Ca2+-induced redistribution of Ca2+/calmodulin-dependent protein kinase II associated with an endoplasmic reticulum stress response in vascular smooth muscle

The relation between CaM kinase II activity and high Ca²⁺-mediated stress responses was studied in cultured vascular smooth muscle cells. Treatment with ionomycin (1 M) for 5 min caused a significant loss of CaM kinase II activity in whole cell homegenates and prominent vesiculation of the endoplasmic reticulum (ER). Similar losses of CaM kinase II activity were observed in the soluble lysate as assessed by activity measurements and Western blotting. Examination of the post-lysate particulate fraction showed that the loss of CaM kinase II from the soluble lysate was accompanied by a redistribution of CaM kinase II to this fraction. The ionomycin-mediated response was limited to this concentration (1 M); lower concentrations of ionomycin as well as stimulation with angiotensin II (1 M) or ATP (100 M) did not cause a shift in CaM kinase II distribution. Treatment with neither the CaM kinase II inhibitor KN-93 nor the phosphatase inhibitor okadaic acid altered the ionomycin-induced redistribution indicating that CaM kinase II activation and/or phosphorylation was not part of the mechanism. The response, however, was eliminated when the cells were treated in Ca²⁺-free medium. Washout of ionomycin led to only a partial restoration of the kinase activity in the soluble fraction after 10 min. Immunofluorescence microscopy of resting cells indicated colocalization of antibodies to CaM kinase II and an ER protein marker. ER vesiculation induced by ionomycin coincided with a parallel redistribution of CaM kinase II and ER marker proteins. These data link ionomycin-induced ER restructuring to a progressive redistribution of CaM kinase II protein to an insoluble particulate fraction and loss of cellular CaM kinase II activity. We propose that redistribution of CaM kinase II and loss of cellular activity are components of a common Ca²⁺-overload induced cellular stress response in cells.     

Phosphorylation of rat brain calmodulin in vivo and in vitro

After injection of [32p]orthophosphate into the third ventricle of rat brain, calmodulin(CaM) was prepared from soluble(S2) and particulate(P2) fractions of the whole brain and analyzed by SDS-PAGE in the presence or absence of Ca2+ followed by autoradiography. CaM from both fractions(S2 and P2) was significantly phosphorylated by endogenous protein kinase(s) of rat brain. The incorporation of radioactive phosphate into membrane-bound CaM from the P2 fraction was much higher than that of soluble CaM from the S2 fraction. CaM was phosphorylated in vitro by casein kinase 2 but not by casein kinase 1 or by cyclic AMP-dependent protein kinase, suggesting that casein kinase 2 may be, at least in part, responsible for the phosphorylation of CaM even in vivo.     

Inactivation and Subcellular Redistribution of Ca2+/ Calmodulin-Dependent Protein Kinase II Following Spinal Cord Ischemia

Abstract: Reversible spinal cord ischemia in rabbits induced a rapid loss of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) activity measured as incorporation of phosphate into exogenous substrates. About 70% of the activity was lost from the cytosolic fraction of spinal cord homogenates after 15 min of ischemia preceding irreversible paraplegia, which takes 25 min in this model. The loss of enzyme activity correlated with a loss of in situ renaturable autophosphorylation activity and a loss of CaM kinase II α and β subunits in the cytosol detected by immunoblotting. CaM kinase II activity in the particulate fraction also decreased but the protein levels of the a and β subunits increased. Thus ischemia resulted in an inactivation of CaM kinase II and a sequential or concurrent subcellular redistribution of the enzyme. However, denaturation and renaturation in situ of the CaM kinase subunits immobilized on membranes partly reversed the apparent inactivation of the enzyme in the particulate fraction. CaM kinase II activity was restored after reperfusion following short (≤25 min) durations of ischemia but not after longer durations (60 min) that result in irreversible paraplegia. The ischemia-induced inactivation of CaM kinase II, which phosphorylates proteins regulating many cellular processes, may be important in the cascade of events leading to delayed neuronal cell death.     

Studies on the regulatory domain of Ca2+/calmodulin-dependent protein kinase II by expression of mutated cDNAs in Escherichia coli.

