Functional Implications of the Subunit Composition of Neuronal CaM Kinase II
1999; Elsevier BV; Volume: 274; Issue: 32 Linguagem: Inglês
10.1074/jbc.274.32.22713
ISSN1083-351X
AutoresLihi Brocke, Lillian W. Chiang, Paul D. Wagner, Howard Schulman,
Tópico(s)Ion channel regulation and function
ResumoThe assembly of 6–12 subunits of Ca2+/calmodulin-dependent kinase II (CaM kinase II) into holoenzymes is an important structural feature of the enzyme and its postulated role as a molecular detector of Ca2+ oscillations. Using single cell reverse transcriptase-polymerase chain reaction, we show that α- and β-CaM kinase II mRNAs are simultaneously present in the majority of hippocampal neurons examined and that co-assembly of their protein products into heteromers is therefore possible. The subunit composition of CaM kinase II holoenzymes was analyzed by immunoprecipitation with subunit-specific monoclonal antibodies. Rat forebrain CaM kinase II consists of heteromers composed of α and β subunits at a ratio of 2:1 and homomers composed of only α subunits. We examined the functional effect of the heteromeric assembly by analyzing the calmodulin dependence of autophosphorylation. Recombinant homomers of α- or β-CaM kinase II, as well as of alternatively spliced β isoforms, have distinct calmodulin dependences for autophosphorylation based on differences in their calmodulin affinities. Half-maximal autophosphorylation of α is achieved at 130 nmcalmodulin, while that for β occurs at 15 nm calmodulin. In CaM kinase II isolated from rat forebrain, however, the calmodulin dependence for autophosphorylation of the β subunits is shifted toward that of α homomers. This suggests that Thr287 in β subunits is phosphorylated by α subunits present in the same holoenzyme. Once autophosphorylated, β-CaM kinase II traps calmodulin by reducing the rate of calmodulin dissociation. The assembly of 6–12 subunits of Ca2+/calmodulin-dependent kinase II (CaM kinase II) into holoenzymes is an important structural feature of the enzyme and its postulated role as a molecular detector of Ca2+ oscillations. Using single cell reverse transcriptase-polymerase chain reaction, we show that α- and β-CaM kinase II mRNAs are simultaneously present in the majority of hippocampal neurons examined and that co-assembly of their protein products into heteromers is therefore possible. The subunit composition of CaM kinase II holoenzymes was analyzed by immunoprecipitation with subunit-specific monoclonal antibodies. Rat forebrain CaM kinase II consists of heteromers composed of α and β subunits at a ratio of 2:1 and homomers composed of only α subunits. We examined the functional effect of the heteromeric assembly by analyzing the calmodulin dependence of autophosphorylation. Recombinant homomers of α- or β-CaM kinase II, as well as of alternatively spliced β isoforms, have distinct calmodulin dependences for autophosphorylation based on differences in their calmodulin affinities. Half-maximal autophosphorylation of α is achieved at 130 nmcalmodulin, while that for β occurs at 15 nm calmodulin. In CaM kinase II isolated from rat forebrain, however, the calmodulin dependence for autophosphorylation of the β subunits is shifted toward that of α homomers. This suggests that Thr287 in β subunits is phosphorylated by α subunits present in the same holoenzyme. Once autophosphorylated, β-CaM kinase II traps calmodulin by reducing the rate of calmodulin dissociation. Multifunctional Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) is a major mediator of Ca2+ action whose activation and autophosphorylation appear to regulate numerous neuronal processes, including two forms of synaptic plasticity, long term potentiation and long term depression (1Bear M.F. Malenka R.C. Curr. Opin. Neurobiol. 