Comparative Analyses of the Three-dimensional Structures and Enzymatic Properties of α, β, γ, and δ Isoforms of Ca2+-Calmodulin-dependent Protein Kinase II
2004; Elsevier BV; Volume: 279; Issue: 13 Linguagem: Inglês
10.1074/jbc.m313597200
ISSN1083-351X
AutoresTara R. Gaertner, Steven J. Kolodziej, Dan Wang, Ryûji Kobayashi, John M. Koomen, James K Stoops, M. Neal Waxham,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoCa2+-calmodulin-dependent protein kinase II (CaM-kinase II) is a ubiquitous Ser/Thr-directed protein kinase that is expressed from a family of four genes (α, β, γ, and δ) in mammalian cells. We have documented the three-dimensional structures and the biophysical and enzymatic properties of the four gene products. Biophysical analyses showed that each isoform assembles into oligomeric forms and their three-dimensional structures at 21–25 Å revealed that all four isoforms were dodecamers with similar but highly unusual architecture. A gear-shaped core comprising the association domain has the catalytic domains tethered on appendages, six of which extend from both ends of the core. At this level of resolution, we can discern no isoform-dependent differences in ultrastructure of the holoenzymes. Enzymatic analyses showed that the isoforms were similar in their Km for ATP and the peptide substrate syntide, but showed significant differences in their interactions with Ca2+-calmodulin as assessed by binding, substrate phosphorylation, and autophosphorylation. Interestingly, the rank order of CaM binding affinity (γ > β > δ > α) does not directly correlate with the rank order of their CaM dependence for autophosphorylation (β > γ > δ > α). Simulations utilizing this data revealed that the measured differences in CaM binding affinities play a minor role in the autophosphorylation of the enzyme, which is largely dictated by the rate of autophosphorylation for each isoform. Ca2+-calmodulin-dependent protein kinase II (CaM-kinase II) is a ubiquitous Ser/Thr-directed protein kinase that is expressed from a family of four genes (α, β, γ, and δ) in mammalian cells. We have documented the three-dimensional structures and the biophysical and enzymatic properties of the four gene products. Biophysical analyses showed that each isoform assembles into oligomeric forms and their three-dimensional structures at 21–25 Å revealed that all four isoforms were dodecamers with similar but highly unusual architecture. A gear-shaped core comprising the association domain has the catalytic domains tethered on appendages, six of which extend from both ends of the core. At this level of resolution, we can discern no isoform-dependent differences in ultrastructure of the holoenzymes. Enzymatic analyses showed that the isoforms were similar in their Km for ATP and the peptide substrate syntide, but showed significant differences in their interactions with Ca2+-calmodulin as assessed by binding, substrate phosphorylation, and autophosphorylation. Interestingly, the rank order of CaM binding affinity (γ > β > δ > α) does not directly correlate with the rank order of their CaM dependence for autophosphorylation (β > γ > δ > α). Simulations utilizing this data revealed that the measured differences in CaM binding affinities play a minor role in the autophosphorylation of the enzyme, which is largely dictated by the rate of autophosphorylation for each isoform. Ca2+-calmodulin-dependent protein kinase II (CaM-kinase II) 1The abbreviations used are: CaM-kinase II, Ca2+/calmodulin-dependent protein kinase II; CaM, calmodulin; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; CaM-C75-IAEDANS, IAEDANS-labeled CaM(C75); IAEDANS, 5-((((2-iodoacetyl)amino)ethyl)amino)napthalene-1-sulfonic acid; MOPS, 4-morpholinepropanesulfonic acid. is a major downstream effector of Ca2+ signaling in eukaryotic cells. A rise in intracellular Ca2+ concentration leads to binding of Ca2+ ions to calmodulin (CaM), which binds to and activates CaM-kinase II. Upon activation, this enzyme has the ability to autophosphorylate, a process that confers Ca2+-independent activity upon the kinase (1Miller S.G. Kennedy M.B. Cell. 1986; 44: 861-870Abstract Full Text PDF PubMed Scopus (641) Google Scholar) and greatly increases its affinity for CaM (2Meyer T. Hanson P.I. Stryer L. Schulman H. Science. 