1. Regulation of brain Ca2+/calmodulin-dependent protein kinase II
T R Soderling, K Fukunaga, D P Rich, Y L Fong, K Smith, R J Colbran Adv Second Messenger Phosphoprotein Res. 1990;24:206-11.
CaM-kinase II is a multifunctional protein kinase highly enriched in neural tissues where it modulates a variety of Ca2(+)-dependent processes. A complex regulatory domain in the kinase within residues 281-309 contains an autoinhibitory sequence, a CaM-binding region, and sites of regulatory autophosphorylation. Autophosphorylation on Thr286 converts the kinase to a Ca2(+)-independent form which could prolong physiological systems controlled by this kinase in response to transient Ca2+ elevations. Such properties appear to exist in dynamic equilibrium in the isolated postsynaptic density and in cultured brain cells. These unique biochemical regulatory properties, coupled with an unusual high concentration in the postsynaptic density of excitatory synapses, makes CaM-kinase II an attractive candidate for involvement in synaptic plasticity.
2. Regulation of Ca2+/calmodulin-dependent protein kinase II by brain gangliosides
K Fukunaga, E Miyamoto, T R Soderling J Neurochem. 1990 Jan;54(1):103-9. doi: 10.1111/j.1471-4159.1990.tb13288.x.
Purified rat brain Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) is stimulated by brain gangliosides to a level of about 30% the activity obtained in the presence of Ca2+/calmodulin (CaM). Of the various gangliosides tested, GT1b was the most potent, giving half-maximal activation at 25 microM. Gangliosides GD1a and GM1 also gave activation, but asialo-GM1 was without effect. Activation was rapid and did not require calcium. The same gangliosides also stimulated the autophosphorylation of CaM-kinase II on serine residues, but did not produce the Ca2+-independent form of the kinase. Ganglioside stimulation of CaM-kinase II was also present in rat brain synaptic membrane fractions. Higher concentrations (125-250 microM) of GT1b, GD1a, and GM1 also inhibited CaM-kinase II activity. This inhibition appears to be substrate-directed, as the extent of inhibition is very dependent on the substrate used. The molecular mechanism of the stimulatory effect of gangliosides was further investigated using a synthetic peptide (CaMK 281-309), which contains the CaM-binding, inhibitory, and autophosphorylation domains of CaM-kinase II. Using purified brain CaM-kinase II in which these regulatory domains were removed by limited proteolysis. CaMK 281-309 strongly inhibited kinase activity (IC50 = 0.2 microM). GT1b completely reversed this inhibition, but did not stimulate phosphorylation of the peptide on threonine-286. These results demonstrate that GT1b can partially mimic the effects of Ca2+/CaM on native CaM-kinase II and on peptide CaMK 281-309.
3. Regulatory domain of calcium/calmodulin-dependent protein kinase II. Mechanism of inhibition and regulation by phosphorylation
R J Colbran, M K Smith, C M Schworer, Y L Fong, T R Soderling J Biol Chem. 1989 Mar 25;264(9):4800-4.
Regulatory mechanisms of rat brain Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) were probed using a synthetic peptide (CaMK-(281-309] corresponding to residues 281-309 (alpha-subunit) which contained the calmodulin (CaM)-binding and inhibitory domains and also the initial autophosphorylation site (Thr286). Kinetic analyses indicated that inhibition of a completely Ca2+/CaM-independent form of CaM-kinase II by CaMK-(281-309) was noncompetitive with respect to peptide substrate (syntide-2) but was competitive with respect to ATP. Interaction of CaMK-(281-309) with the ATP-binding site was independently confirmed since inactivation of proteolyzed CaM-kinase II by phenylglyoxal (t1/2 = 7 min) was blocked by ATP analog plus Mg2+ or by CaMK-(281-309). In the presence of Ca2+/CaM, CaMK-(281-309) no longer protected against phenylglyoxal inactivation, consistent with our previous observations (Colbran, R.J., Fong, Y.-L., Schworer, C.M., and Soderling, T.R. (1988) J. Biol. Chem. 263, 18145-18151) that binding of Ca2+/CaM to CaMK-(281-309) 1) blocks its inhibitory property, and 2) enhances its phosphorylation at Thr 286. The present study also showed that phosphorylation of CaMK-(281-309) decreased its inhibitory potency at least 10-fold without affecting its Ca2+/CaM-binding ability. Thus, CaM-kinase II is inactive in the absence of Ca2+/CaM because an inhibitory domain within residues 281-309 interacts with the catalytic domain and blocks ATP binding. Autophosphorylation of Thr286 results in a Ca2+/CaM-independent form of the kinase by disrupting the inhibitory interaction with the catalytic domain.