1994; 4: 389-399Crossref PubMed Scopus (1059) Google Scholar, 2Mayford M. Wang J. Kandel E.R. O'Dell T.J. Cell. 1995; 81: 891-904Abstract Full Text PDF PubMed Scopus (452) Google Scholar). These two opposing changes in synaptic strength are differentially regulated by the frequency of stimulation of hippocampal neurons. Interestingly, CaM kinase II has been suggested to be a molecular detector of the Ca2+ spike frequency, based on its unique structural and regulatory properties (3De Koninck P. Schulman H. Science. 1998; 279: 227-230Crossref PubMed Scopus (1097) Google Scholar) (reviewed in Refs.4Hanson P.I. Schulman H. Annu. Rev. Biochem. 1992; 61: 559-601Crossref PubMed Scopus (666) Google Scholar and 5Schulman H. Hanson P.I. Meyer T. Cell Calcium. 1992; 13: 401-411Crossref PubMed Scopus (88) Google Scholar). The neuronal CaM kinase II consists of two major subunits of 52 and 60 kDa that are encoded by α- and β-CaM kinase II genes, respectively. Additional isoforms are generated by alternative splicing of these as well as of the ubiquitous γ- and δ-CaM kinase II genes (6Bennett M.K. Kennedy M.B. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1794-1798Crossref PubMed Scopus (216) Google Scholar, 7Brocke L. Srinivasan M. Schulman H. J. Neurosci. 1995; 15: 6797-6808Crossref PubMed Google Scholar, 8Nghiem P. Saati S.M. Martens C.L. Gardner P. Schulman H. J. Biol. Chem. 1993; 268: 5471-5479Abstract Full Text PDF PubMed Google Scholar, 9Edman C.F. Schulman H. Biochim. Biophys. Acta. 1994; 1221: 90-102Google Scholar, 10Tombes R.M. Krystal G.W. Biochim. Biophys. Acta. 1997; 1355: 281-292Crossref PubMed Scopus (64) Google Scholar). All of the subunits share a common organization of functional domains and of an ultrastructural arrangement into holoenzymes (4Hanson P.I. Schulman H. Annu. Rev. Biochem. 1992; 61: 559-601Crossref PubMed Scopus (666) Google Scholar). The C-terminal association domains of 6–12 subunits assemble into a central globular structure from which the N-terminal catalytic/regulatory domains extend radially like petals of a flower (11Kanaseki T. Ikeuchi Y. Sugiura H. Yamauchi T. J. Cell Biol. 1991; 115: 1049-1060Crossref PubMed Scopus (177) Google Scholar). This unusual structure positions the catalytic/regulatory domains for an intersubunit autophosphorylation that is dependent on the frequency of Ca2+ oscillations (3De Koninck P. Schulman H. Science. 1998; 279: 227-230Crossref PubMed Scopus (1097) Google Scholar, 12Hanson P.I. Meyer T. Stryer L. Schulman H. Neuron. 1994; 12: 943-956Abstract Full Text PDF PubMed Scopus (395) Google Scholar). Upon activation by Ca2+/calmodulin, Thr286 in the regulatory domain of α subunits (or Thr287 in β subunits) is rapidly phosphorylated. Autophosphorylation of Thr286 on α subunits has three consequences: (a) calmodulin remains bound to the phosphorylated subunit for extended periods of time even at low Ca2+concentrations (trapped state), because the autophosphorylation greatly reduces the calmodulin disassociation rate (13Meyer T. Hanson P.I. Stryer L. Schulman H. Science. 1992; 256: 1199-1201Crossref PubMed Scopus (518) Google Scholar); (b) autophosphorylated α and β subunits are rendered Ca2+/calmodulin-independent (autonomous) (reviewed in Ref.4Hanson P.I. Schulman H. Annu. Rev. Biochem. 1992; 61: 559-601Crossref PubMed Scopus (666) Google Scholar); and (c) the kinase attains an enhanced affinity for NMDA receptors in postsynaptic densities, a submembranous neuronal specialization (14Strack S. Colbran R.J. J. Biol. Chem. 1998; 273: 20689-20692Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 15Leonard S.A. Lim I.A. Hemsworth D.E. Horne M.C. Hell J.