1992; 256: 1199-1202Crossref PubMed Scopus (513) Google Scholar). Once activated, CaM-kinase II phosphorylates numerous target proteins and is involved in many cellular functions, including synaptic plasticity, synaptic vesicle mobilization, regulation of gene expression, regulation of smooth muscle contractility, and modulation of ion channel function (3Lin J.W. Sugimori M. Llinas R.R. McGuinness T.L. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8257-8261Crossref PubMed Scopus (113) Google Scholar, 4Wu X. McMurray C.T. J. Biol. Chem. 2001; 276: 1735-1741Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 5Silva A.J. Stevens C.F. Tonegawa S. Wang Y. Science. 1992; 257: 201-206Crossref PubMed Scopus (1183) Google Scholar, 6Tansey M.G. Word R.A. Hidaka H. Singer H.A. Schworer C.M. Kamm K.E. Stull J.T. J. Biol. Chem. 1992; 267: 12511-12516Abstract Full Text PDF PubMed Google Scholar, 7Derkach V. Barria A. Soderling T.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3269-3274Crossref PubMed Scopus (693) Google Scholar). The fact that CaM-kinase II has so many potential substrates raises the question of the relationship between its activation and a specific response to a particular Ca2+ signal. Possibly, the regulated expression of the multiple isoforms of CaM-kinase II confers these unique properties. CaM-kinase II is expressed from a family of four closely related genes, α, β, γ, and δ, each of which produces mRNA that can be alternatively spliced, giving rise to at least 30 different proteins (8Tombes R.M. Krystal G.W. Biochim. Biophys. Acta. 1997; 1355: 281-292Crossref PubMed Scopus (63) Google Scholar, 9Hudmon A. Schulman H. Annu. Rev. Biochem. 2002; 71: 473-510Crossref PubMed Scopus (525) Google Scholar). The overall organization of each of the four kinase isoforms is similar: an N-terminal catalytic domain is followed by a regulatory domain that contains an autoinhibitory region and a CaM-binding site, and a C-terminal association domain, through which the subunits interact to assemble into holoenzymes (10Kolodziej S.J. Hudmon A. Waxham M.N. Stoops J.K. J. Biol. Chem. 2000; 275: 14354-14359Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Between the CaM-binding domain and the association domain is a region termed the variable domain, where the majority of the gene product and splice variant differences are found. Combinations of 10 possible sequences can be inserted in this region, leading to a large number of possible splice variants (8Tombes R.M. Krystal G.W. Biochim. Biophys. Acta. 1997; 1355: 281-292Crossref PubMed Scopus (63) Google Scholar). The four CaM-kinase II genes exhibit different expression profiles in mammalian tissues. All four are found in brain, with α and β mRNA expressed at higher levels than γ and δ (11Takaishi T. Saito N. Tanaka C. J. Neurochem. 1992; 58: 1971-1974Crossref PubMed Scopus (30) Google Scholar). αCaM-kinase II is a neuron-specific isoform, and is present at high concentrations in postnatal forebrain neurons (12Erondu N.E. Kennedy M.B. J. Neurosci. 1985; 5: 3270-3277Crossref PubMed Google Scholar). βCaM-kinase is primarily neuronal, although the βM splice variant is found in skeletal muscle (13Bayer K.U. Lohler J. Schulman H. Harbers K. Brain Res. Mol. Brain Res. 1999; 70: 147-154Crossref PubMed Scopus (131) Google Scholar). The γ and δ isoforms are expressed throughout the body (13Bayer K.U. Lohler J. Schulman H. Harbers K. Brain Res. Mol. Brain Res. 1999; 70: 147-154Crossref PubMed Scopus (131) Google Scholar); δ plays an important role in cardiac muscle (14Hoch B. Haase H. Schulze W. Hagemann D. Morano I. Krause E.G. Karczewski P. J. Cell. Biochem. 