Substrate recognition by Ca2+/Calmodulin-dependent protein kinase kinase. Role of the arg-pro-rich insert domain.

Mammalian Ca2+/CaM-dependent protein kinase kinase (CaM-KK) has been identified and cloned as an activator for two kinases, CaM kinase I (CaM-KI) and CaM kinase IV (CaM-KIV), and a recent report (Yano, S., Tokumitsu, H., and Soderling, T. R. (1998) Nature 396, 584-587) demonstrates that CaM-KK can also activate and phosphorylate protein kinase B (PKB). In this study, we identify a CaM-KK from Caenorhabditis elegans, and comparison of its sequence with the mammalian CaM-KK alpha and beta shows a unique Arg-Pro (RP)-rich insert in their catalytic domains relative to other protein kinases. Deletion of the RP-domain resulted in complete loss of CaM-KIV activation activity and physical interaction of CaM-KK with glutathione S-transferase-CaM-KIV (T196A). However, CaM-KK autophosphorylation and phosphorylation of a synthetic peptide substrate were normal in the RP-domain mutant. Site-directed mutagenesis of three conserved Arg in the RP- domain of CaM-KK confirmed that these positive charges are important for CaM-KIV activation. The RP- domain deletion mutant also failed to fully activate and phosphorylate CaM-KI, but this mutant was indistinguishable from wild-type CaM-KK for the phosphorylation and activation of PKB. These results indicate that the RP-domain in CaM-KK is critical for recognition of downstream CaM-kinases but not for its catalytic activity (i.e. autophosphorylation) and PKB activation.     

Differential regulatory mechanism of Ca2+/calmodulin-dependent protein kinase kinase isoforms.

We have previously demonstrated that the alpha isoform of Ca(2+)/calmodulin-dependent protein kinase kinase (CaM-KKalpha) is strictly regulated by an autoinhibitory mechanism and activated by the binding of Ca(2+)/CaM [Tokumitsu, H., Muramatsu, M., Ikura, M., and Kobayashi, R. (2000) J. Biol. Chem. 275, 20090-20095]. In this study, we find that rat brain extract contains Ca(2+)/CaM-independent CaM-KK activity. This result is consistent with an enhanced Ca(2+)/CaM-independent activity (60-70% of total activity) observed with the recombinant CaM-KKbeta isoform. By using various truncation mutants of CaM-KKbeta, we have identified a region of 23 amino acids (residues 129-151) located at the N-terminus of the catalytic domain as an important regulatory element of the autonomous activity. A CaM-KKbeta deletion mutant of this domain shows a significant increase of Ca(2+)/CaM dependency for the CaM-KK activity as well as for the autophosphorylation activity. The activities of CaM-KKalpha and CaM-KKbeta chimera, in which autoinhibitory sequences were replaced by each other, were completely dependent on Ca(2+)/CaM, suggesting that the autoinhibitory regions of CaM-KKalpha and CaM-KKbeta are functional. These results establish for the first time that residues 129-151 of CaM-KKbeta participate in the release of the autoinhibitory domain from its catalytic core, resulting in generation of autonomous activity.     

