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.     

Studies of the regulatory mechanism of Ca2+/calmodulin-dependent protein kinase II. Mutation of threonine 286 to alanine and aspartate

A cDNA clone for the alpha subunit of mouse brain Ca2+/CaM-dependent protein kinase II (CaM-kinase II) was transcribed in vitro and translated in a rabbit reticulocyte lysate system. Inclusion of [35S]methionine in the translation system yielded a single 35S-polypeptide of about 50 kDa. When the translation system was assayed for CaM-kinase II activity, there was a 5-10-fold enrichment of kinase activity which was totally dependent on Ca2+/calmodulin (CaM). Both the 50-kDa 35S-polypeptide and the Ca2+/CaM-dependent protein kinase activity were quantitatively immunoprecipitated by rat brain CaM-kinase II antibody. When the translated wild-type kinase was subjected to autophosphorylation conditions in the presence of Ca2+, CaM, Mg2+, and ATP, the Ca2+-independent activity (assayed in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid) increased from 5.8 +/- 0.7 to 26.5 +/- 2.1% of total activity (assayed in the presence of Ca2+/CaM). These properties confirm the identity of the kinase translated in vitro as CaM-kinase II. The role of Thr-286 autophosphorylation in formation of the Ca2+-independent activity was investigated by site-directed mutation of Thr-286 to Ala (Ala-286 kinase) and to Asp (Asp-286 kinase). The Ala-286 kinase was completely dependent on Ca2+/CaM for activity prior and subsequent to autophosphorylation. The Asp-286 kinase exhibited 21.9 +/- 0.8% Ca2+-independent activity, and this was not increased by autophosphorylation. These results establish that introduction of negative charge(s) at residue 286, either by autophosphorylation of Thr or by mutation to Asp, is sufficient and necessary to generate the partially Ca2+-independent form of CaM-kinase II.     

Generation of the Ca2(+)-independent form of Ca2+/calmodulin-dependent protein kinase II in cerebellar granule cells

Conditions that regulate the generation of the Ca2(+)-independent form of Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) in cultured rat cerebellar granule cells have been investigated. Under basal conditions, 4-5% of total CaM-kinase II activity, assayed in the presence of Ca2+/CaM, was the Ca2(+)-independent form active in the presence of EGTA. Depolarization with 56 mM K+ produced a transient increase to 9% Ca2+ independence within 15 s followed by a decline to 5-6% at 10 min. The divalent cation ionophore ionomycin elicited 10% Ca2+ independence, which remained elevated. Removal of Ca2+ from the Krebs-Ringer medium reduced basal Ca2+ independence to 1-2% and eliminated the elevation in response to K+ depolarization. Inclusion of 5 microM okadaic acid, a protein phosphatase inhibitor, in the incubation medium potentiated the levels of Ca2(+)-independent activity of CaM-kinase II. Additional studies in granule cell extracts indicated that there were both okadiac acid-sensitive and -insensitive protein phosphatases involved in the reversal of the Ca2+ independence of CaM-kinase II. Phosphopeptide mapping of the CNBr-cleaved 32P-labeled 58-60-kDa subunit of CaM-kinase II revealed that under basal conditions, the kinase contained phosphate in many sites. Conditions that promoted formation of the Ca2(+)-independent form of the kinase increased the 32P incorporation into multiple sites of the kinase. However, there was a good temporal correlation between 32P incorporation into CNBr peptide 1, which contains Thr-287, and generation of the Ca2(+)-independent kinase activity. These results indicate that formation of the Ca2(+)-independent species of CaM-kinase II is dynamically regulated in cerebellar granule cells by Ca2(+)-mobilizing agents and by protein phosphatase activity and is correlated with autophosphorylation of Thr-287.     

Autophosphorylation of Calmodulin-Kinase II in Synaptic Junctions Modulates Endogenous Kinase Activity