1998; 68: 259-268Crossref PubMed Scopus (47) Google Scholar, 15Edman C.F. Schulman H. Biochim. Biophys. Acta. 1994; 1221: 89-101Crossref PubMed Scopus (158) Google Scholar), whereas both δ and γ variants are important in smooth muscle (16Singer H.A. Benscoter H.A. Schworer C.M. J. Biol. Chem. 1997; 272: 9393-9400Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 17Zhou Z.L. Ikebe M. Biochem. J. 1994; 299: 489-495Crossref PubMed Scopus (21) Google Scholar, 18Schworer C.M. Rothblum L.I. Thekkumkara T.J. Singer H.A. J. Biol. Chem. 1993; 268: 14443-14449Abstract Full Text PDF PubMed Google Scholar). CaM-kinase II isoforms all assemble into oligomeric complexes. Estimates in the literature for the number of subunits within these complexes have ranged from 8 to 14 (15Edman C.F. Schulman H. Biochim. Biophys. Acta. 1994; 1221: 89-101Crossref PubMed Scopus (158) Google Scholar, 19Miller S.G. Kennedy M.B. J. Biol. Chem. 1985; 260: 9039-9046Abstract Full Text PDF PubMed Google Scholar, 20Caran N. Johnson L.D. Jenkins K.J. Tombes R.M. J. Biol. Chem. 2001; 276: 42514-42519Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 21Nghiem P. Saati S.M. Martens C.L. Gardner P. Schulman H. J. Biol. Chem. 1993; 268: 5471-5479Abstract Full Text PDF PubMed Google Scholar, 22Kolb S.J. Hudmon A. Ginsberg T.R. Waxham M.N. J. Biol. Chem. 1998; 273: 31555-31564Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 23Kanaseki T. Ikeuchi Y. Sugiura H. Yamauchi T. J. Cell Biol. 1991; 115: 1049-1060Crossref PubMed Scopus (175) Google Scholar), with some unusual exceptions (24Shen K. Teruel M.N. Subramanian K. Meyer T. Neuron. 1998; 21: 593-606Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 25Zhou Z.H. Ando S. Furutsuka D. Ikebe M. Biochem. J. 1995; 310: 517-525Crossref PubMed Scopus (12) Google Scholar). We previously showed using three-dimensional reconstructions of single particle electron microscope images that the α isoform of CaM-kinase II is a dodecameric complex with overall dimensions of 200 × 220 Å. We further showed that the N-terminal catalytic domains reside in foot-like appendages extending away from a central core structure formed by the C-terminal association domains. Several other analyses have been accomplished visualizing single particle images (23Kanaseki T. Ikeuchi Y. Sugiura H. Yamauchi T. J. Cell Biol. 1991; 115: 1049-1060Crossref PubMed Scopus (175) Google Scholar, 26Dosemeci A. Reese T.S. Petersen J.D. Choi C. Beushausen S. Biochem. Biophys. Res. Commun. 1999; 263: 657-662Crossref PubMed Scopus (10) Google Scholar, 27Woodgett J.R. Davison M.T. Cohen P. Eur. J. Biochem. 1983; 136: 481-487Crossref PubMed Scopus (99) Google Scholar) and two-dimensional average images (28Morris E.P. Torok K. J. Mol. Biol. 2001; 308: 1-8Crossref PubMed Scopus (72) Google Scholar) and each of these studies identified that CaM-kinase II isoforms assemble into oligomeric complexes. However, inconsistencies remain regarding the subunit composition within the holoenzymes and to date, three-dimensional structural information is available only for the α isoform of CaM-kinase II. Many aspects of CaM-kinase II enzymatic function have been studied with respect to differences between pairs of isoforms or between sets of splice variants; however, there has not yet been a side-by-side comparison of members from each of the four distinct gene products of CaM-kinase II. In this study, we describe such a comparison, looking at potential differences in the structure and enzymatic properties between the α, β, γB, and δA isoforms. Expression of Isoforms—CaM-kinase II isoforms were expressed in Sf21 cells and purified as previously described (29Putkey J.A. Waxham M.N. J. Biol. Chem. 1996; 271: 29619-29623Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). A description of the expression of the α and β isoforms can be found in Ref. 22Kolb S.J. Hudmon A. Ginsberg T.R. Waxham M.N. J. Biol. Chem. 1998; 273: 31555-31564Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar. The cDNAs encoding the full-length rat δA and human γB isoforms were obtained from Dr. Andy Hudmon and Dr. Howard Schulman and were cloned into the EcoRI site of the pFastbac-1 baculovirus expression vector. Bacmid, virus production, protein production, and protein purification were accomplished as described (29Putkey J.A. Waxham M.N. J. Biol. Chem. 1996; 271: 29619-29623Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Expression, Purification, Mutagenesis, and Labeling of CaM—Calmodulin was expressed from the pET23 vector in the BL21 DE3 pLys-S strain of Escherichia coli. To purify CaM, the cell pellets were lysed by multiple cycles of freeze/thaw, followed by resuspension in 50 mm Tris, 10 mm EDTA, and sonication. The lysate was heated to 70 °C and then cell debris was pelleted by centrifugation at 100,000 × g for 1 h. The supernatant was brought to 2.5 m ammonium sulfate, the precipitate was spun out, and the resulting supernatant