The serine/threonine kinases (including the PKC and Raf isoforms) obey the paradigm of auto-inhibition: their regulatory domains sterically obstruct the catalytic domains. In order to permit access to substrates and ATP, the general strategy is to unfold the molecules so as to relieve steric hindrance. A complex set of instructions by regulatory kinases, phosphatases, lipid messengers, interacting proteins, and divalent cations orchestrates the unfolding and catalytic enabling of these proteins. Additionally to this classic activation cycle, these kinases respond to reactive oxygen and, in concert with a similar set of cofactors, acquire full enzymatic potential.
The classic second messenger and redox activation pathways converge on the zinc-finger domains. These are protein folds traditionally thought of as rigid structures, clamped together by 2 chelated zinc ions. Two sets of three cysteine and one histidine residues recruited from a 50 amino acid mini-domain are organized in nonlinear fashion into 2 perfect tetraeders, each with a central zinc ion. Because zinc-chelation depends on thiolate anions of cysteines, it came as no surprise that during redox activation, when thiols are converted to disulfide, zinc ions were freed. We have demonstrated zinc mobilization in PKC in vivo by imaging techniques with zinc-sensitive fluorescent probes, as well as in vitro. Interestingly, phorbol ester, a pharmacologic mimetic known to bind the PKC zinc-finger and to activate PKC, also caused zinc-mobilization in vivo and in vitro. Therefore, whether by oxidation or binding of the classical lipid second messenger, the zinc-coordination centers in PKC are disassembled as a prelude to kinase activation. In agreement, the zinc content of nonactivated native PKC was twice that of PKC activated in vivo either by mild oxidation or by phorbol ester. Far from forming rigid structures, the zinc-finger domains of PKC act as flexible hinges. Removal of the central zinc ions, like pulling a linch pin, allows the opening of the hinge and enables the unfolded protein to become catalytically active.