The cDNAs encoding the alpha and beta subunits of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) were ligated into the bacterial expression vector pET and expressed in Escherichia coli. The bacterially expressed alpha and beta subunits exhibited Ca2+/calmodulin-dependent activity and were easily purified to apparent homogeneity from cell extracts. To determine the minimum size required for catalytic activity and the properties of the calmodulin-binding domain, mutated CaM kinase II cDNAs were expressed in E. coli and the enzymatic property of expressed proteins was examined. The replacement of Thr-286 of the alpha subunit with the negatively charged amino acid Asp or that of Arg-283 with the neutral amino acid Gly induced the partially Ca2+ independent activity. The mutant enzymes alpha-I(delta 283-478) and alpha-II(delta 359-478), which truncated the C-terminal region of the alpha subunit, exhibited CaM kinase II activity and the activities of alpha-I(delta 283-478) and alpha-II(delta 359-478) were completely independent of and partially dependent on Ca2+ and calmodulin, respectively. However, the truncated protein alpha(delta 250-478), which was only 33 amino acids shorter than the alpha-I(delta 283-478) protein had no enzymatic activity, indicating that alpha-I(delta 283-478) was close to the minimum size of the active form. The mutant enzyme alpha(delta 291-315), which lacked the calmodulin-binding domain exhibited Ca2+ independent activity. The molecular mass was, however, smaller than that expected from the amino acid sequence. The mutant enzyme alpha(delta 304-315), which lacked the C-terminal half of the calmodulin-binding domain of the alpha subunit, however, exhibited Ca(2+)-independent activity without a reduction in molecular size, indicating that residues 304-315 of the alpha subunit constituted the core calmodulin-binding domain.     

Calmodulin activation of target enzymes. Consequences of deletions in the central helix

The central helical region of calmodulin (CaM) includes amino acids 65-92 and serves to separate the two pairs of Ca2(+)-binding sites. This region may impart conformational flexibility and also interact with target proteins. The functional effects of deleting two, three, five, or eight amino acids from the central helix were monitored by examining the activation of phosphodiesterase, smooth muscle myosin light chain (MLC) kinase, and Ca2+/CaM-dependent protein kinase II (CaM kinase II). CaMDM(-8), a calmodulin-deletion mutant with 8 amino acids deleted from the middle of the central helix, failed to activate MLC kinase, phosphodiesterase, or CaM kinase II at physiologically significant concentrations of activator but also had altered electrophoretic mobility and tyrosine fluorescence properties suggesting major changes in the structure of this mutant. Deletion of five amino acids (77-81) resulted in an increase in apparent Kact for phosphodiesterase (150-fold), CaM kinase II (25-fold), and MLC kinase (5-fold) relative to CaM. The maximal autophosphorylation activity of CaM kinase II was also diminished 70% with CaMDM(-5). For phosphodiesterase activation, CaMDM(-2) has a 15-fold increase in apparent Kact while CaMDM(-3) had an apparent Kact value only 3-fold higher than native CaM. In contrast, the activation of MLC kinase by the two (79-80)- and three (79-81)-amino acid deletion mutants were indistinguishable from each other or native CaM. CaMDM(-2) and CaMDM(-3) stimulated CaM kinase II autophosphorylation to 85 and 70%, respectively, of native CaM with less than a 2-fold increase in Kact. Therefore, all deletions in the central helix of CaM reduce the efficiency of phosphodiesterase activation as reflected by substantial alterations in Kact. MLC kinase activation, however, is relatively insensitive to small two or three amino acid deletions. CaM kinase II interacts with the central helix deletion mutants in a complex manner with alterations in both the Kact and the maximum activity. The data suggest the central helix of CaM may serve as a flexible tether for MLC kinase (and to a lesser extent CaM kinase II) but that an extended conformation of CaM, as predicted from the crystal structure, may be required for phosphodiesterase activation.     

Calmodulin is involved in the Ca2+-dependent activation of ceramide kinase as a calcium sensor

