Activation of nuclear Ca(2+)/calmodulin-dependent protein kinase II and brain-derived neurotrophic factor gene expression by stimulation of dopamine D2 receptor in transfected NG108-15 cells

We identified the isoforms of Ca(2+) /calmodulin-dependent protein kinase II (CaM kinase II) subunits in rat striatum. All four subunits of CaM kinase II alpha, beta, gamma and delta were detected including the isoforms of alphaB, gammaA, gammaA', gammaA.B, delta3 and delta7 with nuclear localization signal. We established NG108-15 cells with the stably expressed dopamine D2L receptor (D2LR, long form), which is an alternative splicing variant. The cells were termed NGD2L. Immunostaining demonstrated that D2LR was localized in plasma membranes. Calcium imaging with fluo-3 AM revealed that quinpirole, a D2R agonist, increased the intracellular Ca(2+), which was blocked by treatment with sulpiride and pertussis toxin in NGD2L cells, but not in mock cells. Furthermore, stimulation of D2LR with quinpirole in NGD2L cells activated the nuclear isoform of CaM kinase II. Stimulation of D2LR increased the expression of exon III- and IV-BDNF mRNA. Overexpression of CaM kinase II delta3 increased exon IV- but not exon III-BDNF mRNA. These results suggest that D2R is involved in the activation of the nuclear isoform of CaM kinase II and thereby in stimulation of gene expression through Ca(2+) signaling.     

Identification of the isoforms of Ca(2+)/Calmodulin-dependent protein kinase II in rat astrocytes and their subcellular localization

Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) occurs in astrocytes as well as in neurons in brain. We have reported that CaM kinase II is involved in the regulation of cytoskeletal proteins and gene expression in astrocytes. In this study, we identified all isoforms of CaM kinase II in astrocytes and examined their subcellular localization. When we amplified the isoforms of four subunits by RT-PCR followed by the "nested" PCR, totally 10 isoforms were obtained. Immunoblot analyses with five types of antibodies against CaM kinase II indicated that the most abundant isoform was delta2. Immunostaining suggested that the delta2 isoform was localized predominantly at the Golgi apparatus. The localization of the delta2 isoform at the Golgi apparatus was also observed in NG108-15 cells. We overexpressed all isoforms that contained the nuclear localization signal to examine their nuclear targeting in NG108-15 cells. In contrast to the alphaB and delta3 isoforms that entered the nucleus, as reported, the gammaA isoform was excluded from the nucleus in the transfected NG108-15 cells. These results suggest that the 15-amino acid insertion following the nuclear localization signal inhibits the nuclear targeting of the gammaA isoform.     

Role of the N- and C-lobes of calmodulin in the activation of Ca(2+)/calmodulin-dependent protein kinase II

Understanding the principles of calmodulin (CaM) activation of target enzymes will help delineate how this seemingly simple molecule can play such a complex role in transducing Ca (2+)-signals to a variety of downstream pathways. In the work reported here, we use biochemical and biophysical tools and a panel of CaM constructs to examine the lobe specific interactions between CaM and CaMKII necessary for the activation and autophosphorylation of the enzyme. Interestingly, the N-terminal lobe of CaM by itself was able to partially activate and allow autophosphorylation of CaMKII while the C-terminal lobe was inactive. When used together, CaMN and CaMC produced maximal CaMKII activation and autophosphorylation. Moreover, CaMNN and CaMCC (chimeras of the two N- or C-terminal lobes) both activated the kinase but with greater K act than for wtCaM. Isothermal titration calorimetry experiments showed the same rank order of affinities of wtCaM > CaMNN > CaMCC as those determined in the activity assay and that the CaM to CaMKII subunit binding ratio was 1:1. Together, our results lead to a proposed sequential mechanism to describe the activation pathway of CaMKII led by binding of the N-lobe followed by the C-lobe. This mechanism contrasts the typical sequential binding mode of CaM with other CaM-dependent enzymes, where the C-lobe of CaM binds first. The consequence of such lobe specific binding mechanisms is discussed in relation to the differential rates of Ca (2+)-binding to each lobe of CaM during intracellular Ca (2+) oscillations.     