Previous studies have purified from brain a Ca2+/calmodulin-dependent protein kinase II (designated CaM-kinase II) that phosphorylates synapsin I, a synaptic vesicle-associated phosphoprotein. CaM-kinase II is composed of a major Mr 50K polypeptide and a minor Mr 60K polypeptide; both bind calmodulin and are phosphorylated in a Ca2+/calmodulin-dependent manner. Recent studies have demonstrated that the 50K component of CaM-kinase II and the major postsynaptic density protein (mPSDp) in brain synaptic junctions (SJs) are virtually identical and that the CaM-kinase II and SJ 60K polypeptides are highly related. In the present study the photoaffinity analog [alpha-32P]8-azido-ATP was used to demonstrate that the 60K and 50K polypeptides of SJ-associated CaM-kinase II each bind ATP in the presence of Ca2+ plus calmodulin. This result is consistent with the observation that these proteins are phosphorylated in a Ca2+/calmodulin-dependent manner. Experiments using 32P-labeled peptides obtained by limited proteolysis of 60K and 50K polypeptides from SJs demonstrated that within each kinase polypeptide the same peptide regions contain both autophosphorylation and 125I-calmodulin binding sites. These results suggested that the autophosphorylation of CaM-kinase II could regulate its capacity to bind calmodulin and, thus, its capacity to phosphorylate substrate proteins. By using 125I-calmodulin overlay techniques and sodium dodecyl sulfate-polyacrylamide gel electrophoresis we found that phosphorylated 50K and 60K CaM-kinase II polypeptides bound more calmodulin (50-70%) than did unphosphorylated kinase polypeptides. Levels of in vitro CaM-kinase II activity in SJs were measured by phosphorylation of exogenous synapsin I. SJs containing highly phosphorylated CaM-kinase II displayed greater activity in phosphorylating synapsin I (300% at 15 nM calmodulin) relative to control SJs that contained unphosphorylated CaM-kinase II. The CaM-kinase II activity in phosphorylated SJs was indistinguishable from control SJs at saturating calmodulin concentrations (300-1,000 nM). These findings show that the degree of autophosphorylation of CaM-kinase II in brain SJs modulates its in vitro activity at low and possibly physiological calmodulin concentrations; such a process may represent a mechanism of regulating this kinase's activity at CNS synapses in situ.     

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 calcineurin by phosphorylation. Identification of the regulatory site phosphorylated by Ca2+/calmodulin-dependent protein kinase II and protein kinase C

The site in calcineurin, the Ca2+/calmodulin (CaM)-dependent protein phosphatase, which is phosphorylated by Ca2+/CaM-dependent protein kinase II (CaM-kinase II) has been identified. Analyses of 32P release from tryptic and cyanogen bromide peptides derived from [32P]calcineurin plus direct sequence determination established the site as -Arg-Val-Phe-Ser(PO4)-Val-Leu-Arg-, which conformed to the consensus phosphorylation sequence for CaM-kinase II (Arg-X-X-Ser/Thr-). This phosphorylation site is located at the C-terminal boundary of the putative CaM-binding domain in calcinerin (Kincaid, R. L., Nightingale, M. S., and Martin, B. M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8983-8987), thereby accounting for the observed inhibition of this phosphorylation when Ca2+/CaM is bound to calcineurin. Since the phosphorylation site sequence also contains elements of the specificity determinants for Ca2+/phospholipid-dependent protein kinase (protein kinase C) (basic residues both N-terminal and C-terminal to Ser/Thr), we tested calcineurin as a substrate for protein kinase C. Protein kinase C catalyzed rapid stoichiometric phosphorylation, and the characteristics of the reaction were the same as with CaM-kinase II: 1) the phosphorylation was blocked by binding of Ca2+/CaM to calcineurin; 2) phosphorylation partially inactivated calcineurin by increasing the Km (from 9.9 +/- 1.1 to 17.5 +/- 1.1 microM 32P-labeled myosin light chain); and 3) [32P]calcineurin exhibited very slow autodephosphorylation but was rapidly dephosphorylated by protein phosphatase IIA. Tryptic and thermolytic 32P-peptide mapping and sequential phosphoamino acid sequence analysis confirmed that protein kinase C and CaM-kinase II phosphorylated the same site.     

CaM-kinase II dephosphorylates Thr(286) by a reversal of the autophosphorylation reaction

Autophosphorylation of CaM-kinase II produces a form of the enzyme not requiring Ca(2+)/calmodulin for sustained activity. We report that autophosphorylated CaM-kinase II dephosphorylates itself in the presence of ADP (termed autodephosphorylation). The dephosphorylation was unaffected by phosphatase inhibitors and was nucleotide specific, occurring with ADP but not with AMP, GTP, or GDP. (32)P-ATP was produced when ADP was added to (32)P-labeled CaM-kinase II, indicating that the enzyme was undergoing dephosphorylation through a reversal of the autophosphorylation reaction. ATP addition also produced loss of (32)P from the autophosphorylated enzyme while maintaining the kinase in a phosphorylated state. This indicates that the enzyme was undergoing cycles of autophosphorylation and dephosphorylation in the activated state. Autothiophosphorylated CaM-kinase II was resistant to autodephosphorylation. Site-directed mutants were used to show that Thr(286) was the predominant site dephosphorylated. Additionally, CaM-kinase II composed of beta subunits exhibited autodephosphorylation. Ca(2+)/CaM-independent activity expressed by the autophosphorylated alpha and beta holoenzymes was reversed following autodephosphorylation.     