Regulatory mechanism of Dictyostelium myosin light chain kinase A

In this study, we examined the activation mechanism of Dictyostelium myosin light chain kinase A (MLCK-A) using constitutively active Ca2+/calmodulin-dependent protein kinase kinase as a surrogate MLCK-A kinase. MLCK-A was phosphorylated at Thr166 by constitutively active Ca2+/calmodulin-dependent protein kinase kinase, resulting in an approximately 140-fold increase in catalytic activity, using intact Dictyostelium myosin II. Recombinant Dictyostelium myosin II regulatory light chain and Kemptamide were also readily phosphorylated by activated MLCK-A. Mass spectrometry analysis revealed that MLCK-A expressed by Escherichia coli was autophosphorylated at Thr289 and that, subsequent to Thr166 phosphorylation, MLCK-A also underwent a slow rate of autophosphorylation at multiple Ser residues. Using site-directed mutagenesis, we show that autophosphorylation at Thr289 is required for efficient phosphorylation and activation by an upstream kinase. By performing enzyme kinetics analysis on a series of MLCK-A truncation mutants, we found that residues 283-288 function as an autoinhibitory domain and that autoinhibition is fully relieved by Thr166 phosphorylation. Simple removal of this region resulted in a significant increase in the kcat of MLCK-A; however, it did not generate maximum enzymatic activity. Together with the results of our kinetic analysis of the enzymes, these findings demonstrate that Thr166 phosphorylation of MLCK-A by an upstream kinase subsequent to autophosphorylation at Thr289 results in generation of maximum MLCK-A activity through both release of an autoinhibitory domain from its catalytic core and a further increase (15-19-fold) in the kcat of the enzyme.     

Phosphorylation of Numb family proteins. Possible involvement of Ca2+/calmodulin-dependent protein kinases

To search for the substrates of Ca2+/calmodulin-dependent protein kinase I (CaM-KI), we performed affinity chromatography purification using either the unphosphorylated or phosphorylated (at Thr177) GST-fused CaM-KI catalytic domain (residues 1-293, K49E) as the affinity ligand. Proteomic analysis was then carried out to identify the interacting proteins. In addition to the detection of two known CaM-KI substrates (CREB and synapsin I), we identified two Numb family proteins (Numb and Numbl) from rat tissues. These proteins were unphosphorylated and were bound only to the Thr177-phosphorylated CaM-KI catalytic domain. This finding is consistent with the results demonstrating that Numb and Numbl were efficiently and stoichiometrically phosphorylated in vitro at equivalent Ser residues (Ser264 in Numb and Ser304 in Numbl) by activated CaM-KI and also by two other CaM-Ks (CaM-KII and CaM-KIV). Using anti-phospho-Numb/Numbl antibody, we observed the phosphorylation of Numb family proteins in various rat tissue extracts, and we also detected the ionomycin-induced phosphorylation of endogenous Numb at Ser264 in COS-7 cells. The present results revealed that the Numb family proteins are phosphorylated in vivo as well as in vitro. Furthermore, we found that the recruitment of 14-3-3 proteins was the functional consequence of the phosphorylation of the Numb family proteins. Interaction of 14-3-3 protein with phosphorylated Numbl-blocked dephosphorylation of Ser304. Taken together, these results indicate that the Numb family proteins may be intracellular targets for CaM-Ks, and they may also be regulated by phosphorylation-dependent interaction with 14-3-3 protein.     

Studies on the regulatory domain of Ca2+/calmodulin-dependent protein kinase II. Functional analyses of arginine 283 using synthetic inhibitory peptides and site-directed mutagenesis of the alpha subunit

The regulatory role of Arg283 in the autoinhibitory domain of Ca2+/calmodulin-dependent protein kinase II was investigated using substituted inhibitory synthetic peptides and site-directed mutation of the expressed kinase. In the synthetic peptide corresponding to the autoinhibitory domain (residues 281-309) of Ca2+/calmodulin-dependent protein kinase II, substitution of Arg283 by other residues increased the IC50 values of the peptides in the following order: Arg much less than Lys much less than Gln much less than Glu. Site-directed mutations of Arg283 to glutamic acid and glutamine in the kinase alpha subunit cDNA were transcribed and translated in vitro. The expressed enzymes had the same total kinase activities, determined in the presence of Ca2+/CaM, but the Glu283 mutant had a slightly higher Ca2(+)-independent kinase activity (5.46 +/- 0.88%) compared to the wild-type Arg283 (1.86 +/- 0.71%) and the Gln283 mutant (2.15 +/- 0.60%). When the expressed kinases were subjected to limited autophosphorylation on ice to monitor generation of the Ca2(+)-independent activity, the Arg283 kinase attained maximal Ca2(+)-independent activity (about 20%) within 30 s, whereas the Gln283 and Glu283 mutants attained maximal Ca2(+)-independence only after about 40 min of autophosphorylation. The results indicate that Arg283 is a very important determinant for the regulatory autophosphorylation of Thr286 that generates the Ca2(+)-independent activity but is not essential for the other multiple autophosphorylations within Ca2+/calmodulin-dependent protein kinase II, and that Arg283 is only one of several important residues for the inhibitory potency of the autoinhibitory domain.     

