The activation mechanism of Ca(2+)/calmodulin-dependent protein kinase II (alphaCaMKII) is investigated by steady-state and stopped-flow fluorescence spectroscopies. Lys(75)-labeled TA-cal [Török, K., and Trentham, D. R. (1994) Biochemistry 33, 12807-12820] is used to measure binding events, and double-labeled AEDANS,DDP-T34C/T110/C-calmodulin [Drum et al. (2000) J. Biol. Chem. 275, 36334-36340] (DA-cal) is used to detect changes in calmodulin conformation. Fluorescence quenching of DA-cal attributed to resonance energy transfer is related to the compactness of the calmodulin molecule. Interprobe distances are estimated by lifetime measurements of Ca(2+)/DA-cal in complexes with unphosphorylated nucleotide-free, nucleotide-bound, and Thr(286)-phospho-alphaCaMKII as well as with alphaCaMKII-derived calmodulin-binding peptides in the presence of Ca(2+). These measurements show that calmodulin can assume at least two spectrally distinct conformations when bound to alphaCaMKII with estimated interprobe distances of 40 and 22-26 A. Incubation with ATP facilitates the assumption of the most compact conformation. Nonhydrolyzable ATP analogues partially replicate the effects of ATP, suggesting that while the binding of ATP induces a conformational change, Thr(286)-autophosphorylation is probably required for the transition of calmodulin into its most compact conformer. The rate constant for the association of Ca(2+)/TA-cal with alphaCaMKII is estimated as 2 x 10(7) M(-1) s(-1) and is not substantially affected by the presence of ATP. The rate of net calmodulin compaction measured by Ca(2+)/DA-cal is markedly slower, occurring with a rate constant of 2.5 x 10(6) M(-1) s(-1), suggesting that unproductive complexes may play a role in the activation mechanism.

Thr(286) autophosphorylation is important for the role of alphaCaMKII in learning and memory. Phospho-Thr(286)-alphaCaMKII has been described to have two types of activity: Ca(2+)-independent partial activity and Ca(2+)/calmodulin-activated full activity. We investigated the mechanism of switching between the two activities in order to relate them to the physiological functioning of alphaCaMKII. Using a fluorometric coupled enzyme assay and smooth muscle myosin light chain (MLC) as substrate, we found that (1) Ca(2+)-independent activity of phospho-Thr(286)-alphaCaMKII represents 5.0 (+/-3.7)% of the activity measured in the presence of optimal concentrations of Ca(2+) and calmodulin and (2) Ca(2+) in the presence of calmodulin activates the enzyme with a K(m) of 137 (+/-56) nM and a Hill coefficient n = 1.8 (+/-0.3). In contrast, unphosphorylated alphaCaMKII has a K(m) for Ca(2+) in the presence of calmodulin of 425 (+/-119) nM and a Hill coefficient n = 5.4 (+/-0.4). Thus, the activity of phospho-Thr(286)-alphaCaMKII is essentially Ca(2+)/calmodulin dependent with MLC as substrate. In physiological terms, our data suggest that alphaCaMKII is only activated in stimulated neurones whereas Ca(2+)/calmodulin activation of phospho-Thr(286)-alphaCaMKII can occur in resting cells (approximately 100 nM [Ca(2+)]). Stopped-flow experiments using Ca(2+)/TA-cal [Ca(2+)/2-chloro-(epsilon-amino-Lys(75))-[6-[4-(N,N-diethylamino)phenyl]-1,3,5-triazin-4-yl]calmodulin] showed that at 100 nM [Ca(2+)] partially Ca(2+)-saturated Ca(2+)/cal.phospho-Thr(286)-alphaCaMKII complexes existed. These are likely to account for the activity of the phospho-Thr(286)-alphaCaMKII enzyme at resting [Ca(2+)]. Ca(2+) dissociation measurements by a fluorescent Ca(2+) chelator revealed that the limiting Ca(2+) dissociation rate constants were 1.5 s(-1) from the Ca(2+)/cal.alphaCaMKII and 0.023 s(-1) from the Ca(2+)/cal.phospho-Thr(286)-alphaCaMKII complex, accounting for the differences in the Ca(2+) sensitivities of the Ca(2+)/cal.alphaCaMKII and Ca(2+)/cal.phospho-Thr(286)-alphaCaMKII enzymes.