Single molecule analyses of the conformational substates of calmodulin bound to the phosphorylase kinase complex

The four integral delta subunits of the phosphorylase kinase (PhK) complex are identical to calmodulin (CaM) and confer Ca(2+) sensitivity to the enzyme, but bind independently of Ca(2+). In addition to binding Ca(2+), an obligatory activator of PhK's phosphoryltransferase activity, the delta subunits transmit allosteric signals to PhK's remaining alpha, beta, and gamma subunits in activating the enzyme. Under mild conditions about 10% of the delta subunits can be exchanged for exogenous CaM. In this study, a CaM double-mutant derivatized with a fluorescent donor-acceptor pair (CaM-DA) was exchanged for delta to assess the conformational substates of PhKdelta by single molecule fluorescence resonance energy transfer (FRET) +/-Ca(2+). The exchanged subunits were determined to occupy distinct conformations, depending on the absence or presence of Ca(2+), as observed by alterations of the compact, mid-length, and extended populations of their FRET distance distributions. Specifically, the combined predominant mid-length and less common compact conformations of PhKdelta became less abundant in the presence of Ca(2+), with the delta subunits assuming more extended conformations. This behavior is in contrast to the compact forms commonly observed for many of CaM's Ca(2+)-dependent interactions with other proteins. In addition, the conformational distributions of the exchanged PhKdelta subunits were distinct from those of CaM-DA free in solution, +/-Ca(2+), as well as from exogenous CaM bound to the PhK complex as delta'. The distinction between delta and delta' is that the latter binds only in the presence of Ca(2+), but stoichiometrically and at a different location in the complex than delta.     

C2 domains of protein kinase C isoforms alpha,beta, and gamma: activation parameters and calcium stoichiometries of the membrane-bound state

The independently folding C2 domain motif serves as a Ca(2+)-dependent membrane docking trigger in a large number of Ca(2+) signaling pathways. A comparison was initiated between three closely related C2 domains from the conventional protein kinase C subfamily (cPKC, isoforms alpha, beta, and gamma). The results reveal that these C2 domain isoforms exhibit some similarities but are specialized in important ways, including different Ca(2+) stoichiometries. In the absence of membranes, Ca(2+) affinities of the isolated C2 domains are similar (2-fold difference) while Hill coefficients reveal cooperative Ca(2+) binding for the PKC beta C2 domain but not for the PKC alpha or PKC gamma C2 domain (H = 2.3 +/- 0.1 for PKC beta, 0.9 +/- 0.1 for PKC alpha, and 0.9 +/- 0.1 for PKC gamma). When phosphatidylserine-containing membranes are present, Ca(2+) affinities range from the sub-micromolar to the micromolar (7-fold difference) ([Ca(2+)](1/2) = 0.7 +/- 0.1 microM for PKC gamma, 1.4 +/- 0.1 microM for PKC alpha, and 5.0 +/- 0.2 microM for PKC beta), and cooperative Ca(2+) binding is observed for all three C2 domains (Hill coefficients equal 1.8 +/- 0.1 for PKC beta, 1.3 +/- 0.1 for PKC alpha, and 1.4 +/- 0.1 for PKC gamma). The large effects of membranes are consistent with a coupled Ca(2+) and membrane binding equilibrium, and with a direct role of the phospholipid in stabilizing bound Ca(2+). The net negative charge of the phospholipid is more important to membrane affinity than its headgroup structure, although a slight preference for phosphatidylserine is observed over other anionic phospholipids. The Ca(2+) stoichiometries of the membrane-bound C2 domains are detectably different. PKC beta and PKC gamma each bind three Ca(2+) ions in the membrane-associated state; membrane-bound PKC alpha binds two Ca(2+) ions, and a third binds weakly or not at all under physiological conditions. Overall, the results indicate that conventional PKC C2 domains first bind a subset of the final Ca(2+) ions in solution, and then associate weakly with the membrane and bind additional Ca(2+) ions to yield a stronger membrane interaction in the fully assembled tertiary complex. The full complement of Ca(2+) ions is needed for tight binding to the membrane. Thus, even though the three C2 domains are 64% identical, differences in Ca(2+) affinity, stoichiometry, and cooperativity are observed, demonstrating that these closely related C2 domains are specialized for their individual functions and contexts.     

