Calmodulin

HEWL, penta-N-acetylchitopentaose (NAG₅), dithiotreithol (DTT), iodoacetamide, calmodulin, equine cytochrome c, horse heart myoglobin, melittin and ubiquitin were purchased from Sigma-Aldrich (Poole, UK). Trypsin and AspN were obtained from Promega (Southhampton, UK). Lys48 poly-ubiquitin ladder, wild-type USP5 and Lys48 diubiquitin were purchased from Boston Biochem (Cambridge, MA, USA). C335A USP5 was expressed and purified as previously described34 (link). Photoleucine 1 was purchased from Thermo Fisher Scientific, Loughborough, UK, and aryldiazirine 2 synthesized as described above.

MBP was prepared from bovine brains according to [28 ]. The obtained protein was purified by reverse phase HPLC on a C₄ 10/250 column (Macherey-Nagel). Actin from porcine muscle, lysozyme from chicken egg, calmodulin from bovine brain, and BSA were obtained from Sigma. Recombinant histone H1.3 was obtained from E. coli, and recombinant human ubiquitin and recombinant human K48-tetraubiquitin were obtained from Boston Biochem. GA (Copaxone) is a commercially available drug from Teva; for the experiments, it was desalted into 20 mM Tris-HCl pH 7.5 using a HiTrap Desalt column (GE Healthcare Life Sciences).

Bovine aorta endothelial cells (BAECs) and cell culture materials were obtained from Lonza (wakersville, MD). 2', 5’-ADP-Sepharose 4B was the product of Pharmacia Biotech. Inc. (Piscataway, NJ). L-[14C]-Arginine was purchased from DuPont/NEN (Boston, MA). N-methyl-D-glucamine dithiocarbamate/ferrous sulfate (MGD-Fe²⁺) were purchased from Enzo Life Science, Inc (Famingdale, NY). Purified Heat shock protein 90, Calcium Chloride, Calmodulin, NADPH, L-arginine, BH4, N-nitro-L-arginine methyl ester (L-NAME), and other reagents were purchased from Sigma Chemical Co. (St. Louis, MO), unless otherwise indicated. Wild type heat shock protein 90 (WTHSP90) and Dominant negative heat shock protein 90 (D88N-HSP90) plasmids were purchased from addgene (Cambridge, MA).

His-tagged MLC-2v was produced in DH5α Escherichia coli and purified using metal affinity chromatography. Flag-tagged WT ‘active’ and D565A ‘dead’ Δ491 MLCK3 were produced in transiently transfected 293T cells and purified using anti-Flag affinity gel followed by elution with Flag peptide. Nonradioactive reactions contained 500 ng MLC-2v, 20 ng d Δ491 MLCK3, 25 mM Hepes (pH 7.4), 50 mM NaCl, 2 mM MgCl₂, 2 mM DTT, 1 mM Ca²⁺, and 500 μM ATP with or without 1 μg calmodulin (Sigma: Cat#: 2008694). Reactions were run for 10 min at 30 °C and stopped by adding an equal volume of 2× SDS sample buffer containing 50 mM EDTA. Radioactive reactions were run under the same conditions, except cold ATP was reduced to 100 μM and 40 μCi ³²P-γ-ATP was added. Reactions were separated by SDS-PAGE, transferred to Immobilon, and subjected to autoradiography or immunoblotting with the indicated antibodies.

CaMKK2 activity was measured as described previously [22 (link)]. A standard 30 μL assay, 1 ng of recombinant bacterial expressed human CaMKK2 (residues 50–588) was added to assay buffer (50 mM HEPES [pH 7.4], 1 mM DTT, 0.02% (v/v) Brij-35) containing 200 μM CaMKKtide peptide substrate, 50 μM CaCl₂, 1 μM calmodulin (Sigma-Aldrich Corp., St. Louis, MO, USA), 50 μM [γ-³²P]-ATP (Perkin Elmer) and 5 mM MgCl₂, in the presence and absence of different concentrations of small-molecule inhibitors. Reactions were incubated for 10 min at 30 °C, after which they were terminated by spotting 15 μL onto P81 phosphocellulose paper (Whatman, GE Healthcare, Chicago, IL, USA) and washing extensively in 1% phosphoric acid. Radioactivity was quantified by scintillation counting.