We recently demonstrated that the activation of ceramide kinase (CERK) and the formation of its product, ceramide 1-phosphate (C1P), are necessary for the degranulation pathway in mast cells and that the kinase activity of this enzyme is completely dependent on the intracellular concentration of Ca(2+) (Mitsutake, S., Kim, T.-J., Inagaki, Y., Kato, M., Yamashita, T., and Igarashi, Y. (2004) J. Biol. Chem. 279, 17570-17577). Despite the demonstrated importance of Ca(2+) as a regulator of CERK activity, there are no apparent binding domains in the enzyme and the regulatory mechanism has not been well understood. In the present study, we found that calmodulin (CaM) is involved in the Ca(2+)-dependent activation of CERK. The CaM antagonist W-7 decreased both CERK activity and intracellular C1P formation. Additionally, exogenously added CaM enhanced CERK activity even at low concentrations of Ca(2+). The CERK protein was co-immunoprecipitated with an anti-CaM antibody, indicating formation of intracellular CaM.CERK complexes. An in vitro CaM binding assay also demonstrated Ca(2+)-dependent binding of CaM to CERK. These results strongly suggest that CaM acts as a Ca(2+) sensor for CERK. Furthermore, a CaM binding assay using various mutants of CERK revealed that the binding site of CERK is located within amino acids 422-435. This region appears to include a type 1-8-14B CaM binding motif and is predicted to form an amphipathic helical wheel, which is utilized in CaM recognition. The expression of a deletion mutant of CERK that contained the CaM binding domain but lost CERK activity inhibited the Ca(2+)-dependent C1P formation. These results suggest that this domain could saturate the CaM and hence block Ca(2+)-dependent activation of CERK. Finally, we reveal that in mast cell degranulation CERK acts downstream of CaM, similar to CaM-dependent protein kinase II, which had been assumed to be the main target of CaM in mast cells.     

Expression and characterization of calmodulin-dependent protein kinase II from cloned cDNAs in Chinese hamster ovary cells

cDNAs containing the entire coding regions of the alpha and beta subunits of calmodulin-dependent protein kinase II (CaM kinase II) were isolated from a rat cerebrum cDNA library, ligated into an expression vector under the control of SV40 early promoter and introduced into Chinese hamster ovary (CHO) cells. To investigate the role of the alpha and beta subunits and their functional domains in CaM kinase II activity, the properties of the kinases expressed in the transfected cells were studied. CaM kinase II activity was detected in the transfected cells when the alpha and beta cDNAs were introduced into CHO cells simultaneously. RNA transfer blot and protein immunoblot analyses demonstrated the expression of the mRNAs and proteins of both alpha and beta subunits in the cloned cells. When alpha or beta cDNA was introduced into CHO cells separately, a significant level of the enzyme activity was also expressed, indicating that the alpha and beta subunits exhibited enzyme activity individually. The apparent Km values for ATP and MAP 2 were almost the same for the alpha subunit, beta subunit, alpha beta complex, and brain CaM kinase II. However, there was a slight difference in the affinity for calmodulin between the expressed proteins. The alpha and beta subunits expressed in the same cells polymerized to form alpha beta complex of a size similar to that of brain CaM kinase II. The alpha subunit also polymerized to form an oligomer, which showed almost the same S value as that of alpha beta complex and brain CaM kinase II. In contrast, the beta subunit did not polymerize. The alpha subunit, beta subunit, alpha beta complex, and brain CaM kinase II were autophosphorylated with [gamma-32P]ATP in the presence of Ca2+ and calmodulin, which resulted in the appearance of Ca2+-independent activity. The Ca2+-independent activity was 60-75% of the total activity as measured in the presence of Ca2+ plus calmodulin. To examine the functional relationship of peptide domains of the subunits of CaM kinase II, deleted cDNAs were introduced into CHO cells and the properties of the expressed proteins were studied. In cells transfected with alpha or beta cDNA from which the association domain was deleted, a significant level of kinase activity was expressed. However, the expressed proteins showed hardly any autophosphorylation and the appearance of Ca2+-independent enzyme activity was very low, indicating that the association domain was essential for the autophosphorylation and for the appearance of the Ca2+-independent activity.     

Beta-adrenergic receptor subtypes and subcellular compartmentation of cyclic AMP and cyclic AMP-dependent protein kinase in rabbit cardiomyocytes

In purified ventricular myocytes from adult rabbit, beta-adrenergic stimulation causes cyclic AMP accumulation and cyclic AMP-protein kinase activation in both particulate and soluble fractions of the cell, whereas prostaglandin E1 elevates cyclic AMP and cyclic AMP-protein kinase activity in the soluble fraction exclusively. Only activation of particulate cyclic AMP-protein kinase activity results in phosphorylase b----a conversion. Using radioligand binding technics, we have determined whether beta 1- and beta 2-receptor subtypes mediate beta-adrenergic effects in particulate and soluble subcellular compartments, respectively. The non-selective antagonist [125I]iodocyanopindolol binds to intact ventricular myocytes with KD of 25 pM and a Bmax of 2.6 X 10(5) receptors/myocyte. Competition for [125I]iodocyanopindolol binding to intact myocytes by the beta-receptor subtype-specific antagonists practolol (beta 1) and zinterol (beta 2) results in monophasic curves with antagonist KD values of 1 microM and 1.5 microM, respectively. We conclude that adult rabbit cardiac myocytes do not possess detectable beta 2 receptors. Further, the ability of isoproterenol to cause elevation of cyclic AMP in two functionally distinct regions within the myocyte must pertain to the actions of a single subtype of beta-receptor, the beta 1-receptor.     

alphaKAP is an anchoring protein for a novel CaM kinase II isoform in skeletal muscle.

