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• W1989361508 abstract "Autophosphorylation of Thr286in Ca2+/calmodulin-dependent protein kinase II occurs within each holoenzyme by an intersubunit reaction and is essential for kinase function in vivo. In addition to a kinase-directed function of calmodulin to activate the kinase, a second calmodulin is required for the autophosphorylation of each Thr286 (Hanson, P. I., Meyer, T., Stryer, L., and Schulman, H. (1994) Neuron 12, 943–956). We have engineered heteromeric holoenzymes comprising distinct “kinase” and “substrate” subunits to test for kinase- and substrate-directed functions of calmodulin. The obligate kinase subunits have aspartate residues substituted for threonine at positions 286, 305, and 306 (the autophosphorylation and calmodulin-binding sites), making it constitutively active but unable to bind calmodulin. Obligate substrate subunits are catalytically inactive (K42M mutation) but are able to bind calmodulin. Phosphorylation of substrate subunits occurs specifically at Thr286 and is completely dependent upon the presence of calmodulin. Blocking the ability of the substrate subunit to bind calmodulin, either with inhibitor KN-93 or by mutagenesis of the calmodulin-binding domain of the substrate subunit, prevents its phosphorylation, consistent with a substrate-directed function of calmodulin that requires its direct binding to the subunit being phosphorylated. Autophosphorylation of Thr286in Ca2+/calmodulin-dependent protein kinase II occurs within each holoenzyme by an intersubunit reaction and is essential for kinase function in vivo. In addition to a kinase-directed function of calmodulin to activate the kinase, a second calmodulin is required for the autophosphorylation of each Thr286 (Hanson, P. I., Meyer, T., Stryer, L., and Schulman, H. (1994) Neuron 12, 943–956). We have engineered heteromeric holoenzymes comprising distinct “kinase” and “substrate” subunits to test for kinase- and substrate-directed functions of calmodulin. The obligate kinase subunits have aspartate residues substituted for threonine at positions 286, 305, and 306 (the autophosphorylation and calmodulin-binding sites), making it constitutively active but unable to bind calmodulin. Obligate substrate subunits are catalytically inactive (K42M mutation) but are able to bind calmodulin. Phosphorylation of substrate subunits occurs specifically at Thr286 and is completely dependent upon the presence of calmodulin. Blocking the ability of the substrate subunit to bind calmodulin, either with inhibitor KN-93 or by mutagenesis of the calmodulin-binding domain of the substrate subunit, prevents its phosphorylation, consistent with a substrate-directed function of calmodulin that requires its direct binding to the subunit being phosphorylated. Ca2+/calmodulin-dependent protein kinase II bovine serum albumin 1,4-piperazinediethanesulfonic acid. Ca2+/calmodulin-dependent protein kinase II (CaM kinase II)1 regulates a wide variety of neuronal processes, including neurotransmitter synthesis and secretion, carbohydrate metabolism, receptor and ion channel function, and gene expression by phosphorylation of critical enzymes and proteins (reviewed in Ref. 1Braun A.P. Schulman H. Annu. Rev. Physiol. 1995; 57: 417-445Crossref PubMed Scopus (738) Google Scholar). It does so in response to increases in intracellular Ca2+ levels initiated by diverse signal transduction pathways. Accordingly, the kinase is both highly abundant in brain and distributed in diverse subcellular compartments (reviewed in Ref. 2Hanson P.I. Schulman H. Annu. Rev. Biochem. 1992; 61: 559-601Crossref PubMed Scopus (664) Google Scholar). Four genes (α, β, γ, and δ) give rise to many related CaM kinase II isoforms, with the α and β isoforms predominating in brain (1Braun A.P. Schulman H. Annu. Rev. Physiol. 1995; 57: 417-445Crossref PubMed Scopus (738) Google Scholar). Each isoform has an amino-terminal catalytic domain, a central regulatory domain containing autoinhibitory and calmodulin binding segments, and an association domain at its carboxyl terminus. Interactions between association domains of individual subunits allow 6–12 subunits to form a multimeric holoenzyme. Electron microscopy suggests that the catalytic and regulatory domains radiate out from a central core of assembled association domains in a flower-petal-like arrangement (3Kanaseki T. Ikeuchi Y. Sugiura H. Yamauchi T. J. Cell Biol. 1991; 115: 1049-1060Crossref PubMed Scopus (175) Google Scholar). CaM kinase II can undergo distinct steps of autophosphorylation at Thr286 within the autoinhibitory domain and at Thr305 and Thr306 in the calmodulin-binding site. Activation of CaM kinase II in the presence of Ca2+/calmodulin results in rapid autophosphorylation at Thr286 (4Miller S.G. Patton B.L. Kennedy M.B. Neuron. 1988; 1: 593-604Abstract Full Text PDF PubMed Scopus (228) Google Scholar, 5Schworer C.M. Colbran R.J. Keefer J.R. Soderling T.R. J. Biol. Chem. 1988; 263: 13486-13489Abstract Full Text PDF PubMed Google Scholar, 6Thiel G. Czernik A.J. Gorelick F. Nairn A.