Differential autophosphorylation of CaM kinase II from phasic and tonic smooth muscle tissues

Ca+/calmodulin-dependent protein kinase II (CaM kinase II) is regulated by calcium oscillations, autophosphorylation, and its subunit composition. All four subunit isoforms were detected in gastric fundus and proximal colon smooth muscles by RT-PCR, but only the gamma and delta isoforms are expressed in myocytes. Relative gamma and delta message levels were quantitated by real-time PCR. CaM kinase II protein and Ca2+/calmodulin-stimulated (total) activity levels are higher in proximal colon smooth muscle lysates than in fundus lysates, but Ca2+/calmodulin-independent (autonomous) activity is higher in fundus lysates. CaM kinase II in fundus lysates is relatively unresponsive to Ca2+/calmodulin. Alkaline phosphatase decreased CaM kinase II autonomous activity in fundus lysates and restored its responsiveness to Ca2+/calmodulin. Acetylcholine (ACh) increased autonomous CaM kinase II activity in fundus and proximal colon smooth muscles in a time- and dose-dependent manner. KN-93 enhanced ACh-induced fundus contractions but inhibited proximal colon contractions. The different properties of CaM kinase II from fundus and proximal colon smooth muscles suggest differential regulation of its autophosphorylation and activity in tonic and phasic gastrointestinal smooth muscles.     

Chronic exposure to ammonia induces isoform-selective alterations in the intracellular distribution and NMDA receptor-mediated translocation of protein kinase C in cerebellar neurons in culture

Hyperammonemia is responsible for most neurological alterations in patients with hepatic encephalopathy by mechanisms that remain unclear. Hyperammonemia alters phosphorylation of neuronal protein kinase C (PKC) substrates and impairs NMDA receptor-associated signal transduction. The aim of this work was to analyse the effects of hyperammonemia on the amount and intracellular distribution of PKC isoforms and on translocation of each isoform induced by NMDA receptor activation in cerebellar neurons. Chronic hyperammonemia alters differentially the intracellular distribution of PKC isoforms. The amount of all isoforms (except PKC zeta) was reduced (17-50%) in the particulate fraction. The contents of alpha, beta1, and epsilon isoforms decreased similarly in cytosol (65-78%) and membranes (66-83%), whereas gamma, delta, and theta; isoforms increased in cytosol but decreased in membranes, and zeta isoform increased in membranes and decreased in cytosol. Chronic hyperammonemia also affects differentially NMDA-induced translocation of PKC isoforms. NMDA-induced translocation of PKC alpha and beta is prevented by ammonia, whereas PKC gamma, delta, epsilon, or theta; translocation is not affected. Inhibition of phospholipase C did not affect PKC alpha translocation but reduced significantly PKC gamma translocation, indicating that NMDA-induced translocation of PKC alpha is mediated by Ca2+, whereas PKC gamma translocation is mediated by diacylglycerol. Chronic hyperammonemia reduces Ca+2-mediated but not diacylglycerol-mediated translocation of PKC isoforms induced by NMDA.     

A Ca(2+)-dependent global conformational change in the 3D structure of phosphorylase kinase obtained from electron microscopy.