A single amino acid difference between alpha and beta Ca2+/calmodulin-dependent protein kinase kinase dictates sensitivity to the specific inhibitor,STO-609

We recently developed STO-609, a selective inhibitor of Ca(2+)/calmodulin-dependent protein kinase kinase (CaM-KK), and we demonstrated that CaM-KK beta is more sensitive to STO-609 than the CaM-KK alpha isoform (Tokumitsu H., Inuzuka H., Ishikawa Y., Ikeda M., Saji I., and Kobayashi R. (2002) J. Biol. Chem. 277, 15813-15818). By using catalytic chimera and point mutants of both isoforms, we demonstrated that Val(269) in CaM-KK beta/Leu(233) in CaM-KK alpha confers a distinct sensitivity ( approximately 10-fold) to STO-609 on CaM-KK isoforms. Various mutations of Val(269) in CaM-KK beta indicate that substitution by hydrophobic residues with bulky side chains significantly decreases drug sensitivity and that the V269F mutant is the most effective drug-resistant enzyme ( approximately 80-fold higher IC(50) value). These findings are consistent with a result obtained with a full-length mutant expressed in COS-7 cells. Furthermore, suppression of CaM-KK-mediated CaM-KIV activation in transfected HeLa cells by STO-609 treatment was completely abolished by the co-expression of the CaM-KK beta V269F mutant. Based on the results that the distinct sensitivity of CaM-KK isoforms to STO-609 is because of a single amino acid substitution (Val/Leu) in the ATP-binding pocket, we have generated an STO-609-resistant CaM-KK mutant, which might be useful for validating the pharmacological effects and specificity of STO-609 in vivo.     

Regulatory mechanism of Ca2+/calmodulin-dependent protein kinase kinase

Ca(2+)/calmodulin-dependent protein kinase kinase (CaM-KK) is a novel member of the CaM kinase family, which specifically phosphorylates and activates CaM kinase I and IV. In this study, we characterized the CaM-binding peptide of alphaCaM-KK (residues 438-463), which suppressed the activity of constitutively active CaM-KK (84-434) in the absence of Ca(2+)/CaM but competitively with ATP. Truncation and site-directed mutagenesis of the CaM-binding region in CaM-KK reveal that Ile(441) is essential for autoinhibition of CaM-KK. Furthermore, CaM-KK chimera mutants containing the CaM-binding sequence of either myosin light chain kinases or CaM kinase II located C-terminal of Leu(440), exhibited enhanced Ca(2+)/CaM-independent activity (60% of total activity). Although the CaM-binding domains of myosin light chain kinases and CaM kinase II bind to the N- and C-terminal domains of CaM in the opposite orientation to CaM-KK (Osawa, M., Tokumitsu, H., Swindells, M. B., Kurihara, H., Orita, M., Shibanuma, T., Furuya, T., and Ikura, M. (1999) Nat. Struct. Biol. 6, 819-824), the chimeric CaM-KKs containing Ile(441) remained Ca(2+)/CaM-dependent. This result demonstrates that the orientation of the CaM binding is not critical for relief of CaM-KK autoinhibition. However, the requirement of Ile(441) for autoinhibition, which is located at the -3 position from the N-terminal anchoring residue (Trp(444)) to CaM, accounts for the opposite orientation of CaM binding of CaM-KK compared with other CaM kinases.     