Regulation of Ca2+/calmodulin-dependent protein kinase II by brain gangliosides

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.     

Studies on the generation of Ca2+/calmodulin-independent activity of calmodulin-dependent protein kinase II by autophosphorylation. Autothiophosphorylation of the enzyme.

The mechanism for the generation of the Ca2+/calmodulin (CaM)-independent activity of calmodulin-dependent protein kinase II (CaM-kinase II) by autophosphorylation was studied by characterizing the autothiophosphorylated enzyme, which is resistant to hydrolysis. When CaM-kinase II was incubated with adenosine 5'-O-(thiotriphosphate) at 5 degrees C, the incorporation of thiophosphate into the enzyme occurred rapidly, reaching a maximum level within a few minutes, in parallel with increase in Ca2+/CaM-independent activity. The maximum level was 1 mol of thiophosphate per mol of subunit of the enzyme, and the thiophosphorylation occurred exclusively at Thr286 in the alpha subunit and Thr287 in the other subunits of the enzyme. These results, taken together, indicate that the autothiophosphorylation of Thr286/Thr287 of each subunit is involved in the generation of the Ca2+/CaM-independent activity. The activity of the autothiophosphorylated enzyme, when assayed in the presence of Ca2+/CaM, showed the same kinetic properties as did the Ca2+/CaM-dependent activity of the original non-phosphorylated enzyme, but when assayed in the absence of Ca2+/CaM, it showed the same Vmax as the Ca2+/CaM-dependent activity but higher Km values for protein substrates. Thus, the phosphorylation of Thr286/Thr287 of the subunit of the enzyme by autophosphorylation appears to not only enhance the affinity of its substrate-binding site for the protein substrate, although it is lower than that of the enzyme activated by the binding of CaM, but also convert the active site to the fully active state.     

Autophosphorylation of rat brain Ca2+-activated and phospholipid-dependent protein kinase

Ca2+-activated and phospholipid-dependent protein kinase (protein kinase C) isolated from rat brain cytosol undergoes autophosphorylation in the presence of Mg2+, ATP, Ca2+, phosphatidylserine, and diolein. Approximately 2-2.5 mol of phosphate were incorporated per mol of the kinase. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, the phosphorylated kinase showed a single protein band of Mr = 82,000 compared to the Mr = 80,000 of the nonphosphorylated enzyme. Analysis of the 32P-labeled tryptic peptides derived from the autophosphorylated kinase by peptide mapping revealed that multiple sites were phosphorylated. Both serine and threonine residues were found to be labeled with 32P. Limited proteolysis of the autophosphorylated kinase with trypsin resulted in the conversion of the kinase into a phospholipid- and Ca2+-independent form. Two major 32P-labeled fragments, Mr = 48,000 and 38,000, were formed as a result of proteolysis, suggesting that the catalytic domain and possibly the Ca2+- and phospholipid-binding region were both phosphorylated. Protein kinase C autophosphorylation has a Km for ATP (1.5 microM) about 10-fold lower than that for phosphorylation of exogenous substrates. The kinetically preferred autophosphorylation appears to be an intramolecular reaction. The autophosphorylated protein kinase C, unlike the protease-degraded enzyme, still depends on Ca2+ and phospholipid for maximal activity. However, the autophosphorylated form of the kinase has a lower Ka for Ca2+ and a higher affinity for the binding of [3H]phorbol-12, 13-dibutyrate. These findings suggest that autophosphorylation of protein kinase C may be important in the regulation of the enzymic activity subsequent to signal transduction.     

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.     

