-PPAR γ (Ser 112) downregulates GFAP via PPREs in developing rat brain: effect of metal mixture and troglitazone in astrocytes

doi: 10.1038/cddis.2013.514

Figure Lengend Snippet: MM and TZ activate MEK1/2→ JNK→ CDK5 in rat brain astrocytes. Five- μ m-thick cryostat sections of cerebral cortex (coronal section) from vehicle (V)- or MM- or TZ-treated rats were stained for p-ERK1/2 or p-JNK followed by GFAP and nuclear Hoechst co-stain. ( a ) Representative photomicrograph ( × 20 magnification) of p-ERK1/2 (green fluorescence), GFAP (red fluorescence) and nucleus (blue fluorescence), and the three merged in the same field for MM (LHS). The p-ERK1/2/GFAP ratio normalized with nuclear Hoechst (RHS). ( b ) Representative photomicrograph ( × 20 magnification) of p-JNK (green fluorescence), GFAP (red fluorescence) and nucleus (blue fluorescence), and the three merged in the same field (LHS) for MM. The p-JNK/GFAP ratio normalized with nuclear Hoechst (RHS). ( c ) Representative photomicrograph ( × 20 magnification) of p-ERK1/2 (green fluorescence), GFAP (red fluorescence) and nucleus (blue fluorescence), and the three merged in the same field for TZ (LHS). The p-ERK1/2/GFAP ratio normalized with nuclear Hoechst (RHS). ( d ) Representative photomicrograph ( × 20 magnification) of p-JNK (green fluorescence), GFAP (red fluorescence) and nucleus (blue fluorescence), and the three merged in the same field for TZ (LHS). The p-JNK/GFAP ratio normalized with Hoechst (RHS). Sections are representatives of four rats from four different litters and bar diagrams represent means±SE. *** P <0.001 (compared with V). ( e ) Rat primary astrocytes were pre-incubated in serum-free medium and then treated with TZ for the indicated time points. Left panel: Representative western blot and densitometric analysis of p-ERK1/2 relative to ERK1/2. Right Panel: Representative western blot and densitometric analysis of p-JNK relative to JNK. Data represent mean±S.E. of four independent experiments in triplicate. *** P <0.001, ** P <0.01 and * P <0.05 (compared with 0 min). ( f ) Rat primary astrocytes were pre-incubated with PD98059 in serum-free medium and then co-treated with TZ for the indicated time points. Representative western blot and densitometric analysis of p-JNK relative to JNK. Data represent mean±S.E. of four independent experiments in triplicate. ( g ) Rat primary astrocytes were pre-incubated in reduced serum medium, PD98059 in reduced serum medium or SP600125 in reduced serum medium for 60 min and then co-treated with MM for the indicated time points. Left panel: Representative western blot and densitometric analysis of p-CDK5 relative to CDK5 for MM. Middle panel: Representative western blot and densitometric analysis of p-CDK5 relative to CDK5 for PD98059+MM. Right panel: Representative western blot and densitometric analysis of p-CDK5 relative to CDK5 for SP600125+MM. Data represent mean±S.E. of four independent experiments in triplicate. *** P <0.001 and * P <0.05 (compared with 0 min). ( h ) Rat primary astrocytes were pre-incubated in reduced serum medium, PD98059 in reduced serum medium or SP600125 in reduced serum medium for 60 min and then co-treated with TZ for the indicated time points. Left panel: Representative western blot and densitometric analysis of p-CDK5 relative to CDK5, for TZ. Middle panel: Representative western blot and densitometric analysis of p-CDK5 relative to CDK5, for PD98059+TZ. Right panel: Representative western blot and densitometric analysis of p-CDK5 relative to CDK5 for SP600125+TZ. Data represent mean±SE of four independent experiments in triplicate. *** P <0.001 and ** P <0.01 (compared with 0 min)

Article Snippet: PPAR- γ agonist TZ, rosiglitazone and pioglitazone, PPAR- γ antagonist T0070907 and CDK5 inhibitors (R)-CR8 were purchased from Tocris Biosciences (Bristol, UK).

Techniques: Staining, Fluorescence, Incubation, Western Blot

Journal: Cell Death & Disease

Article Title: CDK5-induced p-PPAR γ (Ser 112) downregulates GFAP via PPREs in developing rat brain: effect of metal mixture and troglitazone in astrocytes

doi: 10.1038/cddis.2013.514

Figure Lengend Snippet: Proposed schematic diagram of MM- and TZ-mediated GFAP downregulation in astrocytes of rat brain. The MM and TZ induces sequential activation of ERK1/2, JNK and CDK5. The CDK5 binds to the PPAR γ , enhances p-PPAR γ (S112) that suppresses GFAP

Article Snippet: PPAR- γ agonist TZ, rosiglitazone and pioglitazone, PPAR- γ antagonist T0070907 and CDK5 inhibitors (R)-CR8 were purchased from Tocris Biosciences (Bristol, UK).

Techniques: Activation Assay