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6337-6341Crossref PubMed Scopus (142) Google Scholar). Subsequent dissociation of calmodulin exposes Thr305 and Thr306 for autophosphorylation, which prevents rebinding of Ca2+/calmodulin (7Colbran R.J. Soderling T.R. J. Biol. Chem. 1990; 265: 11213-11219Abstract Full Text PDF PubMed Google Scholar, 8Hanson P.I. Schulman H. J. Biol. Chem. 1992; 267: 17216-17224Abstract Full Text PDF PubMed Google Scholar, 9Hashimoto Y. Schworer C.M. Colbran R.J. Soderling T.R. J. Biol. Chem. 1987; 262: 8051-8055Abstract Full Text PDF PubMed Google Scholar, 10Patton B.L. Miller S.G. Kennedy M.B. J. Biol. Chem. 1990; 265: 11204-11212Abstract Full Text PDF PubMed Google Scholar). Autophosphorylation of Thr286 has significant consequences for some neuronal functions in vivo, as demonstrated with mice in which Thr286 of the endogenous kinase is mutated to Ala, preventing autophosphorylation at this position without disturbing kinase expression or its catalytic site (11Cho Y.H. Giese K.P. Tanila H. Silva A.J. Eichenbaum H. Science. 1998; 279: 867-869Crossref PubMed Scopus (160) Google Scholar, 12Giese K.P. Fedorov N.B. Filipkowski R.K. Silva A.J. Science. 1998; 279: 870-873Crossref PubMed Scopus (891) Google Scholar). The mutant mice are impaired in spatial learning tests requiring the hippocampus and long term potentiation in hippocampal slices cannot be induced (12Giese K.P. Fedorov N.B. Filipkowski R.K. Silva A.J. Science. 1998; 279: 870-873Crossref PubMed Scopus (891) Google Scholar). Conversion of CaM kinase II to its autonomous form is mimicked in mutant T286D-CaM kinase II (13Fong Y.L. Taylor W.L. Means A.R. Soderling T.R. J. Biol. Chem. 1989; 264: 16759-16763Abstract Full Text PDF PubMed Google Scholar, 14Waldmann R. Hanson P.I. Schulman H. Biochemistry. 1990; 29: 1679-1684Crossref PubMed Scopus (102) Google Scholar). Consequently, long term potentiation and spatial learning are altered in transgenic mice expressing T286D mutant kinase (15Bach M.E. Hawkins R.D. Osman M. Kandel E.R. Mayford M. Cell. 1995; 81: 905-915Abstract Full Text PDF PubMed Scopus (386) Google Scholar, 16Mayford M. Wang J. Kandel E.R. O'Dell T.J. Cell. 1995; 81: 891-904Abstract Full Text PDF PubMed Scopus (449) Google Scholar, 17Mayford M. Bach M.E. Huang Y.Y. Wang L. Hawkins R.D. Kandel E.R. Science. 1996; 274: 1678-1683Crossref PubMed Scopus (1110) Google Scholar, 18Rotenberg A. Mayford M. Hawkins R.D. Kandel E.R. Muller R.U. Cell. 1996; 87: 1351-1361Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). There are several possible biochemical consequences of Thr286 phosphorylation. Phospho-Thr286-bearing subunits have a markedly reduced calmodulin off-rate and thereby “trap” calmodulin, perhaps providing a mechanism for CaM kinase II to sequester calmodulin from other enzymes and proteins while prolonging its activated state (19Meyer T. Hanson P.I. Stryer L. Schulman H. Science. 1992; 256: 1199-1202Crossref PubMed Scopus (514) Google Scholar). Even after calmodulin dissociates, the presence of phospho-Thr286 disables the autoinhibitory domain, leaving the kinase 50–70% Ca2+-independent or autonomous (20Lai Y. Nairn A.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4253-4257Crossref PubMed Scopus (216) Google Scholar, 21Lou L.L. Lloyd S.J. Schulman H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9497-9501Crossref PubMed Scopus (136) Google Scholar, 22Miller S.G. Kennedy M.B. Cell. 1986; 44: 861-870Abstract Full Text PDF PubMed Scopus (641) Google Scholar, 23Schworer C.M. Colbran R.J. Soderling T.R. J. Biol. Chem. 1986; 261: 8581-8584Abstract Full Text PDF PubMed Google Scholar). Finally, autophosphorylation makes activation of the kinase sensitive to the frequency of Ca2+ oscillations (24De Koninck P. Schulman H. Science. 1998; 279: 227-230Crossref PubMed Scopus (1088) Google Scholar). During brief Ca2+ transients and with submaximal calmodulin levels, significant occupancy of calmodulin-binding sites and high activation only occur as the frequency of stimulation exceeds a threshold that achieves autophosphorylation (24De Koninck P. Schulman H. Science. 1998; 279: 227-230Crossref PubMed Scopus (1088) Google Scholar). Calmodulin trapping, autonomous activity, and frequency-dependent activation all require the autophosphorylation of Thr286. Given both its mechanistic and functional consequences, it is crucial to understand the mechanism underlying this phenomenon. Autophosphorylation of Thr286 is associated with the multimeric structure of CaM kinase II, yet activation by bound calmodulin is an inherent property of individual subunits, with each subunit binding one molecule of calmodulin (19Meyer T. Hanson P.I. Stryer L. Schulman H. Science. 1992; 256: 1199-1202Crossref PubMed Scopus (514) Google Scholar, 25Katoh T. Fujisawa H. Biochim. Biophys. Acta. 1991; 1091: 205-212Crossref PubMed Scopus (22) Google Scholar). Autophosphorylation of its critical autonomy site is an intraholoenzyme reaction (20Lai Y. Nairn A.