Calcium/calmodulin-independent autophosphorylation sites of calcium/calmodulin-dependent protein kinase II. Studies on the effect of phosphorylation of threonine 305/306 and serine 314 on calmodulin binding using synthetic peptides

Two synthetic peptides containing the previously identified calmodulin (CaM)-binding domain of Ca2+/CaM-dependent protein kinase II (CaM-kinase II) (residues 296-309, Payne, M. E., Fong, Y.-L., Ono, T., Colbran, R. J., Kemp, B. E., Soderling, T. R., and Means, A. R. (1988) J. Biol. Chem. 263, 7190-7195) were phosphorylated by Ca2+/CaM-independent forms of the kinase. In the presence of EGTA, CaMK-(290-309) was phosphorylated exclusively on threonine residues (Km = 13 microM; Vmax = 211 nmol/min/mg). When the phosphorylated product was analyzed by reversed-phase high performance liquid chromatography (HPLC) two radioactive peaks were resolved. The first peak contained CaMK-(290-309) phosphorylated on Thr306, whereas the second peak contained CaMK-(290-309) phosphorylated on Thr305. However, under the same conditions CaMK-(294-319) was phosphorylated predominantly (approximately 70%) on serine residues (Km = 23 microM; Vmax = 99 nmol/min/mg) and HPLC analysis revealed a single major radioactive peak predominantly (more than 90%) phosphorylated at Ser314. Phosphorylation of both peptides was completely blocked in the presence of Ca2+ and a stoichiometric amount of CaM. Samples of each phosphorylated peptide were tested for CaM-binding ability by two procedures and compared to the nonphosphorylated peptides. Phosphorylation of either Thr305 or Thr306 greatly reduced the interaction between CaMK-(290-309) and CaM, whereas phosphorylation of Ser314 did not affect the ability of CaMK-(294-319) to bind CaM. These results indicate that Thr305 and/or Thr306 may be the Ca2+/CaM-independent autophosphorylation site(s) responsible for the loss of ability of CaM-kinase II to bind and be activated by Ca2+/CaM (Hashimoto, Y., Schworer, C. M., Colbran, R. J., and Soderling, T. R., J. Biol. Chem. 262, 8051-8055).     

Evidence that Ser-82 is a unique phosphorylation site on vimentin for Ca2(+)-calmodulin-dependent protein kinase II.

We identified the sites on vimentin that are phosphorylated by Ca2(+)-calmodulin-dependent protein kinase II (CaM-kinase II). Sequential analysis of the purified phosphopeptides demonstrated that the sites are -Thr-Arg-Thr-Tyr-Ser(PO4)38-Leu-Gly-Ser-Ala- and -Val-Arg-Leu-Leu-Gln-Asp-Ser(PO4)82-Val-Asp-, which are located within the amino-terminal head domain of vimentin. For Ser-82 but not Ser-38, the proposed CaM-kinase II recognition amino acid sequence (Arg-X-X-Ser/Thr) was not found. Studies with a series of synthetic peptide analogs corresponding to Ser-82 and its surrounding amino acid sequence indicate that Asp-84 acts as an essential substrate specificity determinant for the Ser-82 phosphorylation by CaM-kinase II. The CaM-kinase II recognition site may be more extensive than heretofore determined.     

Phosphorylation of smooth muscle myosin light chain kinase by Ca2+/calmodulin-dependent protein kinase II: comparative study of the phosphorylation sites

Smooth muscle myosin light chain kinase (MLC-kinase) was rapidly phosphorylated in vitro by the autophosphorylated form of Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) to a molar stoichiometry of 2.77 +/- 0.15 associated with a threefold increase in the concentration of calmodulin (CaM) required for half-maximal activation of MLC-kinase. Binding of CaM to MLC-kinase markedly reduced the phosphorylation stoichiometry to 0.21 +/- 0.05 and almost completely inhibited phosphorylation of sites in two peptides (32P-peptides P1 and P2) with reduced phosphorylation of peptide P3. By analogy, cAMP-dependent protein kinase phosphorylated MLC-kinase to a stoichiometry of 3.0 or greater in the absence of CaM with about a threefold decrease in the apparent affinity of MLC-kinase for CaM. Binding of CaM to MLC-kinase inhibited the phosphorylation to 0.84 +/- 0.13. Complete tryptic digests contained two major 32P-peptides as reported previously. One of the peptides, whose phosphorylation was inhibited in the presence of excess calmodulin, appeared to be the same as P2. Automated Edman sequence analysis suggested that both CaM-kinase II and cAMP-dependent protein kinase phosphorylated this peptide at the second of the two adjacent serine residues located at the C-terminal boundary of the CaM-binding domain. However, the other peptide phosphorylated by cAMP-dependent protein kinase, regardless of whether CaM was bound, was different from P1 and P3. Thus, MLC-kinase has a regulatory phosphorylation site(s) that is phosphorylated by the autophosphorylated form of CaM-kinase II and is blocked by Ca2+/CaM-binding.     