The role of adenosine 5'-triphosphate (ATP) in the activation mechanism of alpha-Ca(2+)/calmodulin-dependent protein kinase II (alphaCaMKII) was investigated using the T286A non-autophosphorylatable mutant of alphaCaMKII. Characterization of the T286A-alphaCaMKII mutant revealed k(cat) = 0.06 +/- 0.02 s(-1) for the T286A mutant, a 6 (+/- 2)-fold lower value compared to wild-type alphaCaMKII with 100 microM smooth muscle myosin light chain (MLC) as substrate. MLC phosphorylation by the T286A mutant and wild-type alphaCaMKII was cooperative, with Hill coefficients 2.3 +/- 0.1 and 2.4 +/- 0.3, respectively. K(m) values for MLC were 96 +/- 28 microM with T286A-alphaCaMKII and 49 +/- 29 microM for wild-type alphaCaMKII. Thus, while the activity of alphaCaMKII was sensitive to mutation of the Thr(286) residue to Ala, the mechanisms of the wild-type and T286A mutant enzyme appeared similar. K(d) for Ca(2+)/calmodulin was 2-fold reduced to 40 nM compared to that of wild-type alphaCaMKII (75 nM). ATP induced a 9-fold stabilization of Ca(2+)/calmodulin binding to the T286A mutant enzyme. Fluorescence stopped-flow kinetic experiments revealed that two Ca(2+)/calmodulin-enzyme complexes were formed, the first, unaffected by ATP, with association and dissociation rate constants of 2 x 10(7) M(-1) s(-1) and 5 s(-1), respectively, containing calmodulin in extended conformation. The second complex, in which calmodulin adopted a compact conformation, was formed with association rate constant 3 x 10(6) M(-1) s(-1) and dissociation at 0.15 s(-1) in the absence and 0.015 s(-1) in the presence of ATP. These data show that ATP is involved in the activation mechanism by forming two classes of Ca(2+)/calmodulin.alphaCaMKII.ATP complex. It is likely that only one of the complexes is on the activation pathway.

Development of neural circuitry relies on precise matching between correct synaptic partners and appropriate synaptic strength tuning. Adaptive developmental adjustments may emerge from activity and calcium-dependent mechanisms. Calcium/calmodulin-dependent protein kinase II (CaMKII) has been associated with developmental synaptic plasticity, but its varied roles in different synapses and developmental stages make mechanistic generalizations difficult. In contrast, we focused on synaptic development roles of CaMKII in a defined sensory-motor circuit. Thus, different forms of CaMKII were expressed with UAS-Gal4 in distinct components of the giant fiber system, the escape circuit of Drosophila, consisting of photoreceptors, interneurons, motoneurons, and muscles.The results demonstrate that the constitutively active CaMKII-T287D impairs development of cholinergic synapses in giant fiber dendrites and thoracic motoneurons, preventing light-induced escape behavior. The locus of the defects is postsynaptic as demonstrated by selective expression of transgenes in distinct components of the circuit. Furthermore, defects among these cholinergic synapses varied in severity, while the glutamatergic neuromuscular junctions appeared unaffected, demonstrating differential effects of CaMKII misregulation on distinct synapses of the same circuit. Limiting transgene expression to adult circuits had no effects, supporting the role of misregulated kinase activity in the development of the system rather than in acutely mediating escape responses. Overexpression of wild-type transgenes did not affect circuit development and function, suggesting but not proving that the CaMKII-T287D effects are not due to ectopic expression. Therefore, regulated CaMKII autophosphorylation appears essential in central synapse development, and particular cholinergic synapses are affected differentially, although they operate via the same nicotinic receptor.

The GAL4/UAS binary system with its recent modifications provides a powerful tool to study gene function in Drosophila enabling control over the timing, tissue specificity, and magnitude of gene expression. GAL4 expression during early embryonic stages has been well determined for certain driver lines, but for some of the commonly used in Drosophila research it is unknown, or partially determined. By monitoring the developmental kinetics of GAL4 expression and transgene transcription, we show that particular GAL4 drivers transiently direct ectopic expression of UAS-linked transgenes at early stages of embryogenesis in a GAL4- dependent manner via a mechanism that involves parental transmission of Gal4 transcripts.

We have used the fluorescently labelled calmodulin TA-CaM to follow calmodulin activation during depolarization of adult rat sensory neurons. Calcium concentration was measured simultaneously using the low affinity indicator Oregon Green BAPTA 5N. TA-CaM fluorescence increased during a 200-ms depolarization but then continued to increase during the subsequent 500 ms, even though total cell calcium was falling at this time. In the next few seconds TA-CaM fluorescence fell, but to a new elevated level that was then maintained for several tens of seconds. During a train of depolarizations that evoked a series of largely independent calcium changes TA-CaM fluorescence was in contrast raised for the duration of the train and for many tens of seconds afterwards. The presence of a peptide corresponding to the calmodulin binding domain of myosin light chain kinase significantly increased the depolarization-induced TA-CaM fluorescence increase and slowed the subsequent fall of fluorescence. We interpret the slow recovery component of the TA-CaM signal as reflecting the slow dissociation of calcium--calmodulin--calmodulin binding protein complexes. Our results show that after brief electrical activity calmodulin's interaction with calmodulin binding proteins persists for approximately one minute.

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