was then brought to saturation with ammonium sulfate. This solution was centrifuged, and the pellet was resuspended in 50 mm Tris, pH 7.5, 200 mm ammonium sulfate, and 1 mm EDTA and loaded onto a phenyl-Sepharose column. The flow-through from this column was brought to 2.5 mm in CaCl2, and then loaded on a second phenyl-Sepharose column. CaM was eluted with 50 mm Tris, pH 7.5, 1 m NaCl, and 2.5 mm EGTA. CaM was mutated at Lys-75 and labeled with IAEDANS as described in Putkey and Waxham (29Putkey J.A. Waxham M.N. J. Biol. Chem. 1996; 271: 29619-29623Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Gel Filtration and Density Gradient Centrifugation—Gel filtration was accomplished using a Superose HR6 10/30 column (Amersham Biosciences) on an AKTA FPLC system and sucrose gradient centrifugation on linear 5–24% gradients were performed as described in Kolb et al. (22Kolb S.J. Hudmon A. Ginsberg T.R. Waxham M.N. J. Biol. Chem. 1998; 273: 31555-31564Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Dynamic Light Scattering—To determine diffusion coefficients (D), the holoenzyme form of each subunit was first isolated by chromatography on a Superose HR6 10/30 column (Amersham Biosciences) in 40 mm HEPES, pH 7.5, 150 mm KCl, and 0.1 mm EGTA and then subjected to dynamic light scattering. The kinases, at concentrations of 0.089 to 0.11 mg/ml, were analyzed using a Molecular Solutions Dyna Pro 801 molecular sizing instrument (instrumentation kindly made available by Dr. Florentine Quiocho, Baylor College of Medicine). The samples were illuminated with 780 nm laser light and scattering was detected at 90° to the input beam using a 50-μl cell at room temperature. Intensity fluctuations were autocorrelated and 20 1-min samplings were obtained for each sample. Each enzyme preparation was analyzed at least three independent times and the reported values are the averages of these three experiments. Close visual inspection of each run was used to eliminate data not conforming to pre-established criteria for monodispersity (modal distribution ranging from 0.998 to 1.002). 2See www.proteinsolutions.com. The fits of the autocorrelation curves return a diffusion coefficient (D) that can be related to the radius of gyration (Rh) through the Stokes-Einstein relationship: D = kT/6πηRh, where k is Boltzmann's constant, T is absolute temperature, and η is solvent viscosity. Calculation of Holoenzyme Mass and Frictional Ratios—The calculated mass was determined from the equation, M = RTS/(D(1 - -Vρ)), where M is the molecular weight (g/mol), R is the gas constant, T is the temperature in K, S is the sedimentation coefficient determined from sucrose gradient analysis, D is the diffusion coefficient determined from dynamic light scattering, V̄ is the partial specific volume (cm3/g), and ρ is the density of the solvent. The frictional ratio f/f0 was calculated using the equations, f = kT/D and f0 = 6πη(3MV̄/4πNA)⅓, where f0 is the friction on a sphere with mass M and NA is Avogadro's number. Electron Microscopy, Digitization, and Particle Extraction—Each CaM-kinase II isoform was purified as described and monodisperse preparations were produced by gel filtration chromatography within 2 h of use. A 6-μl sample of each isoform (40–80 μg/ml) in 40 mm HEPES, 1 mm EGTA, 500 mm KCl, pH 7.4, was applied to the carbon side of a freshly prepared carbon-coated formvar grid (500 mesh copper) and after 30 s the excess was removed by wicking with bibulous paper. The bound molecules were washed 3–4 times with 6 μl of 0.25% methylamine tungstate stain, pH 7.1, containing 10 μg/ml bacitracin as a wetting agent; the excess stain was removed by wicking with bibulous paper. Images of the stained molecules were acquired at ×50,000 with a JEOL JEM 1200 electron microscope operating at 100 kV using conventional irradiation procedures. The images were recorded at 0.3–1.0 μm under focus. Micrographs were digitized using a Zeiss SCAI scanner at a pixel size of 5.6 Å on the specimen scale. Power spectra from the micrographs were analyzed for astigmatism and drift and, if significant, the micrograph was rejected. Image Alignment and Three-dimensional Reconstruction—Average images of the characteristic views were obtained by extracting particles representative of the end views of