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4253-4257Crossref PubMed Scopus (216) Google Scholar, 26Kuret J. Schulman H. J. Biol. Chem. 1985; 260: 6427-6433Abstract Full Text PDF PubMed Google Scholar), which occurs via an intersubunit mechanism (27Hanson P.I. Meyer T. Stryer L. Schulman H. Neuron. 1994; 12: 943-956Abstract Full Text PDF PubMed Scopus (393) Google Scholar, 28Mukherji S. Soderling T.R. J. Biol. Chem. 1994; 269: 13744-13747Abstract Full Text PDF PubMed Google Scholar). Calmodulin exhibits cooperativity in calmodulin trapping and autophosphorylation (24De Koninck P. Schulman H. Science. 1998; 279: 227-230Crossref PubMed Scopus (1088) Google Scholar, 27Hanson P.I. Meyer T. Stryer L. Schulman H. Neuron. 1994; 12: 943-956Abstract Full Text PDF PubMed Scopus (393) Google Scholar, 29Le Vine H.d. Sahyoun N.E. Cuatrecasas P. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2253-2257Crossref PubMed Scopus (22) Google Scholar), suggesting that calmodulin serves a second function in addition to activation. This is consistent with the finding that Thr286 is not constitutively autophosphorylated in holoenzymes in which some or all of the subunits are mutated to be constitutively active (27Hanson P.I. Meyer T. Stryer L. Schulman H. Neuron. 1994; 12: 943-956Abstract Full Text PDF PubMed Scopus (393) Google Scholar, 30Brickey D.A. Bann J.G. Fong Y.L. Perrino L. Brennan R.G. Soderling T.R. J. Biol. Chem. 1994; 269: 29047-29054Abstract Full Text PDF PubMed Google Scholar). The simplest mechanism to explain these findings is for one molecule of calmodulin to bind for activation of a “kinase” subunit (a kinase-directed action) coincident with a second molecule of calmodulin that must be bound to the “substrate” subunit (a substrate-directed function; Ref. 31Hawley S.A. Selbert M.A. Goldstein E.G. Edelman A.M. Carling D. Hardie D.G. J. Biol. Chem. 1995; 270: 27186-27191Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar), e.g. to expose Thr286 for autophosphorylation (27Hanson P.I. Meyer T. Stryer L. Schulman H. Neuron. 1994; 12: 943-956Abstract Full Text PDF PubMed Scopus (393) Google Scholar). However, evidence supporting such a requirement for direct binding of calmodulin to the substrate subunit during autophosphorylation of CaM kinase II holoenzymes has not been demonstrated. In this report, we present such evidence using CaM kinase II heteromultimers in which subunits are mutated to assume obligatory roles, either as a kinase or a substrate subunit. Occupation by calmodulin on each of the two subunits participating in CaM kinase II autophosphorylation explains the cooperative effect of calmodulin (25Katoh T. Fujisawa H. Biochim. Biophys. Acta. 1991; 1091: 205-212Crossref PubMed Scopus (22) Google Scholar,27Hanson P.I. Meyer T. Stryer L. Schulman H. Neuron. 1994; 12: 943-956Abstract Full Text PDF PubMed Scopus (393) Google Scholar), and may underlie the high sensitivity of CaM kinase II to changes in Ca2+/calmodulin concentrations and frequencies of Ca2+ oscillations (24De Koninck P. Schulman H. Science. 1998; 279: 227-230Crossref PubMed Scopus (1088) Google Scholar). Porcine brain calmodulin was purchased from Ocean Biologics (Edmonds, WA). Both KN-93 and immobilized calmodulin were obtained from Calbiochem. CaM kinase II substrate peptide autocamtide-3 and anti-α-CaM kinase II monoclonal CB-α2 antibody were purchased from Life Technologies, Inc. Restriction enzymes and other DNA-modifying enzymes were purchased through New England Biolabs or Life Technologies, Inc. The supplier for [γ-32P]ATP (6000 Ci/mmol) was NEN Life Science Products. The plasmid used for expression of wild type and mutant α-CaM kinase II constructs was the pCD derivative SRα (32Takebe Y. Seiki M. Fujisawa J. Hoy P. Yokota K. Arai K. Yoshida M. Arai N. Mol. Cell. Biol. 1988; 8: 466-472Crossref PubMed Google Scholar). Plasmids encoding wild type, K42M mutant, K42M/T286A double mutant, and tag-CaM kinase II (in which an 18-amino acid sequence from the influenza hemagglutinin HA1 protein was inserted by site-directed mutagenesis into CaM kinase II between Thr3 and Ile4 to generate a “tagged” form of the enzyme) were those as described previously (27Hanson P.I. Meyer T. Stryer L. Schulman H. Neuron. 1994; 12: 943-956Abstract Full Text PDF PubMed Scopus (393) Google Scholar). Three additional constructs in SRα, used as parent plasmids for subcloning to create mutant CaM kinase II constructs described herein, were T(286/305/306)D-αB-CaM kinase II, T(305/306)D-αB-CaM kinase II, and K42M/C289P-αB-CaM kinase II, which were engineered by and obtained from M. Srinivasan and H. Schulman. 