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3239-3244Crossref PubMed Scopus (336) Google Scholar, 16Shen K. Meyer T. Science. 1999; 284: 162-167Crossref PubMed Scopus (544) Google Scholar). Both autonomy and calmodulin trapping enable the phosphorylated kinase subunits to remain active beyond the limited duration of a Ca2+ spike. The multimeric structure of CaM kinase II is important for the regulation of the kinase by autophosphorylation, since the phosphorylation of Thr286/287 occurs by an intersubunit reaction within each holoenzyme (12Hanson P.I. Meyer T. Stryer L. Schulman H. Neuron. 1994; 12: 943-956Abstract Full Text PDF PubMed Scopus (395) Google Scholar, 17Kuret J. Schulman H. J. Biol. Chem. 1985; 260: 6427-6433Abstract Full Text PDF PubMed Google Scholar, 18Mukherji S. Soderling T.R. J. Biol. Chem. 1994; 269: 13744-13747Abstract Full Text PDF PubMed Google Scholar, 19Rich R.C. Schulman H. J. Biol. Chem. 1998; 273: 28434Abstract Full Text Full Text PDF Scopus (131) Google Scholar). The catalytic domain of one activated subunit in the holoenzyme phosphorylates Thr286/287 in the regulatory domain of a neighboring subunit that must also have calmodulin bound. Therefore, assembly into holoenzymes concentrates the subunits and positions them for autophosphorylation. The multimeric structure of CaM kinase II, the intersubunit phosphorylation of Thr286/287, and the resulting calmodulin trapping and autonomous activity are crucial elements that enable the kinase to act as a Ca2+ spike frequency detector (3De Koninck P. Schulman H. Science. 1998; 279: 227-230Crossref PubMed Scopus (1097) Google Scholar, 5Schulman H. Hanson P.I. Meyer T. Cell Calcium. 1992; 13: 401-411Crossref PubMed Scopus (88) Google Scholar,13Meyer T. Hanson P.I. Stryer L. Schulman H. Science. 1992; 256: 1199-1201Crossref PubMed Scopus (518) Google Scholar). Under conditions of limited free calmodulin, each Ca2+spike activates only a subset of CaM kinase II subunits in a holoenzyme, some of which become autophosphorylated and trap their bound calmodulin. If the interval between spikes is brief, subsequent Ca2+ spike will lead to binding of calmodulin to holoenzymes that still retain calmodulin from a previous spike, increasing the calmodulin occupancy and probability of neighboring subunits with calmodulin bound and therefore the probability of autophosphorylation. A threshold frequency is reached, beyond which autophosphorylation and calmodulin trapping become increasingly more efficient and the number of activated subunits increases. Although autophosphorylation is not necessary for activation of the kinasein vitro, it may be necessary for appropriate activation in response to brief repetitive stimulation in vivo. The essential nature of this autophosphorylation was nicely demonstrated with the finding that mice defective in autophosphorylation of Thr286 in α-CaM kinase II because of a point mutation that substituted an Ala286 for Thr286 do not exhibit long term potentiation, are defective in spatial learning, and have unstable hippocampal place cells (20Giese K.P. Fedorov N.B. Filipkowski R.K. Silva A.J. Science. 1998; 279: 870-873Crossref PubMed Scopus (899) Google Scholar,21Cho Y.H. Giese K.P. Tanila H. Silva A.J. Eichenbaum H. Science. 1998; 279: 867-869Crossref PubMed Scopus (160) Google Scholar). In light of these findings, the subunit composition of CaM kinase II holoenzymes becomes very significant. Since intersubunit phosphorylation of Thr286/287 involves two calmodulin-bound subunits, the kinetic properties of the neighboring subunits in the holoenzyme serving as kinase and as substrate in each reaction are important variables. The frequency dependence for activation of homomers of α or β isoforms, for example, is markedly different (3De Koninck P. Schulman H. Science. 