Phosphorylase kinase (PhK), a Ca(2+)-dependent regulatory enzyme of the glycogenolytic cascade in skeletal muscle, is a 1.3 MDa hexadecameric oligomer comprising four copies of four distinct subunits, termed alpha, beta, gamma, and delta, the last being endogenous calmodulin. The structures of both nonactivated and Ca(2+)-activated PhK were determined to elucidate Ca(2+)-induced structural changes associated with PhK's activation. Reconstructions of both conformers of the kinase, each including over 11,000 particles, yielded bridged, bilobal structures with resolutions estimated by Fourier shell correlation at 24 A using a 0.5 correlation cutoff, or at 18 A by the 3sigma (corrected for D(2) symmetry) threshold curve. Extensive Ca(2+)-induced structural changes were observed in regions encompassing both the lobes and bridges, consistent with changes in subunit interactions upon activation. The relative placement of the alpha, beta, gamma, and delta subunits in the nonactivated three-dimensional structure, relying upon previous two-dimensional localizations, is in agreement with the known effects of Ca(2+) on subunit conformations and interactions in the PhK complex.     

Doc2gamma, a third isoform of double C2 protein, lacking calcium-dependent phospholipid binding activity

The Doc2 (double C2) family consists of two isoforms (Doc2alpha and Doc2beta) characterized by an N-terminal Munc13-1 interacting domain (Mid) and two C2 domains that interact with Ca(2+) and phospholipid at the C-terminus. This Ca(2+)-binding property is thought to be important to the regulation of neurotransmitter release. In this paper, we report a third isoform of mouse Doc2, named Doc2gamma. Doc2gamma also contains a putative Mid domain and two C2 domains, and it is 45.6 and 43.2% identical to mouse Doc2alpha and Doc2beta, respectively, at the amino acid level. In contrast to the other Doc2 isoforms, the C2 domains of Doc2gamma impair Ca(2+)-dependent phospholipid binding activity. The highest expression of Doc2gamma mRNA was found in the heart, but occurs ubiquitously, the same as Doc2beta. These findings indicate that Doc2gamma may also function as an effector for Munc13-1 and that it may be involved in the regulation of vesicular trafficking.     

Respiratory syncytial virus infection results in activation of multiple protein kinase C isoforms leading to activation of mitogen-activated protein kinase

Respiratory syncytial virus (RSV) is an important respiratory pathogen that preferentially infects epithelial cells in the airway and causes a local inflammatory response. Very little is known about the second messenger pathways involved in this response. To characterize some of the acute response pathways involved in RSV infection, we used cultured human epithelial cells (A549) and optimal tissue culture-infective doses (TCID(50)) of RSV. We have previously shown that RSV-induced IL-8 release is linked to activation of the extracellular signal-related kinase (ERK) mitogen-activated protein kinase pathway. In this study, we evaluated the upstream events involved in ERK activation by RSV. RSV activated ERK at two time points, an early time point consistent with viral binding and a later sustained activation consistent with viral replication. We next evaluated the role of protein kinase C (PKC) isoforms in RSV-induced ERK kinase activity. We found that A549 cells contain the Ca(2+)-dependent isoforms alpha and beta1, and the Ca(2+)-independent isoforms delta, epsilon, eta, mu, theta, and zeta. Western analysis showed that RSV caused no change in the amounts of these isoforms. However, kinase activity assays demonstrated activation of isoform zeta within 10 min of infection, followed by a sustained activation of isoforms beta1, delta, epsilon, and mu 24-48 h postinfection. A cell-permeable peptide inhibitor specific for the zeta isoform decreased early ERK kinase activation by RSV. Down-regulation of the other PKC isoforms with PMA blocked the late sustained activation of ERK by RSV. These studies suggest that RSV activates multiple PKC isoforms with subsequent downstream activation of ERK kinase.     