Generation of autonomous activity of Ca(2+)/calmodulin-dependent protein kinase kinase β by autophosphorylation

Ca(2+)/calmodulin-dependent protein kinase kinases (CaMKKs) phosphorylate and activate specific downstream protein kinases, including CaMKI, CaMKIV, and 5'-AMP-activated protein kinase, which mediates a variety of Ca(2+) signaling cascades. CaMKKs have been shown to undergo autophosphorylation, although their role in enzymatic regulation remains unclear. Here, we found that CaMKKα and β isoforms expressed in nonstimulated transfected COS-7 cells, as well as recombinant CaMKKs expressed in and purified from Escherichia coli, were phosphorylated at Thr residues. Introduction of a kinase-dead mutation completely impaired the Thr phosphorylation of these recombinant CaMKK isoforms. In addition, wild-type recombinant CaMKKs were unable to transphosphorylate the kinase-dead mutants, suggesting that CaMKK isoforms undergo Ca(2+)/CaM-independent autophosphorylation in an intramolecular manner. Liquid chromatography-tandem mass spectrometry analysis identified Thr(482) in the autoinhibitory domain as one of the autophosphorylation sites in CaMKKβ, but phosphorylation of the equivalent Thr residue (Thr(446)) in the α isoform was not observed. Unlike CaMKKα that has high Ca(2+)/CaM-dependent activity, wild-type CaMKKβ displays enhanced autonomous activity (Ca(2+)/CaM-independent activity, 71% of total activity). This activity was significantly reduced (to 37%) by substitution of Thr(482) with a nonphosphorylatable Ala, without significant changes in Ca(2+)/CaM binding. In addition, a CaMKKα mutant containing the CaMKKβ regulatory domain was shown to be partially phosphorylated at Thr(446), resulting in a modest elevation of its autonomous activity. The combined results indicate that, in contrast to the α isoform, CaMKKβ exhibited increased autonomous activity, which was caused, at least in part, by autophosphorylation at Thr(482), resulting in partial disruption of the autoinhibitory mechanism.     

STO-609, a specific inhibitor of the Ca(2+)/calmodulin-dependent protein kinase kinase

STO-609, a selective inhibitor of Ca(2+)/calmodulin-dependent protein kinase kinase (CaM-KK) was synthesized, and its inhibitory properties were investigated both in vitro and in vivo. STO-609 inhibits the activities of recombinant CaM-KK alpha and CaM-KK beta isoforms, with K(i) values of 80 and 15 ng/ml, respectively, and also inhibits their autophosphorylation activities. Comparison of the inhibitory potency of the compound against various protein kinases revealed that STO-609 is highly selective for CaM-KK without any significant effect on the downstream CaM kinases (CaM-KI and -IV), and the IC(50) value of the compound against CaM-KII is approximately 10 microg/ml. STO-609 inhibits constitutively active CaM-KK alpha (glutathione S-transferase (GST)-CaM-KK-(84-434)) as well as the wild-type enzyme. Kinetic analysis indicates that the compound is a competitive inhibitor of ATP. In transfected HeLa cells, STO-609 suppresses the Ca(2+)-induced activation of CaM-KIV in a dose-dependent manner. In agreement with this observation, the inhibitor significantly reduces the endogenous activity of CaM-KK in SH-SY5Y neuroblastoma cells at a concentration of 1 microg/ml (approximately 80% inhibitory rate). Taken together, these results indicate that STO-609 is a selective and cell-permeable inhibitor of CaM-KK and that it may be a useful tool for evaluating the physiological significance of the CaM-KK-mediated pathway in vivo as well as in vitro.     