1998; 279: 227-230Crossref PubMed Scopus (1097) Google Scholar). In an electron microscopic study, forebrain CaM kinase II holoenzymes were found to exist as homomeric α decamers and homomeric β octamers with no evidence of heteromers (11Kanaseki T. Ikeuchi Y. Sugiura H. Yamauchi T. J. Cell Biol. 1991; 115: 1049-1060Crossref PubMed Scopus (177) Google Scholar). However, other studies reported that α-specific monoclonal antibody (mAb) 1The abbreviations used are: mAb, monoclonal antibody; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-PCR; RU, resonance units; MLCK, myosin light chain kinase; PIPES, 1,4-piperazinediethanesulfonic acid co-immunoprecipitated some β-CaM kinase II together with α-CaM kinase II from either rat forebrain (22Bennett M.K. Erondu N.E. Kennedy M.B. J. Biol. Chem. 1983; 258: 12735-12744Abstract Full Text PDF PubMed Google Scholar) or rat cerebellum (23Miller S.G. Kennedy M.B. J. Biol. Chem. 1985; 260: 9039-9046Abstract Full Text PDF PubMed Google Scholar). Chicken forebrain CaM kinase II purified on an affinity column of α-specific antibody consisted of both α and β isoforms (24Liu N. Cooper N.G.F. J. Mol. Neurosci. 1995; 5: 193-206Crossref Scopus (5) Google Scholar). Co-expression of α and β cDNAs produced heteromers both in transfected CHO cells (25Yamauchi T. Ohsako S. Deguchi T. J. Biol. Chem. 1989; 264: 19108-19116Abstract Full Text PDF PubMed Google Scholar) and in infected Sf9 cells (18Mukherji S. Soderling T.R. J. Biol. Chem. 1994; 269: 13744-13747Abstract Full Text PDF PubMed Google Scholar). Co-assembly of CaM kinase II subunits will have functional consequences if α and β subunits differ in their calmodulin dependence, their rates of autophosphorylation, and/or their capability to trap calmodulin. Differences in orientation of catalytic/regulatory domains between homomeric and heteromeric assembly of association domains may also alter rates of intersubunit autophosphorylation. For example, half-maximal activation and autophosphorylation of recombinant β-CaM kinase II occurs at a 5- and 4-fold lower calmodulin level, respectively, than recombinant α-CaM kinase II (3De Koninck P. Schulman H. Science. 1998; 279: 227-230Crossref PubMed Scopus (1097) Google Scholar, 25Yamauchi T. Ohsako S. Deguchi T. J. Biol. Chem. 1989; 264: 19108-19116Abstract Full Text PDF PubMed Google Scholar). It is not known whether α and β isoforms differ in trapping of calmodulin, since this has been demonstrated for recombinant α-CaM kinase II (13Meyer T. Hanson P.I. Stryer L. Schulman H. Science. 1992; 256: 1199-1201Crossref PubMed Scopus (518) Google Scholar) but has not been assessed for β-CaM kinase II. To study the functional properties of CaM kinase II holoenzymes, we analyzed the subunit composition of CaM kinase II holoenzymes from different preparations by immunoprecipitation with subunit-specific mAbs. Our results indicate that CaM kinase II can be composed of α:β heteromers with variable subunit ratios in addition to homomers of either α or β subunits. We therefore compared the autophosphorylation of homomers and heteromers composed of α, β, and alternatively spliced β subunits. Indeed, the calmodulin dependence is modified based on the subunit composition. Finally, plasmon surface resonance studies showed that autophosphorylated β subunits trap calmodulin and would contribute to prolonging the active state of the enzyme. [γ-32P]ATP (5000 Ci/mmol) was purchased from ICN (Aurora, OH). Bovine brain calmodulin was obtained from Ocean Biologic (Edmonds, WA). CaM kinase II substrate peptide autocamtide-3 was synthesized and purified by David King (University of California, Berkeley). Forebrain CaM kinase II was extracted and purified essentially as described (26Schulman H. J. Cell Biol. 1984; 99: 11-19Crossref PubMed Scopus (135) Google Scholar). Recombinant CaM kinase II proteins were prepared by transient transfections of COS-7 cells with pSRα.BKS containing full-length cDNA for CaM kinase II isoforms (7Brocke L. Srinivasan M. Schulman H. J. Neurosci. 