Alternative splicing introduces a nuclear localization signal that targets multifunctional CaM kinase to the nucleus

Intracellular targeting may enable protein kinases with broad substrate- specificities, such as multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) to achieve a selectivity of action in vivo. We have examined the intracellular targeting of three delta-CaM kinase isoforms. The delta B-CaM kinase isoform is targeted to the nucleus in transfected cells while the delta A- and delta C-CaM kinase isoforms are cytosolic/cytoskeletal. A chimeric construct of alpha-CaM kinase containing the delta B-CaM kinase variable domain is rerouted to the nucleus while the native alpha-CaM kinase and chimeras of alpha-CaM kinase which contain the delta A- or delta C-CaM kinase variable domains are retained in the cytoplasm. Using site-directed mutagenesis, we have defined a nuclear localization signal (NLS) within an 11-amino acid sequence, likely inserted by alternative splicing, in the variable domain of delta B-CaM kinase. Isoform-specific nuclear targeting of CaM kinase is probably a key mechanism in the selective regulation of nuclear functions by CaM kinase. CaM kinase is a multimer that can be composed of several isoforms. We find that when cells express two different isoforms of CaM kinase, cellular targeting is determined by the ratio of the isoforms. When an excess of the cytoplasmic isoform of CaM kinase is coexpressed along with the nuclear isoform, both isoforms are localized in the cytoplasm. Conversely an excess of the nuclear isoform can reroute the cytoplasmic isoform to the nucleus. The nuclear isoform likely coassembles with the cytosolic isoform, to form a heteromultimeric holoenzyme which is transported into the nucleus. These experiments demonstrate isoform-specific targeting of CaM kinase and indicate that such targeting can be modified by the expression of multiple isoforms of the enzyme.     

Retinoic acid-specific induction of a protein kinase C isoform during differentiation of HL-60 cells.

Human promyelocytic leukemia cells (HL-60) were treated with several differentiation inducers, then the changes in the activity of cytosolic protein kinase C (PKC) isoforms were examined by hydroxylapatite chromatography and the species of the isoforms were determined immunologically. In three undifferentiated HL-60 cell lines examined, PKC alpha and beta isoforms were present, but PKC gamma isoform was not detected. When the cells were induced by dimethylsulfoxide, dibutyryl cAMP, or nicotinamide to differentiate into granulocytes, these two PKC isoforms each increased to about 2- to 3-fold. When retinoic acid was used as the inducer, in addition to PKC alpha and beta, a third PKC isoform appeared. This isoform was clearly distinct from rat PKC alpha, beta, and gamma, immunologically. This isoform showed a distinctly lower Ca(2+)-requirement (3 microM) than that of PKC alpha or beta (100 microM) and was more dependent on cardiolipin and phosphatidylethanolamine, compared with PKC alpha, beta, and gamma. These results suggest that while the increases in the activities of PKC alpha and beta isoforms are common in the differentiation program initiated by several inducers, including retinoic acid, the emergence of an unclassified PKC isoform is a retinoic acid-specific process.     

Alternative splicing modulates the frequency-dependent response of CaMKII to Ca(2+) oscillations

Ca(2+) oscillations are required in various signal trans duction pathways, and contain information both in their amplitude and frequency. Remarkably, the Ca(2+)/calmodulin(CaM)-dependent protein kinase II (CaMKII) can decode such frequencies. A Ca(2+)/CaM-stimulated autophosphorylation leads to Ca(2+)/CaM-independent (autonomous) activity of the kinase that outlasts the initial stimulation. This autonomous activity increases exponentially with the frequency of Ca(2+) oscillations. Here we show that three beta-CaMKII splice variants (beta(M), beta and beta(e)') have very similar specific activity and maximal autonomy. However, their autonomy generated by Ca(2+) oscillations differs significantly. A mechanistic basis was found in alterations of the CaM activation constant and of the initial rate of autophosphorylation. Structurally, the splice variants differ only in a variable 'linker' region between the kinase and association domains. Therefore, we propose that differences in relative positioning of kinase domains within multimeric holoenzymes are responsible for the observed effects. Notably, the beta-CaMKII splice variants are differentially expressed, even among individual hippocampal neurons. Taken together, our results suggest that alternative splicing provides cells with a mechanism to modulate their sensitivity to Ca(2+) oscillations.     