Expression of a multifunctional Ca2+/calmodulin-dependent protein kinase and mutational analysis of its autoregulation

Autophosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase converts it from a Ca2(+)-dependent to a Ca2(+)-independent or autonomous kinase, a process that may underlie some long-term enhancement of transient Ca2+ signals. We demonstrate that the neuronal alpha subunit clone expressed in COS-7 cells (alpha-CaM kinase) is sufficient to encode the regulatory phenomena characteristic of the multisubunit kinase isolated from brain. Activity of alpha-CaM kinase is highly dependent on Ca2+/calmodulin. It is converted by autophosphorylation to an enzyme capable of Ca2(+)-independent (autonomous) substrate phosphorylation and autophosphorylation. Using site-directed mutagenesis, we separately eliminate five putative autophosphorylation sites within the regulatory domain and directly examine their individual roles. Ca2+/calmodulin-dependent kinase activity is fully retained by each mutant, but Thr286 is unique among the sites in being indispensable for generation of an autonomous kinase.     

Inhibitory autophosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase analyzed by site-directed mutagenesis.

Initial autophosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) occurs at Thr286 (the "autonomy" site) and converts the kinase from a Ca(2+)-dependent to a partially Ca(2+)-independent or autonomous enzyme. After removal of Ca2+/calmodulin, the autonomous kinase undergoes a "burst" of inhibitory autophosphorylation at sites distinct from the autonomy site which may be masked in the presence of bound calmodulin. This burst of Ca(2+)-independent autophosphorylation blocks the ability of calmodulin to activate the kinase. We have used site-directed mutagenesis to replace putative inhibitory autophosphorylation sites within the calmodulin binding domain of recombinant alpha-CaM kinase with nonphosphorylatable alanines and examined the effects on autophosphorylation, kinase activity, and calmodulin binding. Although prominent Ca(2+)-independent autophosphorylation occurs within the calmodulin binding domain at Thr305, Thr306, and Ser314 in wild-type alpha-CaM kinase, the inhibitory effect on kinase activity and calmodulin binding is retained in mutants lacking any one of these three sites. However, when both Thr305 and Thr306 are converted to alanines the kinase does not display inhibition of either activity or calmodulin binding. Autophosphorylation at either Thr305 or Thr306 is therefore sufficient to block both binding and activation of the kinase by Ca2+/calmodulin. Thr306 is also slowly autophosphorylated in a basal reaction in the continuous absence of Ca2+/calmodulin. Autophosphorylation of Thr306 by the kinase in either its basal or autonomous state suggests that in the absence of bound calmodulin, the region of the autoregulatory domain surrounding Thr306, rather than the region near the autonomy site, lies nearest the peptide substrate binding site of the kinase.     

Regulatory domain of calcium/calmodulin-dependent protein kinase II. Mechanism of inhibition and regulation by phosphorylation

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.     

Regulation of Ca(2+)/calmodulin-dependent protein kinase kinase alpha by cAMP-dependent protein kinase: I. Biochemical analysis

Ca(2+)/calmodulin-dependent protein kinases (CaM-kinases) I and IV are activated upon phosphorylation of their Thr(177) and Thr(196), respectively, by the upstream Ca(2+)/calmodulin-dependent protein kinases CaM-kinase kinase alpha and beta, and deactivated upon dephosphorylation by protein phosphatases such as CaM-kinase phosphatase. Recent studies demonstrated that the activity of CaM-kinase kinase alpha is decreased upon phosphorylation by cAMP-dependent protein kinase (PKA), and the relationship between the inhibition and phosphorylation of CaM-kinase kinase alpha by PKA has been studied. In the present study, we demonstrate that the activity of CaM-kinase kinase alpha toward PKIV peptide, which contains the sequence surrounding Thr(196) of CaM-kinase IV, is increased by incubation with PKA in the presence of Ca(2+)/calmodulin but decreased in its absence, while the activity toward CaM-kinase IV is decreased by incubation with PKA in both the presence and absence of Ca(2+)/calmodulin. Six phosphorylation sites on CaM-kinase kinase alpha, Ser(24) for autophosphorylation, and Ser(52), Ser(74), Thr(108), Ser(458), and Ser(475) for phosphorylation by PKA, were identified by amino acid sequence analysis of the phosphopeptides purified from the tryptic digest of the phosphorylated enzymes. The presence of Ca(2+)/calmodulin suppresses phosphorylation on Ser(52), Ser(74), Thr(108), and Ser(458) by PKA, but accelerates phosphorylation on Ser(475). The changes in the activity of the enzyme upon phosphorylation appear to occur as a result of conformational changes induced by phosphorylation on several sites.     