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) is present in a membrane-bound form that phosphorylates synapsin I on neuronal synaptic vesicles and the ryanodine receptor at skeletal muscle sarcoplasmic reticulum (SR), but it is unclear how this soluble enzyme is targeted to membranes. We demonstrate that alphaKAP, a non-kinase protein encoded by a gene within the gene of alpha-CaM kinase II, can target the CaM kinase II holoenzyme to the SR membrane. Our results indicate that alphaKAP (i) is anchored to the membrane via its N-terminal hydrophobic domain, (ii) can co-assemble with catalytically competent CaM kinase II isoforms and target them to the membrane regardless of their state of activation, and (iii) is co-localized and associated with rat skeletal muscle CaM kinase II in vivo. alphaKAP is therefore the first demonstrated anchoring protein for CaM kinase II. CaM kinase II assembled with alphaKAP retains normal enzymatic activity and the ability to become Ca2+-independent following autophosphorylation. A new variant of beta-CaM kinase II, termed betaM-CaM kinase II, is one of the predominant CaM kinase II isoforms associated with alphaKAP in skeletal muscle SR.     

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.     

Glutamate-Induced Loss of Ca2+/Calmodulin-Dependent Protein Kinase II Activity in Cultured Rat Hippocampal Neurons

Abstract: The exposure of cultured rat hippocampal neurons to 500 µ M glutamate for 20 min induced a 55% decrease in the total Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) activity. The Ca²⁺-independent activity and autophosphorylation of CaM kinase II decreased to the same extent as the changes observed in total CaM kinase II activity, and these decreases in activities were prevented by pretreatment with MK-801, an N -methyl- d -aspartate (NMDA)-type receptor antagonist, and the removal of extracellular calcium but not by antagonists against other types of glutamate receptors and protease inhibitors. Similarly, the decrease in the CaM kinase II activity was induced by a Ca²⁺ ionophore, ionomycin. Immunoblot analysis with the anti-CaM kinase II antibody revealed a significant decrease in the amount of the enzyme in the soluble fraction, in contrast with the inverse increase in the insoluble fraction; thus, the translocation was probably induced during treatment of the cells with glutamate. These results suggest that glutamate released during brain ischemia induces a loss of CaM kinase II activity in hippocampal neurons, by stimulation of the NMDA receptor, and that inactivation of the enzyme may possibly be involved in the cascade of the glutamate neurotoxicity following brain ischemia.     

Molecular characterization of calmodulin trapping by calcium/calmodulin-dependent protein kinase II

Autophosphorylation of alpha-Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) at Thr(286) results in calmodulin (CaM) trapping, a >10,000-fold decrease in the dissociation rate of CaM from the enzyme. Here we present the first site-directed mutagenesis study on the dissociation of the high affinity complex between CaM and full-length CaM kinase II. We measured dissociation kinetics of CaM and CaM kinase II proteins by using a fluorescently modified CaM that is sensitive to binding to target proteins. In low [Ca(2+)], the phosphorylated mutant kinase F293A and the CaM mutant E120A/M124A exhibited deficient trapping compared with wild-type. In high [Ca(2+)], the CaM mutations E120A, M124A, and E120A/M124A and the CaM kinase II mutations F293A, F293E, N294A, N294P, and R297E increased dissociation rate constants by factors ranging from 2.3 to 116. We have also identified residues in CaM and CaM kinase II that interact in the trapped state by mutant cycle-based analysis, which suggests that interactions between Phe(293) in the kinase and Glu(120) and Met(124) in CaM specifically stabilize the trapped CaM-CaM kinase II complex. Our studies further show that Phe(293) and Asn(294) in CaM kinase II play dual roles, because they likely destabilize the low affinity state of CaM complexed to unphosphorylated kinase but stabilize the trapped state of CaM bound to phosphorylated kinase.     

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.     