2M. Srinivasan and H. Schulman, manuscript in preparation. These αB isoforms have an 11-amino acid insert between Lys328 and Glu329 containing a nuclear targeting signal (33Srinivasan M. Edman C.F. Schulman H. J. Cell Biol. 1994; 126: 839-852Crossref PubMed Scopus (238) Google Scholar). This insert was removed from each construct by subcloning the 985-base pair fragment between the two AccIII sites into SRα-α-CaM kinase II, thus creating plasmids containing T(286/305/306)D triple mutant or T(305/306)D or K42M/C289P double mutant kinases. To form tag-T(286/305/306)D mutant, thePstI/EagI fragment from SRα-tag-CaM kinase II was subcloned into SRα-T(286/305/306)D-CaM kinase II. The calcium phosphate precipitation method of Chen and Okayama (34Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4824) Google Scholar) was used to transiently transfect or cotransfect COS-7 cells with 15–20 μg of total DNA/10-cm plate as described previously (35Hanson 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) using the modifications for harvesting cell lysates described by Brocke et al. (36Brocke L. Srinivasan M. Schulman H. J. Neurosci. 1995; 15: 6797-6808Crossref PubMed Google Scholar). Purification of wild type and mutant kinase was performed using a two-column procedure (phosphocellulose followed by calmodulin-agarose) as described (27Hanson P.I. Meyer T. Stryer L. Schulman H. Neuron. 1994; 12: 943-956Abstract Full Text PDF PubMed Scopus (393) Google Scholar) based on an original three column purification procedure (8Hanson P.I. Schulman H. J. Biol. Chem. 1992; 267: 17216-17224Abstract Full Text PDF PubMed Google Scholar). All phosphorylation reactions were conducted on ice using 50-μl reaction volumes. A primary reason for performing reactions at this temperature is that it effectively limits autophosphorylation to Thr286in CaM kinase II when used in conjunction with the other reaction conditions as described (37Lou L.L. Schulman H. J. Neurosci. 1989; 9: 2020-2032Crossref PubMed Google Scholar, 38Ikeda A. Okuno S. Fujisawa H. J. Biol. Chem. 1991; 266: 11582-11588Abstract Full Text PDF PubMed Google Scholar). Kinase activity was assayed for 1 min and contained at final concentrations 50 mm PIPES (pH 7.0), 10 mmMgCl2, 0.1 mg/ml bovine serum albumin (BSA), 20 μm autocamtide-3, 25 μm[γ-32P]ATP (1 Ci/mmol), with either 0.5 mmCaCl2 plus 10 μg/ml calmodulin (for Ca2+-stimulated activity) or else 0.5 mm EGTA (for Ca2+-independent activity). Reactions were initiated by addition of enzymes, stopped by adding 10 μl of 30% trichloroacetic acid, and 32P incorporation into peptide was quantified as described (14Waldmann R. Hanson P.I. Schulman H. Biochemistry. 1990; 29: 1679-1684Crossref PubMed Scopus (102) Google Scholar) using Cerenkov radiation at an efficiency of 52%. Autophosphorylation reactions were performed for 15 s and, unless otherwise indicated, contained 50 mm PIPES (pH 7.0), 10 mm MgCl2, 0.1 mg/ml BSA, 200 μm[γ-32P]ATP (1–4 Ci/mmol), with either 0.5 mm CaCl2 plus 10 μg/ml calmodulin (for Ca2+-stimulated autophosphorylation) or else 0.5 mm EGTA (for Ca2+-independent autophosphorylation). Reactions were started by addition of enzymes and terminated with the addition of 25 μl of SDS electrophoresis sample buffer. Due to variability between wild type and mutant CaM kinase II expression in COS cells, equivalent total kinase activity in COS lysates was achieved by diluting lysates expressing higher levels of kinase activity with mock-transfected COS lysate. Routinely, 5 μl of COS lysate was used in reactions. Dilution buffer for purified kinases consisted of 2 mm PIPES (pH 7.0) with 0.1 mg/ml BSA, and these dilutions were adjusted to ensure comparable total activity between wild type and mutant CaM kinase II in phosphorylation reactions. Proteins were separated by electrophoresis on 9% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Schleicher & Schüll). CaM kinase II subunits on membranes were visualized by using calmodulin overlay assay, autoradiography (for 32P-labeled samples), or immunodetection. The calmodulin overlay assay was performed with biotinylated calmodulin and enhanced chemiluminescence as described (39Nghiem P. Saati S.M. Martens C.L. Gardner P. Schulman H. J. Biol. Chem. 1993; 268: 5471-5479Abstract Full Text PDF PubMed Google Scholar). Immunodetection specific for α-CaM kinase II was conducted with anti-CaM kinase II monoclonal CB-α2 antibody as described (40Scholz W.K. Baitinger C. Schulman H. Kelly P.T. J. Neurosci. 1988; 8: 1039-1051Crossref PubMed Google Scholar). For purified kinases, autophosphorylated as described above except that nonradioactive ATP was used in place of [γ-32P]ATP, immunodetection was also performed with anti-phospho-Thr286CaM kinase II rabbit polyclonal antisera generously supplied from D. D. Ginty. This antisera selectively recognizes CaM kinase II phosphorylated at Thr286. Immunoblots were prepared by blocking membranes in buffer A (20 mm Tris-HCl, pH 7.4, 0.9% (w/v) NaCl, 0.1% (v/v) Tween 20) plus 5% (w/v) BSA for 1 h at room temperature, and then incubating in primary antibody diluted 1:500 in buffer A plus 1% BSA for 2.5 h. Bound antibodies were detected by incubation for 1 h with donkey anti-rabbit IgG conjugated to horseradish peroxidase (Amersham Pharmacia Biotech) diluted 1:2500 in buffer A plus 1% BSA and visualized using the Enhanced Chemiluminescence detection kit and film from