Dual role of calmodulin in autophosphorylation of multifunctional cam kinase may underlie decoding of calcium signals

Neuron,

Vol. 12, 943-956, May, 1994, Copyright

0 1994 by Cell Press

Dual Role of Calmodulin in Autophosphorylation of Multifunctional CaM Kinase May Underlie Decoding of Calcium Signals Phyllis I. Hanson,* Tobias Meyer,+ Lubert Stryer, and Howard Schulman Department of Neurobiology Stanford University School of Medicine Stanford, California 94305-5401

Summary Autophosphorylation of multifunctional Ca*+/calmodulin-dependent protein kinase makes it Ca*+ independent by trapping bound calmodulin and by enabling the kinase to remain partially active even after calmodulin dissociates. We show that autophosphorylation is an intersubunit reaction between neighbors in the multimeric kinase which requires two molecules of calmodulin. Caz+/calmodulin acts not only to activate the “kinase” subunit but also to present effectively the “substrate” subunit for autophosphorylation. Conversion of the kinase to the potentiated or trapped state is a cooperative process that is inefficient at low occupancy of calmodulin. Simulations show that repetitive Ca*+ pulses at limiting calmodulin lead to the recruitment of calmodulin to the holoenzyme, which further stimulates autophosphorylation and trapping. This cooperative, positive feedback loop will potentiate the response of the kinase to sequential Ca*+ transients and establish a threshold frequency at which the enzyme becomes highly active. Introduction Ca2+ spikes and oscillations due to repetitive action potentials and receptor-triggered activation of the phosphoinositide cascade play key roles in many signal transduction processes (Berridge, 1993; Meyer and Stryer, 1991). Information may be encoded digitally by the number and frequency of Ca*+ spikes and decoded by appropriate effector systems. A number of neuronal processes, including synaptic plasticity and release, exhibit frequency-dependent modulation (Ip and Zigmond, 1984; Mulkey et al., 1993). Calmodulindependent enzymes are attractive targets for Ca2+ spikes because calmodulin is cooperatively activated by the binding of three or four Ca2+ ions, enabling it to detect small changes in the cytoso1i.c Ca*+ level. The activity of target enzymes may further be controlled by the level of free calmodulin, since much of calmodulin may be bound to proteins such as neuromodulin (or GAP-43), MARCKS, calcineurin, and neurogranin (or RC3) (Estep et al., 1989; Skene, 1990; Klee, 1991). *Present address: Howard Hughes Medical Institute, versity School of Medicine, New Haven, Connecticut +Present address: Department of Cell Biology, Duke Medical Center, Durham, North Carolina 27710.

Yale Uni06510. University

A number of properties would facilitate the functioning of a molecular Ca2+ frequency detector that suppresses subthreshold frequencies: multiple activity states per detector unit would allow it to function as a spike counter, with individual spikes producing submaximal outputs; a rate of deactivation of the detector that is slow compared with the interval between spikes would permit repetitive stimuli to produce additive effects; and finally, cooperativity in generating or retaining the active state would enable tuning the threshold for activation to a narrow range of frequencies. A candidate for such a molecular decoder of Ca*+ spikes is multifunctional Ca*+/calmodulin-dependent protein kinase (CaM kinase or CaM kinase II), a ubiquitous mediator of Ca2+ effects on cellular targets involved in neurotransmission, neuronal plasticity, gene expression, ion channels, and the cell cycle (for reviews see Hanson and Schulman, 199213; Colbran and Soderling, 1990). CaM kinase exhibits several physical and regulatory properties that might enable it to detect Ca2+ spikes. Closely related isoforms ranging in size from 54 to 61 kDa (from a-, B-, y-, and 8-CaM kinase genes) assemble into multimeric holoenzymes containing 8-10 subunits. Every isoform contains an amino-terminal catalytic domain, acentral autoinhibitory and calmodulin-binding domain, and a carboxyterminal domain responsible for oligomerization and association (for reviews see Hanson and Schulman, 1992b; Colbran and Soderling, 1990). Each of the subunits in a holoenzyme is individually regulated by Ca*+/calmodulin (Yamauchi et al., 1989). A range of kinase activity can therefore be elicited by varying the extent of calmodulin occupancy per holoenzyme. Each kinase subunit exists in multiple functional states that differ in their activity and rate of deactivation following a brief Ca2+ spike (Schulman et al., 1992). The critical determinant of these functional states is autophosphorylation. Prior to autophosphorylation, the kinase is in a state referred to as CaMKO, in which it has essentially no activity (0%) in the absence of Ca2+/calmodulin because of the intrinsic autoinhibitory segment in its regulatory domain. Binding of Ca*+/calmodulin to the autoinhibitory segment producesafullyactivestate(lOO%)inwhichthe kinase can phosphorylate substrates as well as itself. At the end of a Caz+ spike, all nonphosphorylated subunits deactivate rapidly as calmodulin dissociates (tofr, 170 ms; Meyer et al., 1992). Ca*+/calmodulin-dependent autophosphorylation of ThrW (within the autoinhibitory domain of the a subunit) produces a new state of the kinase (CaMKP) with two interesting regulatory properties. First, autoslows the rate of phosphorylation of Thr*% markedly dissociation of calmodulin, thereby trapping calmodulin even at subthreshold Ca*+ concentrations and maintainingactivityat maximal,Ca*+-stimulated levels

NfXlr0Il 944

A. cc-CaMK* 271 catalytic domam

reg.