Immunohistochemical Localization of Ca2+/Calmodulin-Dependent Protein Kinase II in Rat Brain and Various Tissues

Polyclonal antibodies against Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) of rat brain were prepared by immunizing rabbits and then purified by antigen-affinity column. The antibodies which recognized both subunits of the enzyme with Mrs 49K and 60K were used for the study on the distribution of CaM kinase II in formalin-fixed, paraffin-embedded tissues. In the brain, a light-microscopic study demonstrated strong immunoreactivity in neuronal somata and dendrites and weak immunoreactivity in nuclei. The densely stained regions included cerebral cortex, hippocampal formation, striatum, substantia nigra, and cerebellar cortex. In substantia nigra, neurites were stained, but not neuronal somata. Electron microscopy revealed that the immunoreactive product was highly concentrated at the postsynaptic densities. In addition to neurons, weak immunoreactivity was also demonstrated in glial cells, such as astrocytes and ependymal cells of ventricles and epithelial cells of choroid plexus. In other tissues, strong immunoreactivity was observed in the islet of pancreas and moderate immunoreactivity in skeletal muscle and kidney tubules. Immunoreactivity was demonstrated in all of the tissues tested. The results suggest that CaM kinase II is widely distributed in the tissues.     

Identification of membrane-bound calcium, calmodulin-dependent protein kinase II in canine heart

Phospholamban, the putative regulatory proteolipid of the Ca2+/Mg2+ ATPase in cardiac sarcoplasmic reticulum, was selectively phosphorylated by a Ca2+/calmodulin (CaM)-dependent protein kinase associated with a cardiac membrane preparation. This kinase also catalyzed the phosphorylation of two exogenous proteins known to be phosphorylated by the multifunctional Ca2+/CaM-dependent protein kinase II (Ca2+/CaM-kinase II), i.e., smooth muscle myosin light chains and glycogen synthase a. The latter protein was phosphorylated at sites previously shown to be phosphorylated by the purified multifunctional Ca2+/CaM-kinase II from liver and brain. The membrane-bound kinase did not phosphorylate phosphorylase b or cardiac myosin light chains, although these proteins were phosphorylated by appropriate, specific calmodulin-dependent protein kinases added exogenously. In addition to phospholamban, several other membrane-associated proteins were phosphorylated in a calmodulin-dependent manner. The principal one exhibited a Mr of approximately 56,000, a value similar to that of the major protein (57,000) in a partially purified preparation of Ca2+/CaM-kinase II from the soluble fraction of canine heart that was autophosphorylated in a calmodulin-dependent manner. These data indicate that the membrane-bound, calmodulin-dependent protein kinase that phosphorylates phospholamban in cardiac membranes is not a specific calmodulin-dependent kinase, but resembles the multifunctional Ca2+/CaM-kinase II. Our data indicate that this kinase may be present in both the particulate and soluble fractions of canine heart.     

Staurosporine: An Effective Inhibitor for Ca2+/Calmodulin-Dependent Protein Kinase II

We investigated the effect of staurosporine on Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) purified from rat brain. (a) Staurosporine (10-100 nM) inhibited the activity of CaM kinase II. The half-maximal and maximal inhibitory concentrations were 20 and 100 nM, respectively. (b) The inhibition with staurosporine was of the noncompetitive type with respect to ATP, calmodulin, and phosphate acceptor (beta-casein). (c) Staurosporine suppressed the auto-phosphorylation of alpha- and beta-subunits of CaM kinase II at concentrations similar to those at which the enzyme activity was inhibited. (d) Staurosporine also attenuated the Ca2+/calmodulin-independent activity of the autophosphorylated CaM kinase II. These results suggest that staurosporine inhibits CaM kinase II by interacting with the catalytic domain, distinct from the ATP-binding site or substrate-binding site, of the enzyme and that staurosporine is an effective inhibitor for CaM kinase II in the cell system.     

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.     

Structural features of Ca2+/calmodulin-dependent protein kinase II revealed by electron microscopy

The molecular conformation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) from the rat forebrain and cerebellum was studied by means of EM using a quick-freezing technique. Each molecule appeared to be composed of two kinds of particles, with one larger central particle and smaller peripheral particles and had shapes resembling that of a flower with 8 or 10 "petals". A favorable shadowing revealed that each peripheral particle had a thin link to the central particle. We predicted that the 8-petal molecules and 10-petal molecules were octamers and decamers of CaM kinase II subunits, respectively, each assembled with the association domains of subunits gathered in the center, and the catalytic domains in the peripheral particles. Binding of antibodies to the enzyme molecules suggested that molecules with 8 and 10 peripheral particles were homopolymers composed only of beta subunit and of alpha subunit, respectively, specifying that CaM kinase II consists of homopolymer of either alpha or beta subunits.