1995; 15: 6797-6808Crossref PubMed Google Scholar, 27Hanson P.I. Kapiloff M.S. Lou L.L. Rosenfeld M.G. Schulman H. Neuron. 1989; 3: 59-70Abstract Full Text PDF PubMed Scopus (238) Google Scholar, 28Srinivasan M. Edman C. Schulman H. J. Cell Biol. 1994; 126: 839-852Crossref PubMed Scopus (238) Google Scholar). Protein extracts of transfected COS-7 cells were prepared, and the recombinant CaM kinase II was purified as described (29Hanson P.I. Schulman H. J. Biol. Chem. 1992; 267: 17216-17224Abstract Full Text PDF PubMed Google Scholar). Myosin light chain kinase (MLCK) from gizzard smooth muscle was purified as described (30Adelstein R.S. Klee C.B. J. Biol. Chem. 1981; 256: 7501-7509Abstract Full Text PDF PubMed Google Scholar). In visualized rat hippocampal slice preparations, the single cell cytoplasms of CA1 pyramidal neurons, identified by morphology and electrophysiological recording, were aspirated though the recording pipette and subjected to RT-PCR analysis as described previously (31Chiang L.W. Schweizer F.E. Tsien R.W. Schulman H. Mol. Brain Res. 1994; 27: 183-188Crossref PubMed Scopus (27) Google Scholar). For each CaM kinase II isoform, two sets of nested PCR primers were designed. These primers span the variable domain and can be used to distinguish between alternatively spliced isoforms based upon the size of the PCR products. The outer and inner set of primers are as follows: α, base pairs 898–923 to 1342–1368 and 907–930 to 1146–1170 (32Lin C.R. Kapiloff M.S. Durgerian S. Tatemoto K. Russo A.F. Hanson P. Schulman H. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5962-5966Crossref PubMed Scopus (218) Google Scholar); β, base pairs 901–923 to 1561–1534 and 910–933 to 1338–1362 (6Bennett M.K. Kennedy M.B. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1794-1798Crossref PubMed Scopus (216) Google Scholar); γ, base pairs 901–926 to 1489–1515 and 910–933 to 1293–1317 (33Tobimatsu T. Kameshita I. Fujisawa H. J. Biol. Chem. 1988; 263: 16082-16086Abstract Full Text PDF PubMed Google Scholar); δ, base pairs 901–926 to 1444–1470 and 910–933 to 1248–1272 (34Tobimatsu T. Fujisawa H. J. Biol. Chem. 1989; 264: 17907-17912Abstract Full Text PDF PubMed Google Scholar). The first round of amplification included all four outer primer pairs, for α, β, γ, and δ isoforms. The entire cDNA produced from a single cytosol was subjected to 30 cycles of capillary PCR (Idaho Technologies, Idaho Falls, ID). The product amplified in the first round was diluted 100-fold into a second round of amplification involving separate capillary PCR reactions for each of the four isoforms using the inner primer pair. PCR products were separated on 2% agarose gel and visualized by ethidium bromide staining. CBα2 and CBβ1 are subunit-specific mAbs for α- and β-CaM kinase II, respectively (35Baitinger C. Alderton J. Poenie M. Schulman H. Steinhardt R.A. J. Cell Biol. 1990; 111: 1763-1773Crossref PubMed Scopus (139) Google Scholar). All immunoprecipitations were carried out in radioimmune precipitation buffer (50 mm Tris/HCl, pH 8, 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) containing 10 μg/ml leupeptin. Approximately 100 ng of purified CaM kinase II was incubated with the mAbs indicated in the text in a final volume of 0.5 ml for 1 h at 4 °C. Both CBα2 and CBβ1 mAbs were diluted 1:2000. Subsequently, 40 μl of protein A beads (Repligen, Cambridge, MA) were added, and the incubation was continued for another 1 h at 4 °C. The beads were centrifuged and the