the complex and subjecting them to a reference-free alignment using the SPIDER software (30Frank J. Radermacher M. Penczek P. Zhu J. Li Y. Ladjadj M. Leith A. J. Struct. Biol. 1996; 116: 190-199Crossref PubMed Scopus (1808) Google Scholar). The average images were 6-fold symmetric. Three-dimensional projection alignment and iterative reconstruction was employed to compute the structures (31Kolodziej S.J. Penczek P.A. Stoops J.K. J. Struct. Biol. 1997; 120: 158-167Crossref PubMed Scopus (27) Google Scholar). A set of quasi-uniformly distributed reference projections using the αCaM-kinase II reconstruction as the initial model was generated within θ = 0–90° and Φ = 0–360° with angular steps of 2°. After the initial angular alignments, a reference-based translational alignment of the boxed images was performed by cross-correlating the particle images with the projection of the preliminary structure to the orientation of the particle image. The newly computed structures, which appeared to have 622 symmetry, were used in the subsequent alignment and iterative reconstructions. Two to three passes of refinement achieved a consistent structure and a stable resolution value. The resolution values of the reconstructions were measured by the Fourier shell correlation with a Fourier ring criterion of 0.5 (32Saxton W.O. Baumeister W. J. Microsc. 1982; 127: 127-138Crossref PubMed Scopus (696) Google Scholar). The α, β, γ, and δ isoforms consisted of 3003, 3061, 5085, and 5313 images, and had resolution values of 21, 23, 22, and 25 Å, respectively. The solid-shaded structures were thresholded to a volume that corresponds to their approximate molecular weight, and the images were rendered using the Explorer software (NAG, Inc., Downer's Grove, IL). A test for the 6-fold symmetry of the particles was accomplished by starting with the 6-fold symmetric model to generate reference projections at 5° increments setting the range of Φ to 73 and 52.1°, to produce 5- and 7-fold models, respectively. During the backprojection to render the new volumes either 5- or 7-fold symmetry was imposed. These newly rendered structures were used as the reference volumes for the next set of alignments using projections at 3° increments. Three further sets of alignments were run at 2° increments using the previously generated structure as the model. The artificially generated model was also used to test the structural symmetry of the β and δ isozymes. In each case the refinements were evaluated by degree of convergence between subsequent steps, the value of the resolution estimation, and visual inspection of the final structures for obvious artifacts. MALDI Mass Spectrometry—For analysis of isoform subunit mass, kinase samples were diluted 1:10 in H2O with 10% acetonitrile and 0.1% formic acid. Dried droplet deposits were prepared by mixing the diluted samples 1:1 with sinapinic acid (10 mg/ml) dissolved in 100% acetonitrile with 0.1% formic acid. Positive ion MALDI mass spectra were acquired in linear mode on an Applied Biosystems Voyager DE-STR instrument using bovine serum albumin as an external calibrant. In some experiments, glutaraldehyde cross-linking was performed to produce covalent bonds between subunits. Glutaraldehyde (25%) was diluted in deionized water and mixed with the protein sample (0.3–0.6 mg/ml final protein concentration) to a final glutaraldehyde concentration of 0.125 or 0.25%. Aliquots were removed at 0.5, 1, 2, 5, and 10 min for analysis. Dried droplet deposits were prepared by mixing the protein solution 1:1 with saturated sinapinic acid or ferulic acid in 40% aqueous acetonitrile with 0.1% trifluoroacetic acid or 0.1% formic acid, respectively. After crystal formation, the samples were washed repeatedly with cold water to remove buffer salts and glycerol prior to MALDI mass spectrometry analysis. Spectra were obtained on the Voyager DE-STR instrument for positive ions in linear mode. Parameters were optimized for immunoglobulin G (25 kV accelerating voltage, 90% grid voltage, and 900–1500 ns delay time). The mass spectra are accumu lations of 1000–2000 laser shots, and all samples were analyzed with the same laser power. Measurement of CaM Affinity—Fluorimetry measurements were made at room temperature in a PTI fluorimeter using 5-nm slit widths for