Antibodies and reagents Polyclonal antibody against HO-1 was obtained from Assay Designs

the catalytic site. In summary, the regulatory region, the activation segment, and the nucleotide-binding site of CaMKI are correlated together, and upon the binding of ATP and CaM these regions interplay in a concerted way to regulate the activity states of the kinase. Comparison with helical conformations of the activation segment in other kinases As written by Noble et al. in paraphrase of the opening sentence of Anna Karenina, “all active kinases are alike but an inactive kinase is inactive after its own fashion”. The CaMKI structures presented here unexpectedly reveal a rare helical DHMEQ chemical information conformation of the activation segment. Among.5000 kinase Structures of Human CaMKIa structures in Protein Data Bank, there are only very few in which the activation segment contains an a-helix, including serine/ threonine kinases CDKs and Nek2 and tyrosine kinases Src/Hck and EGFR. In most of these structures, the N-terminal part of the activation segment folds into a short a-helix which interacts with helix aC to constrain it in an inactive position. Hydrophilic interactions of this part with helix aC, particularly with an invariant Glu, have been suggested to be critical for maintenance of the inactive conformation of the kinase. In addition, hydrophobic interactions between this part and helix aC have also been demonstrated to be important. For example, in the inactive EGFR, a Leu immediately following the DFG motif and an adjacent Leu on the short a-helix of the activation segment pack against the hydrophobic side of helix aC to stabilize its inactive conformation. Mutations of these two residues to polar ones have been found in lung cancer patients, and the EGFR kinase domain carrying the L834R mutation displays a substantially increased activity, underscoring the importance of the two residues in the inhibition of the kinase activity. In the CaMK family, only the Leu residue equivalent to Leu834 of EGFR is conserved, corresponding to Leu165 of CaMKI. In the apo CaMKI320, although the N-terminal part of the activation segment takes a loop conformation rather than a helical conformation, the side chain of Leu165 is also oriented towards helix aC and forms hydrophobic interactions with residues of helix aC, contributing to the stabilization of helix aC in the inactive conformation. The specific conformation of Leu165 is apparently associated with the aT-containing activation segment, as in the rat CaMKI320 and CaMKI320-ATP and CaMKI315-ATP structures, Leu165 is disordered, while in the CaMK293-ATP structure, the side chain of Leu165 points to an opposite direction towards the catalytic site. In some Nek2 structures, the C-terminal part of the activation segment adopts a helical conformation and occupies a position similar to that of helix aT in CaMKI320, including the wild-type Nek2 in complex with ADP, and the T175A mutant Nek2 in apo form and in complex with an ATP analog, indicating that the helical conformation is formed independent of Thr175 and nucleotide binding. Consistently, in the wild-type Nek2, Thr175 is exposed to the solvent; whereas in CaMKI320, Thr177 points to the catalytic site to interact with Tyr195 and hence is inaccessible to CaMKK. Considering the previous report that Thr177 of the full-length CaMKI cannot be phosphorylated in the absence of CaM, the helical conformation of the activation segment that is characterized by the sequestration of Thr177 is likely to be adopted by the autoinhibited full-length CaMKI. Drugs that spe