Baculovirus-mediated overexpression of the phosphorylase b kinase holoenzyme and alpha gamma delta and gamma delta subcomplexes

Recombinant baculoviruses were created and used to coexpress rat phosphorylase kinase (Phk) alpha, gamma, and delta subunits and rabbit beta subunit in insect cells. Coexpression allowed creation of the (alphabetagammadelta)4 hexadecamer, the alphagammadelta heterotrimer, and the gammadelta heterodimeric subcomplexes. Neither the individual alpha, beta, or gamma subunit nor any complex containing the beta subunit other than the hexadecameric holoenzyme was obtained in soluble form. The expressed complexes exhibited pH- and [Ca2+]-dependent specific activities that were similar to those of the Phk holoenzyme purified from rabbit skeletal muscle (SkM Phk). SkM Phk, expressed Phk, and the alphagammadelta subcomplex were activated by exogenous calmodulin and underwent Ca(2+)-dependent autophosphorylation. In some of these features there were subtle differences that could likely be attributed to differences in the covalent modification state of the baculovirus-driven expressed protein. Our results provide an important avenue to probe the detailed characterization of the structure of Phk and the function of the individual domains of the subunits using baculovirus-mediated expression of Phk and Phk subcomplexes.     

Calmodulin regulation and identification of calmodulin binding region of type-3 ryanodine receptor calcium release channel

Ryanodine receptors (RyRs) are a family of intracellular Ca(2+) channels that are regulated by calmodulin (CaM). At low Ca(2+) concentrations (<1 microM), CaM activates RyR1 and RyR3 and inhibits RyR2. At elevated Ca(2+) concentrations (>1 microM), CaM inhibits all three RyR isoforms. Here we report that the regulation of recombinant RyR3 by CaM is sensitive to redox regulation. RyR3 in the presence of reduced glutathione binds CaM with 10-15-fold higher affinity, at low and high Ca(2+) concentrations, compared to in the presence of oxidized glutathione. However, compared to RyR1 assayed at low Ca(2+) concentrations under both reducing and oxidizing conditions, CaM binds RyR3 with reduced affinity but activates RyR3 to a greater extent. Under reducing conditions, RyR1 and RyR3 activities are inhibited with a similar affinity at [Ca(2+)] > 1 microM. Mutagenesis studies demonstrate that RyR3 contains a single conserved CaM binding site. Corresponding amino acid substitutions in the CaM binding site differentially affect CaM binding and CaM regulation of RyR3 and those of the two other isoforms. The results support the suggestion that other isoform dependent regions have a major role in the regulation of RyRs by CaM [Yamaguchi et al. (2004) J. Biol. Chem. 279, 36433-36439].     

Regulation of the RYR1 and RYR2 Ca2+ release channel isoforms by Ca2+-insensitive mutants of calmodulin

Calmodulin (CaM) may function as a regulatory subunit of ryanodine receptor (RYR) channels, modulating both channel activation and inhibition by Ca2+; however, mechanisms underlying differences in CaM regulation of the RYR isoforms expressed in skeletal muscle (RYR1) and cardiac muscle (RYR2) are poorly understood. Here we use a series of CaM mutants deficient in Ca2+ binding to compare determinants of CaM regulation of the RYR1 and RYR2 isoforms. In submicromolar Ca2+, activation of the RYR1 isoform by each of the single-point CaM mutants was similar to that by wild-type apoCaM, whereas in micromolar Ca2+, RYR1 inhibition by Ca2+CaM was abolished by mutations targeting CaM's C-terminal Ca2+ sites. In contrast to the RYR1, no activation of the cardiac RYR2 isoform by wild-type CaM was observed, but rather CaM inhibited the RYR2 at all Ca2+ concentrations (100 nM to 1 mM). Consequently, whereas the apparent Ca2+ sensitivity of the RYR1 isoform was enhanced in the presence of CaM, the RYR2 displayed the opposite response (RYR2 Ca2+ EC50 increased 7-10-fold in the presence of 5 microM wild-type CaM). CaM inhibition of the RYR2 was nonetheless abolished by each of four mutations targeting individual CaM Ca2+ sites. Furthermore, a mutant CaM deficient in Ca2+ binding at all four Ca2+ sites significantly activated the RYR2 and acted as a competitive inhibitor of RYR2 regulation by wild-type Ca2+CaM. We conclude that Ca2+ binding to CaM determines the effect of CaM on both RYR1 and RYR2 channels and that isoform differences in CaM regulation reflect the differential tuning of Ca2+ binding sites on CaM when bound to the different RYRs. These results thus suggest a novel mechanism by which CaM may contribute to functional diversity among the RYR isoforms.     

