Drosophila CaMKII is essentially identical to mammalian CaMKII in terms of its regulation and autophosphorylation, the impressive biochemical properties that have made this enzyme an intense focus for studies of learning and memory (Wang et al., 1998). In previous studies we found functional diversity among the alternatively spliced isoforms of the fly kinase (GuptaRoy and Griffith, 1996; GuptaRoy et al., 1998; GuptaRoy et al., 2000). We also identified a number of substrates for the kinase that are involved in its developmental role (Koh et al., 1999; GuptaRoy et al., 2000) and its role in regulating excitability (Griffith et al., 1994; Zhou et al., 1999; Wang et al., 2002). The current biochemical focus of the lab is understanding how CaMKII, an abundant enzyme with a broad substrate specificity, can act to precisely regulate particular biochemical pathways.

Regulation of Ion Channel Substrates

The neuromuscular junction phenotype of animals expressing the ala peptide CaMKII inhibitor looks very much like that of animals mutant for the voltage-gated potassium channel eag (Griffith et al. 1994) and phosphorylation of Eag by CaMKII can regulate channel activity (Wang et al., 2002). The interaction of these two proteins, however, goes beyond a simple kinase/substrate relationship. Using co-immunoprecipitation from head extracts and in vitro binding assays, we have shown that CaMKII and Eag form a stable complex and that association with Eag activates CaMKII independent of CaM and autophosphorylation. Ca²⁺/CaM is necessary to initiate binding of CaMKII to Eag but not to sustain association since binding persists when CaM is removed. Eag's CaMKII-binding domain has homology to CaMKII's autoregulatory region and the constitutively active CaMKII mutant, T287D, binds Eag Ca²⁺-independently in vitro and in vivo. These results favor a model in which the CaMKII-binding domain of Eag displaces CaMKII's autoinhibitory region. Displacement results in autophosphorylation-independent activation of CaMKII that persists even when Ca²⁺ levels have dropped. Activity-dependent binding to this potassium channel substrate allows CaMKII to remain locally active even when Ca²⁺ levels have dropped, providing a novel mechanism by which CaMKII can regulate excitability locally over long time scales.

Regulation of CaMKII by dCASK

Drosophila CASK (also known as Camguk or Caki), the fly homolog of CASK/Lin-2, associates in an ATP-regulated manner with CaMKII to catalyze formation of a pool of calcium-insensitive CaMKII (Lu et al, 2003). In the presence of Ca²⁺/CaM, CaMKII complexed to dCASK can autophosphorylate at T287 and become constitutively active. In the absence of Ca²⁺/CaM, ATP hydrolysis results in phosphorylation of T306 and inactivation of CaMKII. dCASK coexpression suppresses CaMKII activity in transfected cells, and the level of dCASK expression in Drosophila modulates postsynaptic T306 phosphorylation. These results suggest that dCASK, in the presence of Ca²⁺/CaM, can provide a localized source of active kinase. When Ca²⁺/CaM or synaptic activity is low, dCASK promotes inactivating autophosphorylation, producing CaMKII that requires phosphatase to reactivate. This interaction provides a mechanism by which the active postsynaptic pool of CaMKII can be controlled locally to differentiate active and inactive synapses.

The interaction of dCASK with CaMKII also regulates the ability of CaMKII to become Ca²⁺-independent by autophosphorylation of T287, acting as a gain controller on the transition to Ca²⁺-independence in vivo. Phosphorylation of T287 occurs between subunits within the CaMKII holoenzyme. dCASK control of the CaMKII switch occurs via its ability to induce autophosphorylation of T306 in the kinase's CaM-binding domain. Phosphorylation of T306 blocks Ca²⁺/CaM binding, lowering the probability of intersubunit T287 phosphorylation.

In animals mutant for dCASK, synapse-specific, activity-dependent autophosphorylation of CaMKII T287 is increased. In wild type adult animals, visual stimuli cause optic circuit-specific increases in pT287 and decreases in pT306. dCASK-deficient adults have a reduced dynamic range for activity-dependent T287 phosphorylation and have circuit-level defects that result in inappropriate activation of the kinase in regions that only receive indirect visual information. dCASK is the first CaMKII-interacting protein other than CaM found to regulate the ability of CaMKII to act as a molecular switch.

Activity Regulated Processes

We are also interested in the effects of activity on neuronal function. The Drosophila genome encodes two homologs of the mammalian Arc (activity-regulated cytoskeletal-associated protein) gene. In collaboration with Troy Littleton's lab, we have generated mutations in the dArc locus by excision of a P-element from the dArc1 5'UTR. In the larval CNS, immunocytochemistry shows DARC1 expression is restricted to a small number of neurons in the brain hemispheres and more broadly expressed in the ventral ganglion. In the adult brain, DARC1 expression again shows a restricted expression in the brain hemispheres including the pars intercerebralis and wider expression through the thoracic ganglion. One of the most interesting characteristics of the mammalian Arc gene is its induction in response to neuronal activity. This property appears to be conserved in dArc. Using a variety of fly lines with genetic mutations that elevate neuronal activity we find elevated levels of DARC1 protein in adult heads. These results suggest that the regulation of neuronal response to activity in Drosophila may use biochemical mechanisms similar to those found in mammals.

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