Phosphorylation within an autoinhibitory domain in endothelial nitric oxide synthase reduces the Ca(2+) concentrations required for calmodulin to bind and activate the enzyme

We have investigated the effects of phosphorylation at Ser-617 and Ser-635 within an autoinhibitory domain (residues 595-639) in bovine endothelial nitric oxide synthase on enzyme activity and the Ca (2+) dependencies for calmodulin binding and enzyme activation. A phosphomimetic S617D substitution doubles the maximum calmodulin-dependent enzyme activity and decreases the EC 50(Ca (2+)) values for calmodulin binding and enzyme activation from the wild-type values of 180 +/- 2 and 397 +/- 23 nM to values of 109 +/- 2 and 258 +/- 11 nM, respectively. Deletion of the autoinhibitory domain also doubles the maximum calmodulin-dependent enzyme activity and decreases the EC 50(Ca (2+)) values for calmodulin binding and calmodulin-dependent enzyme activation to 65 +/- 4 and 118 +/- 4 nM, respectively. An S635D substitution has little or no effect on enzyme activity or EC 50(Ca (2+)) values, either alone or when combined with the S617D substitution. These results suggest that phosphorylation at Ser-617 partially reverses suppression by the autoinhibitory domain. Associated effects on the EC 50(Ca (2+)) values and maximum calmodulin-dependent enzyme activity are predicted to contribute equally to phosphorylation-dependent enhancement of NO production during a typical agonist-evoked Ca (2+) transient, while the reduction in EC 50(Ca (2+)) values is predicted to be the major contributor to enhancement at resting free Ca (2+) concentrations.     

Mutational analysis of sites in sepiapterin reductase phosphorylated by Ca2+/calmodulin-dependent protein kinase II

Sepiapterin reductase (SPR) catalyzes the last step in the pathway of tetrahydrobiopterin biosynthesis in tissues. SPR is phosphorylated by Ca2+-dependent protein kinases, which indicates that Ca2+-activated protein kinases may play a role in the regulation of SPR in vivo. Phosphorylation sites of rat sepiapterin reductase (rSPR) by Ca2+/calmodulin-dependent protein kinase II were determined in the present study. Using specific monoclonal anti-phospho-Ser and -Thr antibodies, we found that only Ser residues of rSPR were phosphorylated. We constructed several point mutants of SPR by systematically replacing the three Ser residues by Ala ones. These mutants showed that all three Ser residues, i.e. S46, S196, and S214, of rSPR were phosphorylated. We also recognized that only Ser-213 of human SPR was phosphorylated. Each of these serine residues in SPR was found in the consensus sequence (Arg-X-X-Ser/Thr) of the phosphorylation site.     

