CaMKII (Calcium/calmodulin-dependent protein kinase II) is one of the most important regulators of calcium signaling. It is a ubiquitous mediator of Ca2+-linked signaling that phosphorylates a wide range of substrates to co-ordinate and regulate Ca2+-mediated alterations in cellular functions. CaMKII is a multifunctional serine/threonine kinase, which plays a critical role in the survival, proliferation, invasion, and differentiation of various cancer cells by activating multiple signaling pathways, such as the extracellular signal-regulated kinase (ERK), protein kinase B (AKT), the signal transducer and activator of transcription 3 (STAT3), and Wnt/β-catenin signaling pathways. It also plays a critical role in the survival, proliferation, and maintenance of cancer stem cells. According to research findings, the ϒ isoform of CaMKII (CaMKIIϒ) is required for the maintenance of stem-like and tumorigenic properties in the blood, lung, liver and prostate cancer cells. Each CaMKII monomer consists of three subunits: a catalytic, a regulatory and an association one that is responsible the assembly into the holoenzyme.
CaMKIIϒ acts as a major molecular switch for regulating the NF-kB, Wnt/β-Catenin, Notch, STAT3, and AKT signaling pathways, which are essential for cancer stem-like features in these cancer cells. The CaMKIIϒ inhibitor, berbamine, supresses leukemia stem cells and liver cancer-initiating cells by inhibiting the activity of CaMKIIϒ. In a recent research, Hydrazinobenzoylcurcumin (HBC), a synthetic derivative of curcumin and a Ca2+/CaM antogonist, inhibited not only the self-renewal capacity but also the metastatic potential of GSCs by blocking the CaM/CaMKII/c-Met signaling pathway.
A selective CaMKII inhibitor, KN93, also inhibited the growth of GSCs and the expression of GSC stemless markers. Additionally, CaMKIIγ knockdown decreased the stem-like features of GBM cells. Therefore, CaMKII has attracted attention as an emerging target for eliminating cancer stem cells.
The transmission of information by the kinase from extracellular stimuli and the intracellular Ca2+ rise is not passive. Rather, its multimeric structure and autoregulation enable this enzyme to participate actively in the sensitivity, timing and location of its action.
CaMKII can: (i) be activated in a Ca2+-spike frequency-dependent manner; (ii) become independent of its initial Ca2+}CaM activators; and (iii) undergo a ‘molecular switch-like’ behaviour, which is crucial for certain forms of learning and memory
CaMKII activation and inhibition
Activity of each Calcium/calmodulin-dependent protein kinase II subunit is stimulated individually by direct binding of Ca2+/CaM to their regulatory domains. Autophosphorylation at Threo286 within the regulatory domain transforms CaMKII from one of the lowest to one of the highest affinity CaM binders within the cell. This autophosphorylation also generates autonomous (Ca2+-independent) kinase activity.
The autoregulatory domain of Calcium/calmodulin-dependent protein kinase II ensures, via a pseudosubstrate type of interaction, that the basal kinetic acivity levels remain 100-1000 fold below the maximal Ca2+/CaM-stimulated values. In this mode of inhibition, the autoinhibitory domain has residues that mimic aprotein substrate or nucleotide to interact with the catalytic domain, thereby blocking access to the substrate-binding pockets. However, a binding of an allosteric activator alters the conformation of the autoinhibitory domain and decreases its inhibitory potency; thereby permitting substrates’ access to the catalytic site.
Like all known Ca2+/caMKs, the autoregulatory domain of CaMKII is N-terminal and contiguous with the CaM-binding domain.
CaMKII Structure and Mechanism of activation
Each of the subunits that comprise CaMKII multimeric enzyme has a conserved structure among the different isoforms; an amino terminal catalytic domain, followed by a regulatory domain that contains a self-inhibitory region and a variable sequence and finally an associative domain in the Carboxy-terminal end, which allows assembly between the different subunits.
The entry of calcium into the cell leads to the formation of the Ca2+/CaM complex, which binds approximately 3-4 calcium ions per CaM in a cooperative form. This complex binds to the regulatory region of CaMKII and produces a conformational change, which not only leads to the phosphorylation of its subunits, but also to an inter-subunits, intra-holoenzyme autophosphorylation at threonine 286 in the alpha isoform and threonine 287 in the β, d and ϒ isoforms.
Autophosphorylation on these sites prevent the enzyme from reverting to its inactive conformation and decreases the dissociation rate of the bound CaM.
Without requirement of any Ca2+ or CaM, Calcium/calmodulin-dependent protein kinase II can be directly activated by gangliosides, especially GTIb, Zn2+ or α-actinin. Α-actinin selectively stimulates phoshorylation of CaMKII substrates that leads to the T-site; however, this activation by α-actinin is not as effective as Ca2+/CaM complex.
Since α-actinin competes with Ca2+/CaM for binding to CaMKII, its net effect in the presence of Ca2+/CaM is inhibitory for all substrates in addition to participation in mechanisms of substrate selection.
Negative regulation of CaMKII activity
Calcium/Camodulin-dependent protein kinase can be negatively regulated by α-actinin, by the inhibitory protein CaM-KII or by Threo305/306 autophosphorylation.
Binding of CaM-KIINα or β isoforms to CaMKII involves the T-site of the kinase and thus, prior displacement of the CaMKII regulatory domain is required. However, in contrast to GluN2B, CaM-KIIN binding completely blocks substrate access. CaM-KIIN can be regulated by its expression level, and possibly by phosphorylation.
Negative regulation by Threo305/306
Threo305/306 phosphorylation inhibits CaM-binding and, vice versa, CaM-binding inhibits Threo305/306 phophorylation. As a result, efficient Threo305/306 autophosphorylation requires autonomous CaMKII with no bound CaM. Hence, dissociation of CaM from Threo286-phosphorylated CaMKII triggers Threo305/306 autophosphorylation. The resulting state of the kinase would be autonomous but without the ability to be further stimulated.
CaMKII and Oxidation
It has been shown that CaMKII is activated by both angiotensinII (AngII) and endothelin-1(ET1) through a primary oxidation-dependent pathway. Oxidation of Methionine281/282 (M281/282) of CaMKII was shown to activate the kinase, thus underscoring the potential of oxidative stress to affect both pathologic and physiologic pathways in excitable cells, such as the cardiomyocyte.
Upon Ca2+/CaM binding at the regulatory CaMKII domain, oxidation of M281/282 leads to activation of the enzyme. Metheonine oxidation inhibits re-association between regulatory and catalytic subunits of CaMKII and thus enables perpetuation of its activity.