Calmodulin (CaM), the small and ubiquitous protein expressed from three identical genes in higher organisms, is the main cellular calcium (Ca2+) sensor. Important effectors are the calcium/calmodulin dependent protein kinases (CAMKs) and the only calcium/calmodulin dependent protein phosphatase calcineurin. Some CAMKs have unique targets while others are multifunctional, effectively orchestrating signaling pathways that exert many and important cellular functions. These pathways are represented
by CAMK2 signaling and the CAMK1 and CAMK4 cascades. Of these, the CAMK2 exerts a plethora of functions. There are four Camk2 genes - alpha, beta, gamma, delta with distinct expression patterns. Alpha and beta are abundant in the brain while gamma and delta are expressed in many tissues but at comparatively lower levels with delta particularly abundant in the myocardium. The Camk2 signaling plays central roles in excitation-contraction (ECC) and excitation-transcription (E-T) coupling in the heart and in long-term potentiation (LTP) and memory in the brain. Along with the Camk1 cascade, it also plays important roles in cell cycle (not shown). Structurally, all genes are characterized by the presence of N-terminal catalytic domain, the internal regulatory domain that harbors the autoinhibitory segment, the Ca2+/CaM binding site and the critical threonine phosphorylation sites, and the C-terminal association domain also known as the hub; a variable length linker separates the regulatory and the hub regions. The variable length gives rise to more than 30 splice variants in mammals with differing responses to Ca2+ oscillations. The structure of the holoenzyme and of a calmodulin-bound delta isomer have been solved by X-ray crystallography and offer insights into the mechanisms of kinase activation. In the absence of Ca2+/CaM, the autoinhibitory segment forms a helix that block the substrate binding site. There is a compact overall arrangement of the holoenzyme complex; the N-terminal kinase domain is tethered to C-terminal association domain via the regulatory region organizing the protein into ring-shaped hexamers that form the dodecameric structure. click to access the PDB page . The Ca2+/calmodulin/delta isoform complex contains the catalytic and regulatory domains of the enzyme. Within, the inhibitory region adopts an extended conformation, rather than helical while allowing for the interaction with an adjacent catalytic domain suggestive of an enzyme 'in transit' towards transphosphorylation. Click to access the PDB page for the calmodulin-bound delta isoform . Binding of Ca2+/CaM displaces the inhibitory segment and the kinase autophosphorylates critical threonine (Thr) residues within the regulatory domain. Phosphorylation of Thr286/287 (286 in alpha, 287 in the other isoforms) dramatically increases the affinity of the kinase for Ca2+CaM and also renders the kinase active even after the Ca2+/Cam has dissociated, hence the autonomous, constitutive activity of Camk2. On the other hand, phosphorylation of Thr305/306 and Thr306/307 interfere with Ca2+CaM binding. These residues are within the Ca2+/CaM binding region; residues 296 and 316 in the enzyme are thought to be involved in the interaction with Ca2+/CaM. The three phosphorylation sites and the regulatory region are invariant across metazoans and there is a high degree of conservation for the hub residues involved in oligomerization. Reactive Oxygen Species (ROS) can activate the kinase in a Ca2+/CaM independent manner by modifying a pair of methionine (Met) residues within the regulatory domain which blocks its re-associatiation with the catalytic domain and preserves the active form of the kinase. The modification is viewed as a marker for oxidative stress and can be reversed by methionine sulfoxide reductases. Dephosphorylation and inactivation of Camk2 is carried out by many phosphatases; the Ca2+/CaM-dependent calcineurin does not directly dephosphorylate Camk2; it acts indirectly by inhibiting an inhibitor of a directly acting phosphatase. Two endogenous inhibitory proteins Camk2n1 and Camk2n2 have been identified in rodent brain and in human dendritic cells. They are specific for the active form of Camk2 and sterically inhibit the kinase; yet, the exact role they exert remains to be established.
In the heart, Camk2 targets many voltage-gated ion channels and excessive activation of the kinase can give rise to arrhythmia. Well established targets include the L-type Ca2+ channel Cacna1c (Cav1.2) and the regulatory beta2 subunit and the sodium channel Scn5a (Nav1.5). The sarcoplasmic reticulum (SR) ryanodine receptor Ryr2 and the regulator of Atp2a2 Ca2+ pump, phospholamban (Pln), are also targets. Camk2 promotes Ca2+ influx and release via Cav1.2 and Ryr2 while promoting Ca2+ uptake via Pln. Pln interacts with the SR pump and inhibits its function; phosphorylation relieves it and renders the pump active. Drugs that target the ion channels have been developed but they have powerful adverse effects. A splice variant of delta gene contains a nuclear localization signal; nuclear targets of Camk2d are histone deacetylases Hdac4 and 5, with the former being the preferred substrate. In the dephosphorylated state, the two bind and repress hypertrophic transcription factors such as Mef2. Phosphorylation relives the repression, allowing for the activation of Mef2-dependent transcription.
The two abundant neuronal subunits of Camk2 are alpha and beta that in addition to homomers, also form heteromers . Activated Camk2 translocates from the cytoplasm to the synapse and interacts with proteins in the postsynaptic density (PSD); a prominent member is Grin2b (NR2B) subtype of ionotropic NMDA receptor. While Camk2 can bind other PSD members, the interaction with Grin2b appears to be crucial for the synaptic translocation of the kinase. This places Camk2 in proximity of ionotropic AMPA receptor Gria1 and stargazin (Cacng2) - a gamma subunit of L-type Ca2+ channel and an auxiliary subunit of AMPA. The phosphorylation of these two substrates enhances the conductance of AMPA receptor and concomitantly leads to the trafficking of additional ones, respectively. These events provide a mechanistic explanation of the early LTP phase. The later phases and LTP maintenance and the exact role Camk2 plays, are far less well understood. Possibly, the binding reactions of Camk2 rather than its catalytic activity are important. Phosphorylation of Thr205/306 may underlie the role of Camk2 in long term depression (LTD), also a feature of the excitatory synapse. To see the ontology report for annotations, Gviewer and download, click here ....(less)