Regulation of Ca2+/Calmodulin-Dependent Protein Kinase II by Brain Gangliosides

Abstract: 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 μ M . 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 μ M ) 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 μ M ). 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.     

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.     

Molecular cloning of Ca2+/calmodulin-dependent protein kinase phosphatase.

Calmodulin-dependent protein kinase (CaM-kinase) phosphatase dephosphorylates and concomitantly deactivates CaM-kinase II activated upon autophosphorylation, and CaM-kinases IV and I activated upon phosphorylation by CaM-kinase kinase [Ishida, I., Okuno, S., Kitani, T., Kameshita, I., and Fujisawa, H. (1998) Biochem. Biophys. Res. Commun. 253, 159-163], suggesting that CaM-kinase phosphatase plays important roles in the function of Ca2+ in the cell, because the three multifunctional CaM-kinases (CaM-kinases I, II, and IV) are thought to be the key enzymes in the Ca2+-signaling system. In the present study, cDNA for CaM-kinase phosphatase was cloned from a rat brain cDNA library. The coded protein consisted of 450 amino acids with a molecular weight of 49, 165. Western blot analysis showed the ubiquitous tissue distribution of CaM-kinase phosphatase. Immunocytochemical analysis revealed that CaM-kinase phosphatase is evenly distributed outside the nucleus in a cell.     

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

We previously reported that rat brain Ca(2+)/calmodulin-dependent protein kinase (CaM-kinase) IV is inactivated by cAMP-dependent protein kinase (PKA) [Kameshita, I. and Fujisawa, H. (1991) Biochem. Biophys. Res. Commun. 180, 191-196]. In the preceding paper, we demonstrated that changes in the activity of CaM-kinase IV by PKA results from the phosphorylation of CaM-kinase kinase alpha by PKA and identified six phosphorylation sites, Ser(24) for autophosphorylation, and Ser(52), Ser(74), Thr(108), Ser(458), and Ser(475) for phosphorylation by PKA. In the present study, a causal relationship between the phosphorylation and change in the activity toward PKIV peptide has been studied using mutant enzymes with amino acid substitutions at the six phosphorylation sites. The following conclusions can be drawn from the experimental results: (i) Phosphorylation of Ser74 and/or unidentified sites causes an increase in activity; (ii) phosphorylation of Thr(108) or Ser(458) causes a decrease in the activity; (iii) the inhibitory effect of the phosphorylation of Thr(108) is canceled by the stimulatory effect of the phosphorylation, but that of Ser(458) is not; and (iv) the inhibitory effects of Thr(108) and Ser(458) are synergistic. In contrast to the activity toward PKIV peptide, the activity toward CaM-kinase IV appears to be decreased by the phosphorylation of Thr(108), but not significantly affected by the phosphorylation of Ser(458).     

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.     

Autophosphorylation of Ca2+/calmodulin-dependent protein kinase II. Effects on total and Ca2+-independent activities and kinetic parameters

Incubation of purified rat brain Ca2+/calmodulin-dependent protein kinase II for 2 min in the presence of Ca2+, calmodulin (CaM), Mg2+, and ATP converted the kinase from a completely Ca2+-dependent kinase to a substantially Ca2+-independent form with little loss of total activity. Subsequent addition of EGTA to the autophosphorylation reaction enhanced further autophosphorylation of the kinase which was associated with a suppression of total kinase activity to the Ca2+-independent value. Protein phosphatase 1 rapidly increased the suppressed total activity back to the control value and slowly decreased the Ca2+-independent activity. Kinetic analysis showed that the kinase not previously autophosphorylated had a Km for the synthetic peptide syntide-2 of 7 microM and Vmax of 9.8 mumol/min/mg when assayed in the presence of Ca2+ and CaM. The partially Ca2+-independent species, assayed in the presence of EGTA, had a Km of 21 microM and Vmax of 6.0. In the presence of Ca2+ and CaM the Km decreased and the Vmax increased to approximately control nonphosphorylated values. The completely Ca2+-independent form generated by sequential autophosphorylation first in the presence of Ca2+ and then EGTA had similar kinetic parameters to the partially independent species when assayed in the presence of EGTA, but addition of Ca2+ and CaM (up to 1 mg/ml) had little effect. These results suggest that separate autophosphorylation sites in the Ca2+/CaM-dependent protein kinase II are associated with formation of Ca2+-independent activity and suppression of total activity.