supernatant was transferred to a fresh tube. A second, similar round of immunoprecipitation was initiated by the addition of mAb to the supernatant of the first round, followed by protein A beads as described above. The beads were washed three times with cold PBS containing 0.25% Nonidet P-40. After the final wash, the beads were resuspended in SDS-containing sample buffer and heated at 85 °C for 4 min, and the supernatants were loaded on 10% SDS-polyacrylamide gels. Proteins were transferred to a nitrocellulose membrane (0.45 μm; Schleicher & Shuell), and CaM kinase II isoforms were visualized by calmodulin overlay assay using biotinylated calmodulin and enhanced chemiluminescence (8Nghiem P. Saati S.M. Martens C.L. Gardner P. Schulman H. J. Biol. Chem. 1993; 268: 5471-5479Abstract Full Text PDF PubMed Google Scholar). The film was scanned and analyzed for the relative intensities of the bands using MacBas software (FUJIX, Stamford, CT). Purified CaM kinase II aliquots were thawed just before use and diluted in ice-cold buffer containing 50 mm PIPES, pH 7.0, 1 mm EGTA, 150 mm NaCl, 0.05% Tween 20. Autophosphorylation reactions were started with the addition of 5 μl of diluted kinase to a final concentration of 5–10 nm calmodulin binding sites into prewarmed reaction tubes. In addition to the kinase, each reaction tube (25 μl) also contained 50 mm PIPES, pH 7.0, 10 mm MgCl2, 0.5 mm CaCl2, 0.1 mg/ml BSA, 200 μm [γ-32P]ATP (2 Ci/mmol), and variable amounts of calmodulin (0–400 nm). After incubation for 15 s at 30 °C, the reactions were stopped by the addition of 12.5 μl of SDS-containing sample buffer. The samples were heated at 85 °C for 4 min and loaded in their entirety onto 10% SDS-polyacrylamide gels (1.5 mm, Novex, San Diego, CA). After electrophoresis, the gels were incubated for 30 min in Coomassie stain and destained overnight for removal of unincorporated radiolabeled ATP by diffusion. The gels were dried and exposed to a phosphor-imaging plate at room temperature for 24 h. Gray scale images of the autophosphorylated kinase were obtained using the BAS2000 system (FUJIX), and band intensities were analyzed using MacBas software (FUJIX). The extent of CaM kinase II autophosphorylation at each calmodulin concentration was calculated as a percentage of the maximum intensity value obtained for each isoform at 200–400 nmcalmodulin. It is important to note that the level of autophosphorylation of a given CaM kinase II isoform at intermediate calmodulin concentration was dependent on the reaction time. CaM kinase II activity assays were performed in identical conditions as the autophosphorylation assays, except 20 μm autocamtide-3 was added. After incubation at 30 °C for 15 s, the reactions were stopped with the addition of ice cold trichloroacetic acid to a final concentration of 5%. The reactions were processed as described (7Brocke L. Srinivasan M. Schulman H. J. Neurosci. 1995; 15: 6797-6808Crossref PubMed Google Scholar) and counted. The calmodulin dependence of each isoform according to the autophosphorylation and activity assays was calculated and plotted as described (9Edman C.F. Schulman H. Biochim. Biophys. Acta. 1994; 1221: 90-102Google Scholar). The interaction between immobilized calmodulin and several CaM kinase II isoforms was measured using a BIAcore instrument (Amersham Pharmacia Biotech). Calmodulin was covalently coupled to a CM5 sensor chip (Amersham Pharmacia Biotech) using the amide reaction kit according to instructions provided by the manufacturer. Briefly, the immobilization procedure was carried out at 25 °C in a buffer containing 10 mm HEPES, pH 7.4, 150 mm NaCl, 3.4 mm EGTA, 0.05% surfactant P20 (Amersham Pharmacia Biotech). Following activation withN-hydroxysuccinimide