both excitation and emission. The excitation wavelength was set at 345 nm and emission was measured at 465 nm. 25 or 100 nm CaM C75-IAEDANS in 25 mm MOPS, pH 7.0, 150 mm KCl, 0.5 mm CaCl2, and 0.1 mg/ml bovine serum albumin was titrated with CaM-kinase II, and the difference in fluorescence intensity was plotted against the calculated concentration of free Cam-kinase II. Binding curves were fitted with the Hill equation, y = axh/(KDh + xh), using SigmaPlot software. Syntide Phosphorylation Assay—The calmodulin dependence of kinase activity was assessed in 25-μl reactions containing 25 mm HEPES, 70 mm KCl, 0.5 mm CaCl2, 0.4 mm dithiothreitol, 10 mm MgCl2, 100 μm [γ-32P]ATP (2 μCi/reaction), 10 ng/reaction kinase, 50 μm syntide, and 0.39 nm to 3.2 μm CaM. Reactions were preincubated for 1 min at 30 °C, started by addition of kinase and then incubated for 30 s at 30 °C. Reactions were terminated by spotting 20 μl onto Whatman P-81 filters and immersing in 75 mm PO4 acid. Reactions to determine the Km for ATP or syntide contained 3.2 μm CaM and varied ATP from 3.0 to 200 μm or varied syntide from 0.8 to 50 μm. Autophosphorylation Assay—Autophosphorylation was assessed in 25-μl reactions containing 50 mm HEPES, 70 mm KCl, 0.5 mm CaCl2, 0.4 mm dithiothreitol, 10 mm MgCl2, 100 μm [γ-32P]ATP (2 μCi/reaction), 100 ng/reaction kinase, and 3.0 nm to 3.2 μm CaM. Reactions were carried out on ice, and were started by addition of kinase. After 15 s, reactions were terminated by adding an equal volume of 100% trichloroacetic acid. Stopped reactions were incubated on ice a further 60 min, and then the precipitated protein was pelleted, washed with 50% trichoroacetic acid, and the incorporated radioactivity was quantified using Cerenkov counting. Simulations of CaM-kinase II Autophosphorylation—CaM-kinase II autophosphorylation in response to pulses of Ca2+ at different frequencies was simulated using a system of 8 differential equations that describe Ca2+ binding to the two lobes of CaM and the interaction of CaM with phosphorylated and unphosphorylated CaM-kinase II. Ca2+ was assumed to bind to CaM cooperatively within a lobe, and was therefore modeled with only two binding steps, using association rate constants 300 μm-1 s-1 and 10 μm-1 s-1 and dissociation rate constants 1600 and 8 s-1 for the N- and C-terminal lobes, respectively. Ca4CaM interacts with inactive CaM-kinase II (Wi) to form active CaM-kinase II (Wa) (association rate constant 21 μm-1 s-1, and dissociation rate constants 1.31, 0.54, 0.15, and 0.70 s-1 for α, β, γ, and δ, respectively), which can then autophosphorylate, trapping CaM on the kinase (Wt). Dissociation of Ca4CaM from autophosphorylated CaM-kinase II is very slow (2Meyer T. Hanson P.I. Stryer L. Schulman H. Science. 1992; 256: 1199-1202Crossref PubMed Scopus (513) Google Scholar) and forms Wp at a rate of 2 × 10-5 s-1. Ca2+ was assumed to dissociate from Wp and Wt at reduced rates (10 and 0.4 s-1), and these steps induce the fast dissociation of CaM from CaM-kinase II, converting it to Wi or Wp. The autophosphorylation step was modeled as in Dupont et al. (33Dupont G. Houart G. De Koninck P. Cell Calcium. 2003; 34: 485-497Crossref PubMed Scopus (105) Google Scholar) except that in our simulations autophosphorylation was a first-order reaction; briefly, the rate of autophosphorylation was dependent on a third-order polynomial of the fraction of active subunits. The parameters for this autophosphorylation step were fit to our data for the CaM dependence of autophosphorylation and are 0.018, 0.015, and 0.033 for α, 0.043, 0.0062, and 0.019 for β, 0.0053, 0.00092, and 0.066 for γ, and 0.055, 0.0074, and 0.015 for δ. To simulate the frequency dependence of autophosphorylation, we imposed step pulses of Ca2+ of 0.1 s duration and 1 μm amplitude, from a baseline of 0.1 μm Ca2+, with frequencies from 0.5 to 8 Hz. The concentrations of reactants in the simulations were 5 μm total CaM and 5 μm total subunits of CaM-kinase II. Expression of Isoforms—A diagram of the domain structure of the α, β, γ, and δ isoforms of CaM-kinase II is shown in Fig. 1. Their most significant difference is because of a variable length insert found just following the regulatory domain of each subunit termed the unique domain (lower part of panel A, Fig. 