Histone h1 protein

Plasmids encoding N-terminally His₆-tagged kinase domains of individual NAKs or GST-tagged CLINT1(N), CLINT1(C) were electroporated into Rosetta strain BL21 E. coli cells using Gene Pulser Xcell Electroporation Systems (Bio-Rad) with 1800V. Protein expression was induced with 0.5 mM IPTG at 20 °C overnight. Cells were then harvested and resuspended in lysis buffer composed of 50 mM Hepes (pH 7.5), 500 mM NaCl, 5 mM imidazole, 5% glycerol, and 0.5 mM TCEP [tris-(2-carboxyethyl) phosphine]. Following sonication and spinning at 48,400g at 4 °C for 60 min, the supernatant was harvested. NAKs were purified via Ni-affinity followed by tobacco etch virus protease digestion to remove the HIS₆ tag. NAKs were further purified by size-exclusion chromatography via Superdex S75 10/60 column on AKTA pure (GE Lifesciences). Proteins were concentrated via 10-kDa Amicon centrifugal filters (Merck) and suspended in storage buffer composed of 10 mM Hepes (pH 7.5), 300 mM NaCl, 5% glycerol, and 0.5 mM TCEP at −80 °C. Clint1(N) and Clint1(C) were purified by glutathione sepharose beads and eluted with 10 mM glutathione. His₆-tagged recombinant RbC (aa 771-928) was purified by cobalt–sepharose affinity chromatography (GE Sepharose cat#17-0575-01) and eluted with 200 mM imidazole. Histone H1 protein was from EMD Millipore (cat#14-155).

In vitro phosphorylation assays were performed as previously described^{60 (link)}. Briefly, purified recombinant His-CDK5 WT or His-Ac-CDK5 (1 µg) was incubated at 30 °C for 30 min with 1 µg of histone H1 protein (Merck, #382150) and 1 µg of p25-His in 1 × kinase buffer (40 mM Tris-HCl pH 7.6, 2 mM DTT, 5 mM MgCl₂, 1 mM NaF, 50 µM unlabeled ATP) in the presence of 5 µCi [γ-³²P]ATP or cold ATP. Phospho-H1 signals were visualized by autoradiography or immunoblotting with a rabbit polyclonal anti-phospho-histone H1 antibody (1:1,000; Millipore, #06-597). Transiently transfected HEK293 cells and hippocampal neurons were lysed, immunoprecipitated with anti-CDK5 antibody and processed for in vitro phosphorylation assays. To assess ATP binding affinity, purified recombinant His-CDK5 WT and His-Ac-CDK5 (1 μg) were reacted for 2 hrs with 20 μl of ATP-conjugated agarose (Jena Bioscience, #AC-101) as previously described^{61 (link)}. For the in vitro deacetylation assay, immunoprecipitated FLAG-tagged SIRT1 or SIRT2 was incubated with 0.5 µg of purified recombinant Ac-CDK5 supplemented with 5 mM β-nicotinamide adenine dinucleotide (NAD⁺, Sigma, #N7004) at 30 °C for 2 hrs as previously described^{62 (link)}.