Amersham Pharmacia Biotech following the manufacturer's directions. We engineered heteromultimers of CaM kinase II composed of subunits that can function as either kinase or substrate subunits in order to test for obligatory binding of calmodulin to the substrate subunit during autophosphorylation. The requisite feature of the kinase subunit is that it be unable to bind calmodulin while being active,i.e. that it be constitutively active. These features are those of wild type CaM kinase II that is autophosphorylated at Thr286 (which makes it autonomous of Ca2+/calmodulin) and at Thr305 and Thr306 (which disables calmodulin binding; Refs. 8Hanson P.I. Schulman H. J. Biol. Chem. 1992; 267: 17216-17224Abstract Full Text PDF PubMed Google Scholar and 27Hanson P.I. Meyer T. Stryer L. Schulman H. Neuron. 1994; 12: 943-956Abstract Full Text PDF PubMed Scopus (393) Google Scholar), which can be mimicked by engineering aspartate in place of the three threonine residues. To distinguish these subunits immunologically and on SDS-polyacrylamide gels, we epitope-tagged the kinase subunits near the amino terminus (see “Experimental Procedures”) to increase their size by 2 kDa without affecting its activity, as previously reported (see Fig. 1 in Ref. 27Hanson P.I. Meyer T. Stryer L. Schulman H. Neuron. 1994; 12: 943-956Abstract Full Text PDF PubMed Scopus (393) Google Scholar). This kinase subunit is termed tag-T(286/305/306)D. Obligate substrate subunits should retain Thr286 and the ability to bind calmodulin, but should not be able to autophosphorylate. This was previously generated by inactivating the catalytic function of the kinase through replacement of Lys42, a conserved residue near the ATP-binding sites of all kinases, with Met (27Hanson P.I. Meyer T. Stryer L. Schulman H. Neuron. 1994; 12: 943-956Abstract Full Text PDF PubMed Scopus (393) Google Scholar). Wild type and mutant CaM kinase II subunits were expressed in COS-7 cells by transfection with corresponding DNAs, and expression levels examined by immunoblot with α-CaM kinase II specific monoclonal antibody CB-α2 and for the ability to bind calmodulin by calmodulin overlay (Fig. 1). Homomultimers composed of either kinase (tag-T(286/305/306)D, apparent molecular mass of 56 kDa) or substrate (K42M, apparent molecular mass of 54 kDa) subunits or heteromultimers composed of both subunits achieved by cotransfecting a mixture of corresponding DNAs were well expressed and distinguishable by mobility on SDS-polyacrylamide gels (Fig. 1 A). Calmodulin overlay assay of identical lysates reveal bands corresponding to K42M mutant (Fig. 1 B, lanes 1, 3, and 4) and to wild type kinase (Fig. 1 B, lane 5) but not to the tag-T(286/305/306)D mutant (Fig. 1 B, lanes 2, 3, and 4). Thus, replacement of Thr305 and Thr306 with aspartate has the intended effect of mimicking autophosphorylation of these sites by similarly blocking binding of calmodulin (7Colbran R.J. Soderling T.R. J. Biol. Chem. 1990; 265: 11213-11219Abstract Full Text PDF PubMed Google Scholar, 8Hanson P.I. Schulman H. J. Biol. Chem. 1992; 267: 17216-17224Abstract Full Text PDF PubMed Google Scholar, 9Hashimoto Y. Schworer C.M. Colbran R.J. Soderling T.R. J. Biol. Chem. 1987; 262: 8051-8055Abstract Full Text PDF PubMed Google Scholar, 10Patton B.L. Miller S.G. Kennedy M.B. J. Biol. Chem. 1990; 265: 11204-11212Abstract Full Text PDF PubMed Google Scholar). The activities of expressed kinases were found to be appropriate for the substrate and kinase subunits needed for our studies (Fig. 2 C). Lysates with expressed homomultimers of K42M mutant (Fig. 2 C, lanes 1 and 2) exhibited little or no Ca2+/calmodulin-stimulated activity, similar to that found in mock-transfected cells (data not shown). By contrast, lysates of cells expressing homomultimers of tag-T(286/305/306)D exhibited high activity in either the presence or absence of added Ca2+/calmodulin (Fig. 2 C, lanes 7 and 8). Comparison of expressed kinase subunit migrating at 56 kDa and substrate subunit at 54 kDa (Fig. 2 B) with activities of comparable samples (Fig. 2 C) indicates that kinase activities in lysates expressing both types of subunits are largely due to the presence of tag-T(286/305/306)D, the kinase subunit. The tag-T(286/305/306)D mutant is therefore an appropriate obligate kinase subunit for our studies, with high Ca2+-independent activity and no calmodulin binding (Fig. 1). Previous studies showed that Thr286 autophosphorylation in CaM kinase II occurs via an intraholoenzyme reaction (20Lai Y. Nairn A.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4253-4257Crossref PubMed Scopus (216) Google Scholar, 26Kuret J. Schulman H. J. Biol. Chem. 1985; 260: 6427-6433Abstract Full Text PDF PubMed Google Scholar) and involves an intersubunit mechanism (27Hanson P.I. Meyer T. Stryer L. Schulman H. Neuron. 