andN-ethyl-N-(3-diethylaminopropyl) carbodiimide, a solution containing 25 μg/ml calmodulin in 10 mm sodium acetate, pH 3.7, was injected into the flow cell, followed by ethanolamine wash. At the end of the immobilization procedure, the temperature was set at 10 °C, since CaM kinase II is a temperature-sensitive enzyme. All of the binding experiments were performed in flow cells containing 90–150 resonance units (RU) of immobilized calmodulin (equivalent to about 0.09–0.15 ng of protein/mm2). Kinase binding experiments were performed in buffer A (20 mm PIPES, pH 7.0, 5 mmMgCl2, 150 mm NaCl, 0.4 mmCaCl2, 0.05% surfactant P20) at a flow rate of 5 μl/min. The co-inject feature of the machine was set to inject 50 μl of purified recombinant CaM kinase II (100–400 ng) diluted in buffer A, followed by 15 μl of 500 nm free calmodulin in buffer A. The free calmodulin in this first dissociation phase should decrease the rebinding of dissociating CaM kinase II to the chip (36Edlund M. Blikstad I. Obrink B. J. Biol. Chem. 1996; 271: 1393-1399Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). In some experiments, we included 200 μm ATP during the first dissociation phase to create autophosphorylation conditions. Next, we started the second dissociation phase by the injection of a low Ca2+ buffer, containing 20 mm PIPES, pH 7.0, 5 mm MgCl2, 150 mm NaCl, 2 mm EGTA, 0.05% surfactant P-20 and calculated amounts of CaCl2 to yield free Ca2+ concentrations of 50, 100, 200, or 300 nm. The remaining bound kinase was later washed away by a regeneration solution containing 2 mmEGTA, 1 m NaCl. The RU readings usually returned to a level of 20–40 RU above base line. Each flow cell was used for total of 3–5 cycles of CaM kinase II binding. Although α and β subunits have been reported to reside on separate homomers in rat brain (11Kanaseki T. Ikeuchi Y. Sugiura H. Yamauchi T. J. Cell Biol. 1991; 115: 1049-1060Crossref PubMed Scopus (177) Google Scholar), in vitroco-transfections of α and β cDNAs into mammalian cells resulted in heteromeric CaM kinase II (18Mukherji S. Soderling T.R. J. Biol. Chem. 1994; 269: 13744-13747Abstract Full Text PDF PubMed Google Scholar, 25Yamauchi T. Ohsako S. Deguchi T. J. Biol. Chem. 1989; 264: 19108-19116Abstract Full Text PDF PubMed Google Scholar). In light of these conflicting data, we examined the expression pattern of CaM kinase II isoforms in individual hippocampal neurons by single cell RT-PCR to determine whether CaM kinase II mRNAs are co-expressed in vivo. PCR primers that span the variable domain of each of the four CaM kinase II subunits were used in order to identify alternatively spliced CaM kinase II isoforms with different insertions and deletions in their variable domain. As shown in Fig. 1, multiple CaM kinase II isoforms were detected in rat brain RNA: two α isoforms (αB and α) (7Brocke L. Srinivasan M. Schulman H. J. Neurosci. 1995; 15: 6797-6808Crossref PubMed Google Scholar), four β isoforms (β, β′, βe, β′e) (7Brocke L. Srinivasan M. Schulman H. J. Neurosci. 1995; 15: 6797-6808Crossref PubMed Google Scholar); see Fig. 5 a), six γ isoforms (8Nghiem P. Saati S.M. Martens C.L. Gardner P. Schulman H. J. Biol. Chem. 1993; 268: 5471-5479Abstract Full Text PDF PubMed Google Scholar, 10Tombes R.M. Krystal G.W. Biochim. Biophys. Acta. 1997; 1355: 281-292Crossref PubMed Scopus (64) Google Scholar), and three δ isoforms (δA, δB, δC) (9Edman C.F. Schulman H. Biochim. Biophys. Acta. 1994; 1221: 90-102Google Scholar). We next studied the expression of CaM kinase II isoforms in single neurons. The expression pattern of CaM kinase II mRNAs in all the single neurons tested is summarized in Table I. In all of the individual neurons tested, mRNAs were detected for either α- or β-CaM kinase II but not for γ- or δ-CaM kinase II (Fig. 1 and Table I). Neuron PP18 and PP1 in Fig. 1 represent expression patterns A and F, respectively. In some single neurons, only α-CaM kinase mRNA was detected (neuron PP1), while in others we detected both α- and β-CaM kinase mRNAs (neuron PP18). More than 90% of the neurons expressed a detectable amount of α mRNA. The alternatively spliced isoform, αB, that contains a 33-base pair insertion and therefore appears as a slightly larger band than α-CaM kinase II (7Brocke L. Srinivasan M. Schulman H. J. Neurosci. 