1). In addition, δA has an extra 21-amino acid sequence extending from the C terminus of the protein. Pairwise alignments of the amino acid sequences show a high degree of similarity, ranging from 82 to 87%. To examine the structure and function of these four gene products, each CaM-kinase II isoform was expressed from its cDNA using the baculovirus system in Sf21 cells. Because of the high level of expression it was straightforward to obtain highly purified protein preparations. A Coomassie-stained gel of the four isoforms is shown in Fig. 1, panel B. The expected molecular weights predicted by the sequence information are 54,110 (α), 60,397 (β), 58,361 (γ), and 60,076 (δ). These are largely consistent with the migration of the purified isoforms on SDS-PAGE, with the relative molecular weights of the expressed isoforms being calculated at ∼52,200 (α), ∼60,500 (β), ∼61,000 (γ), and ∼60,000 (δ). Note also that there is an additional protein band in the β preparation with a lesser amount in the δ preparation (arrowheads in Fig. 1) and none visible in either the α or γ preparations. The calculated mass of this additional band is ∼64,700 and ∼64,600, for β and δ, respectively, and this protein in the β preparation cross-reacted with β-specific monoclonal antibodies. The origin or nature of this higher molecular weight product is presently unknown. To provide a more accurate mass measurement of each purified preparation, MALDI-TOF mass spectrometry was performed. This analysis led to a molecular mass of 53,885 ± 97 kDa for α, 60,265 ± 66 kDa for β, 58,325 ± 15 kDa for γ, and 59,856 ± 63 kDa for δ (Table I).Table IHydrodynamic analysis of CaM-kinase II isoforms The subunit mass was determined by MALDI-TOF mass spectrometry, the sedimentation coefficient was determined by sedimentation velocity centrifugation, and the diffusion coefficient (D) was determined by dynamic light scattering as described under "Experimental Procedures." The Rh values were calculated from D, the calculated mass values were calculated from the sedimentation coefficient and D, and f/f0 was calculated from the mass and D, all as described under "Experimental Procedures." The reported values are mean ± SD.IsoformSubunit massSedimentation coefficientDRhCalculated massf/f0kDas × 1013cm2/s × 107nmkDaα53.917.2 ± 0.4aData from Kolb et al. (22)2.53 ± 0.039.2 ± 0.16851.58β60.314.5 ± 1.1aData from Kolb et al. (22)1.84 ± 0.0212.7 ± 0.19461.95γ58.316.7 ± 1.22.09 ± 0.0211.4 ± 0.18191.83δ59.917.3 ± 0.22.00 ± 0.0411.9 ± 0.28881.87a Data from Kolb et al. (22Kolb S.J. Hudmon A. Ginsberg T.R. Waxham M.N. J. Biol. Chem. 1998; 273: 31555-31564Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) Open table in a new tab Hydrodynamic Analysis—Sucrose gradient centrifugation, dynamic light scattering, and gel filtration chromatography were used to determine the physical characteristics of each CaM-kinase II isoform. The sedimentation coefficient was determined by centrifugation of the preparations on a 5–24% sucrose gradient. The β isoform had the lowest sedimentation coefficient of 14.5 S, whereas the other three isoforms all had sedimentation coefficients of about 17 S (see Table I). Dynamic light scattering measures the diffusion coefficient of molecules, which is proportional to their hydrodynamic radius Rh, calculated through the Stokes-Einstein relationship. Using such an analysis, the D and Rh values were determined for each CaM-kinase II isoform. α has the smallest Rh of 9.22 ± 0.13 nm, β has an Rh of 12.73 ± 0.07 nm, γ has an Rh of 11.35 ± 0.12 nm, and δ has an Rh of 11.90 ± 0.17 nm. The 9.22 nm value for αCaM-kinase II is very similar to that reported by Bradshaw et al. (34Bradshaw J.M. Hudmon A. Schulman H. J. Biol. Chem. 2002; 277: 20991-20998Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), who used dynamic light scattering to determine the Rh of the αCaM-kinase II enzyme complex to be 9.1 nm. The rank order of these Rh values is the same as the rank order of the molecular weights of the subunits of each isoform (α < γ < δ < β). Similar results were found using gel filtration chromatography (data not shown), however, accurate estimates of Rh values were difficult because of the fact that these large complexes elute near th
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