1994; 12: 943-956Abstract Full Text PDF PubMed Scopus (393) Google Scholar, 28Mukherji S. Soderling T.R. J. Biol. Chem. 1994; 269: 13744-13747Abstract Full Text PDF PubMed Google Scholar). We therefore reasoned that the phosphorylation of K42M by tag-T(286/305/306)D should be a likely indicator of whether the two subunits were coassembled into heteromultimers. Indeed, Fig. 2 A illustrates that phosphorylation of the K42M subunit only occurred in COS lysates in which the tag-T(286/305/306)D mutant was coexpressed (and in the presence of Ca2+/calmodulin; see lane 6). No subunit 32P incorporation was seen in cell lysates expressing homomultimers of either subunit (lanes 1 and 2 for K42M, and lanes 7 and 8 for tag-T(286/305/306)D), nor when these lysates were mixed together (lanes 3 and 4). Phosphorylation of K42M does not occur even though the amount of kinase protein and activity in the two preparations (mixed versus coexpressed) is similar. These data suggest that the tag-T(286/305/306)D and K42M subunits coassemble to form heteromeric holoenzymes. This conclusion is also supported by the observation that tag-T(286/305/306)D is copurified with K42M using a procedure (see “Experimental Procedures”) involving immobilized calmodulin chromatography (lanes 1–4 in Fig. 3 B). In addition to mutant K42M, all other substrate subunits used in the experiments below formed heteromeric holoenzymes when coexpressed with tagged kinase subunits, as judged by co-immunoprecipitation using an antibody specific to the hemagglutinin epitope tag (data not shown). We purified heteromeric CaM kinase II composed of obligate kinase (tag-T(286/305/306)D) and substrate (K42M) subunits in order to test the possible requirement for calmodulin binding to the substrate subunit with Thr286. Heteromeric (lanes 1–4) or wild type (lanes 5–8) CaM kinase II was incubated under autophosphorylation conditions in either the absence (−) or presence (+) of Ca2+/calmodulin (Fig. 3). In this experiment, phosphorylation of Thr286 was detected by immunoblot with polyclonal antibody that selectively detects phospho-Thr286 CaM kinase II kindly provided by D. Ginty. Although the heteromeric kinase expressed both kinase and substrate subunits (Fig. 3 B, lanes 1and 2) and was constitutively active when presented with an exogenous substrate with no difference in activity in the absence or presence of Ca2+/calmodulin (Fig. 3 C, lanes 1 and 2), it was unable to phosphorylate itself, i.e. its substrate subunit, unless Ca2+/calmodulin was present (Fig. 3 A, comparelanes 1 and 2). Thus, autophosphorylation, but not phosphorylation of exogenous substrates, by the constitutively active heteromultimer is conditional on the presence of Ca2+/calmodulin. By contrast, Ca2+/calmodulin was required for both substrate phosphorylation and autophosphorylation by wild type CaM kinase II (Fig. 3 C, lanes 5 and 6; Fig. 3A, lanes 5 and 6). We took advantage of KN-93, an inhibitor that blocks binding of calmodulin to the kinase without interfering with the Ca2+/calmodulin-independent activity of the autophosphorylated enzyme (41Sumi M. Kiuchi K. Ishikawa T. Ishii A. Hagiwara M. Nagatsu T. Hidaka H. Biochem. Biophys. Res. Commun. 1991; 181: 968-975Crossref PubMed Scopus (461) Google Scholar) to further test the requirement for calmodulin binding. KN-93 has the same differential effect on kinase made Ca2+/calmodulin-independent by the Thr286to aspartate mutation; it did not block the constitutive activity of the heteromultimer (Fig. 3 C, lanes 1–4) but did block activation of wild type kinase (Fig. 3 C, lanes 5–8). However, despite its inability to block phosphorylation of exogenous substrates by the heteromultimer, KN-93 completely inhibited autophosphorylation of the K42M subunit in the multimer (Fig. 3 A, comparelanes 2 and 4), suggesting that the calmodulin-binding site of the substrate subunit must be occupied in order for it to be phosphorylated by the kinase subunit. We further examined the requirement for calmodulin binding by disabling the calmodulin-binding site of the substrate subunit using the double T305D/T306D point mutation shown previously to block Ca2+/calmodulin binding (Fig. 1 B). Indeed, substrate subunits consisting of mutant T(305/306)D, which are unable to bind calmodulin (data not shown), cannot be phosphorylated when coexpressed and coassembled with either tag-T(286/305/306)D (Fig. 4, lanes 1 and 2) or tag-wild type CaM kinase II (Fig. 4, lanes 3 and 4). Wild type CaM kinase II subunits in the heteromultimer can phosphorylate themselves (Fig. 4 A) or an exogenous substrate (Fig. 4 C) in the presence of Ca2+/calmodulin but cannot phosphorylate coassembled substrate T(305/306)D subunits, which are incapable of binding calmodulin (Fig. 4 A). Similar results were achieved when triple mutant K42M/T305D/T306D CaM kinase II subunits were substituted for T(305/306)D subunits in heteromultimers (data not shown). Since CaM kinase II can be autophosphorylated at multiple sites, it was important to determine whether all of the substrate-directed incorporation of 32P was occurring at Thr286, the only site whose phosphorylation generates autonomous activity (13Fong Y.L. Taylor W.L. Means A.R. Soderling T.R. J. Biol. Chem. 1989; 