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.     

Intrinsically active variants of all human p38 isoforms

The p38 mitogen-activated protein kinases are activated in response to various extracellular signals in eukaryotic cells and play a critical role in the cellular responses to these signals. The four mammalian isoforms (p38alpha, p38beta, p38gamma, and p38delta) are coexpressed and coactivated in the same cells. The exact role of each p38 isoform has not been entirely identified, in part due to the inability to activate each member individually. This could be resolved by the use of intrinsically active mutants. Based on previous studies on yeast p38/Hog1 [Bell M, Capone R, Pashtan I, Levitzki A & Engelberg D (2001) J Biol Chem276, 25351-2538] and human p38alpha[Diskin R, Askari N, Capone R, Engelberg D & Livnah O (2004) J Biol Chem279, 47040-47049] we have generated intrinsically active p38beta, p38gamma and p38delta mutants. In addition, we have identified a new activating mutation site in p38alpha. Most of the activating mutations are located in the L16 loop, in which conformational changes were shown to induce activation. We show that these changes impose substantial autophosphorylation activity, providing a mechanistic explanation for the intrinsic activity of the mutants. The new active variants maintain specificity towards substrates and inhibitors similar to that of the parental wild-type proteins, and are phosphorylated by mitogen-activated protein kinase kinase 6, their upstream activator. Thus, we have completed the development of a series of intrinsically active mutants of all p38 isoforms. These active variants could now become powerful tools for the elucidating the activation mechanism and specific biological roles of each p38 isoform.     

Phosphorylation of dystrophin and alpha-syntrophin by Ca(2+)-calmodulin dependent protein kinase II.

A Ca(2+)-calmodulin dependent protein kinase activity (DGC-PK) was previously shown to associate with skeletal muscle dystrophin glycoprotein complex (DGC) preparations, and phosphorylate dystrophin and a protein with the same electrophoretic mobility as alpha-syntrophin (R. Madhavan, H.W. Jarrett, Biochemistry 33 (1994) 5797-5804). Here, we show that DGC-PK and Ca(2+)-calmodulin dependent protein kinase II (CaM kinase II) phosphorylate a common site (RSDS(3616)) within the dystrophin C terminal domain that fits the consensus CaM kinase II phosphorylation motif (R/KXXS/T). Furthermore, both kinase activities phosphorylate exactly the same three fusion proteins (dystrophin fusions DysS7 and DysS9, and the syntrophin fusion) out of a panel of eight fusion proteins (representing nearly 100% of syntrophin and 80% of dystrophin protein sequences), demonstrating that DGC-PK and CaM kinase II have the same substrate specificity. Complementing these results, anti-CaM kinase II antibodies specifically stained purified DGC immobilized on nitrocellulose membranes. Renaturation of electrophoretically resolved DGC proteins revealed a single protein kinase band (M(r) approximately 60,000) that, like CaM kinase II, underwent Ca(2+)-calmodulin dependent autophosphorylation. Based on these observations, we conclude DGC-PK represents a dystrophin-/syntrophin-phosphorylating skeletal muscle isoform of CaM kinase II. We also show that phosphorylation of the dystrophin C terminal domain sequences inhibits their syntrophin binding in vitro, suggesting a regulatory role for phosphorylation.