1995; 15: 6797-6808Crossref PubMed Google Scholar) was detected in only one of the neurons. We detected alternatively spliced β isoforms in 58% of the neurons (14/24); nine cells expressed the β- isoform, three expressed βe, and two expressed β′ (Table I). Thus, while some of the neurons contain little or no β and are likely to express predominantly α-CaM kinase II homomers, many cells express both α- and β-CaM kinase II and have the potential for forming α:β heteromers.Figure 5Calmodulin dependence of autophosphorylation in alternatively spliced CaM kinase II isoforms. a, schematic representation of alternatively spliced β-CaM kinase II isoforms. The regulatory domain shared by all β isoforms is composed of exons XI and X. The variable domain is composed of four small exons, IX–VI (54Karls U. Muller U. Gilbert D.J. Copeland N.G. Jenkins N.A. Harbers K. Mol. Cell. Biol. 1992; 12: 3644-3652Crossref PubMed Google Scholar). β-CaM kinase II contains all four exons, while β′, βe, and β′e each lack the indicated exon(s) (dashed lines). b, purified, recombinant preparations of α (●), β (▪), β′ (■) (n = 3,), βe (⋄, n = 3) and β′e (▵, n = 3) were autophosphorylated at increasing concentrations of calmodulin, and phosphate incorporation was quantified as described under "Experimental Procedures."View Large Image Figure ViewerDownload (PPT)Table IEight expression pattern groups of α- and β-CaM kinase II isoforms in single neuronsGene expression pattern (PCR signal)No. of single neurons with each patternααBββ′βeβ′eA+−+−−−7B+−−−+−2C+−+−+−1D+++−−−1E+−−+−−1F+−−−−−10G−−−+−−1H−−−−+−1Total22/241/249/242/244/240/2424The expression of the six neuronal CaM kinase II isoforms can be grouped into eight expression pattern groups, designated A to H, with the number of neurons in each group indicated on the right. Open table in a new tab The expression of the six neuronal CaM kinase II isoforms can be grouped into eight expression pattern groups, designated A to H, with the number of neurons in each group indicated on the right. We analyzed the subunit composition of forebrain CaM kinase II preparation using subunit-specific mAbs in order to assess whether α and β subunits assemble as homomers or heteromers. The specificity of the mAbs under our immunoprecipitation conditions was first verified with purified, recombinant α and β homomers. α-mAb (CBα2) immunoprecipitated α protein but not β protein, whereas β-mAb (CBβ1) immunoprecipitated only β protein (Fig.2 a). Furthermore, mixing the two recombinant homomeric isoforms before the addition of the mAbs did not lead to subunit exchange or nonspecific immunoprecipitation, since each antibody precipitated only its cognate subunit (Fig.2 b, lanes 1 and 3). This control experiment demonstrates that successive use of the antibodies can clearly separate the two subunits when each isoform resides on a distinct homomeric holoenzyme. Purified rat forebrain CaM kinase II preparation was subjected to immunoprecipitation with the subunit-specific mAbs. The α-mAb immunoprecipitated both α and β, with a subunit ratio of 3:1 (Fig.3 a, lanes 1 and 3), consistent with previous estimations (22Bennett M.K.
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