264: 16759-16763Abstract Full Text PDF PubMed Google Scholar,35Hanson 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). We used tag-T(286/305/306)D as the obligate kinase subunit and compared its phosphorylation of coexpressed mutants K42M or K42M/T286A as substrate (Fig. 5). In fact, no32P incorporation was detected when the substrate subunit contained the T286A mutation (Fig. 5 A, comparelanes 2 and 4). These data, along with the demonstration that Thr286 is phosphorylated, using phosphoselective antisera (Fig. 2), strongly indicate that the substrate-directed effect of calmodulin is to enable phosphorylation of Thr286. Results presented above are consistent with the idea that Thr286 is shielded from autophosphorylation in the native conformation of CaM kinase II when Ca2+/calmodulin is not bound to the enzyme (28Mukherji S. Soderling T.R. J. Biol. Chem. 1994; 269: 13744-13747Abstract Full Text PDF PubMed Google Scholar, 30Brickey D.A. Bann J.G. Fong Y.L. Perrino L. Brennan R.G. Soderling T.R. J. Biol. Chem. 1994; 269: 29047-29054Abstract Full Text PDF PubMed Google Scholar). Soderling and colleagues (30Brickey D.A. Bann J.G. Fong Y.L. Perrino L. Brennan R.G. Soderling T.R. J. Biol. Chem. 1994; 269: 29047-29054Abstract Full Text PDF PubMed Google Scholar, 42Mukherji S. Brickey D.A. Soderling T.R. J. Biol. Chem. 1994; 269: 20733-20738Abstract Full Text PDF PubMed Google Scholar) have suggested that mutation of Cys289 to proline disrupts the autoinhibitory domain to expose Thr286 and makes the enzyme Ca2+-independent. Therefore, we examined whether this mutation in substrate subunit would obviate the need for Ca2+/calmodulin binding to this subunit during autophosphorylation. We analyzed the autophosphorylation of mutant K42M/C289P, as the obligate substrate subunit with a potentially altered conformation near Thr286, in heteromultimers with either tag-T(286/305/306)D as obligate kinase or tag-wild type as both Ca2+-dependent kinase and substrate (Fig. 5,lanes 5–8). Autophosphorylation of K42M/C289P occurred in a Ca2+/calmodulin-independent fashion (Fig. 5 A, lanes 5 and 6) in contrast to the requirement for Ca2+/calmodulin seen when substrate does not have a proline residue to disturb the secondary structure near Thr286 (Fig. 5 A, lanes 1 and 2). When K42M/C289P is coassembled with wild type CaM kinase II subunits, its phosphorylation does require Ca2+/calmodulin, but in this case calmodulin is providing a kinase-directed function to activate the kinase subunits whose activation and autophosphorylation require Ca2+/calmodulin (Fig. 5, A and C, lanes 7and 8). These data support a substrate-directed function for calmodulin in CaM kinase II Thr286 autophosphorylation, a function that is not necessary when Thr286 of the substrate subunit is already exposed, e.g. by a nearby mutation. It has been suggested that Ca2+/calmodulin has two independent functions during CaM kinase II autophosphorylation (27Hanson P.I. Meyer T. Stryer L. Schulman H. Neuron. 1994; 12: 943-956Abstract Full Text PDF PubMed Scopus (393) Google Scholar). Ca2+/calmodulin serves a conventional kinase-directed function in which it relieves the inhibitory interaction between the autoinhibitory domain and the catalytic domain, thereby activating the kinase (43Colbran R.J. Smith M.K. Schworer C.M. Fong Y.L. Soderling T.R. J. Biol. Chem. 1989; 264: 4800-4804Abstract Full Text PDF PubMed Google Scholar). The observation that phosphorylation of inactive truncated CaM kinase II mutants by previously activated CaM kinase II monomers still required calmodulin suggested a requirement for a second calmodulin (27Hanson P.I. Meyer T. Stryer L. Schulman H. Neuron. 1994; 12: 943-956Abstract Full Text PDF PubMed Scopus (393) Google Scholar). We have now delineated a second function for calmodulin in intact holoenzymes of the kinase by demonstration that the calmodulin must bind directly to a substrate subunit in order for Thr286 autophosphorylation to proceed, a substrate-directed function. As shown in Fig. 3, phosphorylation of Thr286remains Ca2+/calmodulin-dependent in heteromultimers composed of a constitutively active kinase that is unable to bind calmodulin coassembled with an obligate substrate subunit. Autophosphorylation within mutant heteromultimers appears to be similar to that in wild type CaM kinase II holoenzymes, i.e. it occurs through an intraholoenzyme and intersubunit mechanism (Fig. 2). Although CaM kinase II is able to autophosphorylate at a number of distinct sites, we employed reaction conditions that limit autophosphorylation to Thr286 (see “Experimental Procedures”). This is illustrated by the lack of 32P incorporation into substrate subunits containing a T286A mutation (Fig. 5). Even when the substrate subunit contains Thr286, this residue is not phosphorylated if the calmodulin-binding site is disabled by substitution of aspartate for threonines 305 and 306 (Fig. 4). Thus, calmodulin binding is likely an inherent requirement for both the substrate and kinase subunits involved in