Identification of an autoinhibitory domain in calcineurin

The hypothesis that calcineurin, the Ca2+/calmodulin-dependent protein phosphatase, contains an autoinhibitory domain was tested using synthetic peptides corresponding to regions of the carboxyl-terminus of calcineurin. Of the several peptides analyzed, one, containing residues I-T-S-F-E-E-A-K-G-L-D-R-I-N-E-R-M-P-P-R-R-D-A-M-P, gave complete inhibition of its protein phosphatase activity. Using [32P]myosin light chain as substrate an IC50 of about 10 microM was obtained with either native calcineurin, assayed in the presence of Ca2+/calmodulin, or with calcineurin subjected to partial proteolysis which converts it to a fully active phosphatase when assayed in the presence of [ethylenebis (oxyethylenenitrilo)]tetraacetic acid. With 50 mM p-nitrophenylphosphate as substrate an IC50 of about 40 microM was observed. Studies with overlapping peptides suggested that the sequence P-P-R-R-D-A-M-P was essential but not sufficient for the observed inhibition. Kinetic analysis indicated that the inhibition of phosphatase activity was not competitive with respect to [32P]myosin light chain. This peptide did not show significant inhibition of the catalytic subunits of protein phosphatases type I or type IIA or of Ca2+/calmodulin-dependent protein kinase II. These results indicate that amino acids within this sequence of calcineurin constitute a unique autoinhibitory domain which interacts with the active site and is responsible for the low basal phosphatase activity in the absence of Ca2+/calmodulin.     

Regulatory Properties of Calcium/Calmodulin-Dependent Protein Kinase II in Rat Brain Postsynaptic Densities

Calcium/calmodulin (CaM)-dependent protein kinase II (CaM-kinase II) contained within the postsynaptic density (PSD) was shown to become partially Ca2+-independent following initial activation by Ca2+/CaM. Generation of this Ca2+-independent species was dependent upon autophosphorylation of both subunits of the enzyme in the presence of Mg2+/ATP/Ca2+/CaM and attained a maximal value of 74 +/- 5% of the total activity within 1-2 min. Subsequent to the generation of this partially Ca2+-independent form of PSD CaM-kinase II, addition of EGTA to the autophosphorylation reaction resulted in further stimulation of 32PO4 incorporation into both kinase subunits and a loss of stimulation of the kinase by Ca2+/CaM. Examination of the sites of Ca2+-dependent autophosphorylation by phosphoamino acid analysis and peptide mapping of both kinase subunits suggested that phosphorylation of Thr286/287 of the alpha- and beta-subunits, respectively, may be responsible for the transition of PSD CaM-kinase II to the Ca2+-independent species. A synthetic peptide 281-309 corresponding to a portion of the regulatory domain (residues 281-314) of the soluble kinase inhibited syntide-2 phosphorylation by the Ca2+-independent form of PSD CaM-kinase II (IC50 = 3.6 +/- 0.8 microM). Binding of Ca2+/CaM to peptide 281-309 abolished its inhibitory property. Phosphorylation of Thr286 in peptide 281-309 also decreased its inhibitory potency. These data suggest that CaM-kinase II in the PSD possesses regulatory properties and mechanisms of activation similar to the cytosolic form of CaM-kinase II.     

Activation of SAD kinase by Ca2+/calmodulin-dependent protein kinase kinase

To search for the downstream target protein kinases of Ca (2+)/calmodulin-dependent protein kinase kinase (CaMKK), we performed affinity chromatography purification of a rat brain extract using a GST-fused CaMKKalpha catalytic domain (residues 126-434) as the affinity ligand. Proteomic analysis was then carried out to identify the CaMKK-interacting protein kinases. In addition to identifying the catalytic subunit of 5'-AMP-activated protein kinase, we identified SAD-B as interacting. A phosphorylation assay and mass spectrometry analysis revealed that SAD-B was phosphorylated in vitro by CaMKK at Thr (189) in the activation loop. Phosphorylation of Thr (189) by CaMKKalpha induced SAD-B kinase activity by over 60-fold. In transfected COS-7 cells, kinase activity and Thr (189) phosphorylation of overexpressed SAD-B were significantly enhanced by coexpression of constitutively active CaMKKalpha (residues 1-434) in a manner similar to that observed with coexpression of LKB1, STRAD, and MO25. Taken together, these results indicate that CaMKKalpha is capable of activating SAD-B through phosphorylation of Thr (189) both in vitro and in vivo and demonstrate for the first time that CaMKK may be an alternative activating kinase for SAD-B.