each autophosphorylation reaction. Our results agree with the previous suggestion by Soderling and colleagues (30Brickey D.A. Bann J.G. Fong Y.L. Perrino L. Brennan R.G. Soderling T.R. J. Biol. Chem. 1994; 269: 29047-29054Abstract Full Text PDF PubMed Google Scholar, 42Mukherji S. Brickey D.A. Soderling T.R. J. Biol. Chem. 1994; 269: 20733-20738Abstract Full Text PDF PubMed Google Scholar) that Thr286 is shielded from phosphorylation in the absence of bound calmodulin. This conclusion stemmed from their observation that mutant C289P CaM kinase II homomultimers displayed some activity and autophosphorylated at Thr286 in the absence of Ca2+/calmodulin (42Mukherji S. Brickey D.A. Soderling T.R. J. Biol. Chem. 1994; 269: 20733-20738Abstract Full Text PDF PubMed Google Scholar). Our findings provide a mechanistic basis for requiring concurrent calmodulin binding to at least two proximate subunits of CaM kinase holoenzymes for initial autophosphorylation to occur. The requirement explains the Hill coefficients of ∼1.6 for generating autonomous CaM kinase II activity in response to increasing levels of calmodulin (24De Koninck P. Schulman H. Science. 1998; 279: 227-230Crossref PubMed Scopus (1088) Google Scholar,29Le Vine H.d. Sahyoun N.E. Cuatrecasas P. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2253-2257Crossref PubMed Scopus (22) Google Scholar) whereas kinase activation toward exogenous substrates is not cooperative (24De Koninck P. Schulman H. Science. 1998; 279: 227-230Crossref PubMed Scopus (1088) Google Scholar). It is likely that features of the kinase that take advantage of its multimeric structure, such as its dual requirements for Ca2+/calmodulin in autophosphorylation, which in turn alters calmodulin binding kinetics, termed calmodulin trapping (19Meyer T. Hanson P.I. Stryer L. Schulman H. Science. 1992; 256: 1199-1202Crossref PubMed Scopus (514) Google Scholar), and produce an autonomous activity, contribute to its ability to act as a frequency detector of Ca2+ oscillations with high sensitivity to frequency and calmodulin levels (24De Koninck P. Schulman H. Science. 1998; 279: 227-230Crossref PubMed Scopus (1088) Google Scholar). Our findings also refine the molecular basis for a second feature of frequency-dependent activation, i.e. a molecular switch in sensitivity to Ca2+ oscillations such that conversion of some subunits to an autonomous species facilitate subsequent increments in autonomous activity or sensitize the enzyme to lower frequencies of stimulation (24De Koninck P. Schulman H. Science. 1998; 279: 227-230Crossref PubMed Scopus (1088) Google Scholar). As suggested (24De Koninck P. Schulman H. Science. 1998; 279: 227-230Crossref PubMed Scopus (1088) Google Scholar), phosphorylation of Thr286 in one subunit relieves it of a requirement for calmodulin and thereby facilitates Thr286phosphorylation in a second subunit, creating a functional cooperativity. Moreover, our results confirm that a constitutive kinase subunit does not require interaction with calmodulin to phosphorylate Thr286 on a neighboring subunit. For example, removal of the calmodulin-binding requirement in the substrate subunit K42M/C289P, which may expose Thr286 without binding calmodulin, enables constitutive autophosphorylation of this subunit (Fig. 5). The need for calmodulin binding onto two distinct subunits for CaM kinase II autophosphorylation is somewhat reminiscent of systems where multiple effects of Ca2+/calmodulin are exerted on both an upstream (e.g. CaM kinase I kinase) and a downstream kinase (in this instance, CaM kinase I), thus bringing acute sensitivity to the system in the presence of the ligand (31Hawley S.A. Selbert M.A. Goldstein E.G. Edelman A.M. Carling D. Hardie D.G. J. Biol. Chem. 1995; 270: 27186-27191Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). As these authors point out, however, the effects of CaM kinase I phosphorylation and CaM kinase II autophosphorylation are different. To demonstrate that the mechanism of Thr286 phosphorylation requires both subunits to have bound calmodulin, we created heteromultimers with distinct kinase and substrate subunits. In vivo, of course, any subunit of wild type CaM kinase II should be able to assume either role. It would be interesting to determine whether the mechanism of subunit activation and subunit presentation, the kinase- and substrate-directed steps of autophosphorylation, involve distinct conformations or interactions with calmodulin. It has recently been shown using theDrosophila homologue of CaM kinase II that autophosphorylation of Thr287 (equivalent to Thr286 in rat α CaM kinase II) requires bound calmodulin acting in a substrate-directed mechanism (Wang, Z., Palmer, G., and Griffith, L. C. (1998) J. Neurochem. 71, 378–387)." @default.
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• W1989361508 title "Substrate-directed Function of Calmodulin in Autophosphorylation of Ca2+/Calmodulin-dependent Protein Kinase II" @default.
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