47R

314 association domain

Figure 1. Introduction of an Antigenic Tag Sequence to Produce Distinguishable a-CaM Kinase Subunits

(A) An 18 amino acid sequence derived from the influenza hemagglutinin HA1 protein was introduced by site-directed mutagenesis into a-CaM kinase between Thr3 and lle4 to generate a-CaMK*, a tagged GAPYPYDVPDYAGPGAQL form of the enzyme. Nine amino acids constituting the antigenic epitope for the C. D. B. monoclonal antibody 12CA5 are underlined (Wilson et al., 1984; Field et al., 1988). 205 (B) Samples of purified a-CaM kinase and E. F. 116 a-CaMK* (700 ng each) were resolved on 97 9% SDS-polyacrylamide gels and stained with Coomassie brilliant blue. a-CaMK* (56 66 kDa) is 2 kDa larger than wild-type a-CaM ,a-CaMK’ .a-CaMK* ,.-. i .- = -a-CaMK 1?LZ kinase (54 kDa). ‘a-CaMK’ (C) a-CaMK* is selectively recognized by 45 the tag-specific monoclonal antibody (12CAS). Samples of a-CaMK and a-CaMK* (700 ng) were resolved on 9% SDS gels, trans29 ferred electrophoretically onto nitrocellucalmodulin blot lose, and analyzed by immunoblotting using the tag-specific monoclonal antibody CB-a-2 12CA5 Coomassie blue (12CA5) and alkaline phosphatase-coupled secondary reagents (Hanson et al., 1989). A similar result was seen using unpurified cytosol from transfected cells as the source ot kinase (data not shown). (D) Both a-CaM kinase and a-CaMK* are detected on an immunoblot developed with a monoclonal antibody recognizing a-CaM kinase (CB-a-2). The 35 kDa band in the a-CaM kinase lane is a proteolytic degradation product of aCaM kinase that was prominent in this sample tested after several months in storage. (E)Distinguishable”substrate”and”kinase”subunitsareexpressedaftercotransfectionofDNAencodinga-CaMK’(j4 kDa)and a-CaMK* (56 kDa) into COS cells. Cytosolic proteins (15 ug) were resolved on a 9% SDS gel, transferred to nitrocellulose, and probed with biotinylated calmodulin in the presence of Ca2+. Bound calmodulin was visualized using Vectastain ABC reagents (Vector Labs) and an Enhanced Chemiluminescence kit (Amersham). Both subunits (a-CaMK’ and a-CaMK*) are expressed at equivalent levels in cotransfected cytosols. (F) Tagged kinase was immunoprecipitated from the cytosol shown in (E) using the tag-specific monoclonal antibody (12CAS). Immunoprecipitation was performed under nondenaturing conditions (see Experimental Procedures), and the precipitated proteins were resolved on a SDS gel and probed with biotinylated calmodulin as in (E). Both tagged and untagged subunits were precipitated.

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(100%) (Meyer et al., 1992). Second, once calmodulin dissociates from a subunit in the CaMKP state, that subunitdoes notfullydeactivateand remains partially active (20%-80%) in the absenceof Ca2+(for review see Hanson and Schulman, 1992b). This Ca*+-independent activity is referred to as the autonomous activity. Both the trapped and autonomous states provide for potentiation of brief Ca2+ transients. Site-directed mutagenesis studies have demonstrated that autophosphorylation at a unique site (Thr286 in the a subunit) is both necessary and sufficient for the transition of the enzyme to the CaMKP state (Hanson et al., 1989; Fong et al., 1989; Waxham et al., 1990; Waldmann et al., 1990). This autophosphorylation is an intramolecular reaction within CaM kinase holoenzymes (Kuret and Schulman, 1985; Lai et al., 1986). The autonomous kinase (CaMKP) phosphorylates substrates but also continues to autophosphorylate. The ability to propagate the autonomous state by autophosphorylation of CaMKO subunits in the absence of Ca2+/calmodulin has been a key feature of previous “memory” models of CaM kinase (Lisman, 1985; Miller and Kennedy, 1986; Lisman and Goldring, 1988), but direct evidence for this is lacking. Ca2+/calmodulin-independent autophosphorylation occurs at new sites that are within the calmodulin-binding domain

(Thr305 and Thr306 in the a subunit) rather than at Thr28G (Patton et al., 1990; Hanson and Schulman, l992a). This generates a Ca2+/calmodulin-insensitive state (CaMKPjP) in which kinase activity is capped at the level of the autonomous kinase. CaMKp’P is not responsive toCa2+/ calmodulin because autophosphorylation within the calmodulin-binding site disables it. In the present study, we analyze how autophosphorylation and calmodulin trapping are controlled within and between the subunits of CaM kinase. We find that, whereas activation by Ca2’/calmodulin is an inherent property of individual subunits, autophosphorylation of the “autonomy” site (Thr286) is a property of the oligomeric enzyme. The autophosphorylation responsible for autonomy and calmodulin trapping is an intermolecular reaction between subunits. Further analysis reveals a dual role for calmodulin; it not only activates kinase subunits, but must be bound to the subunit serving as “substrate” for effective autophosphorylation to occur. The dual requirement for calmodulin provides a molecular basis for cooperativityof autophosphorylation and calmodulin trapping. Intersubunit autophosphorylation of Thr286 will therefore be favored by stimuli that activate multiple subunits in each holoenzyme. Simulations based on experimental results demonstrate that phos-

Decoding

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calmodulin blot

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Figure 2. ThP

Autophosphorylation

,CaMK*

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.286A

-CaMK’

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calmodulin blot Reaction

in Holoenzymes

Each panel shows an autoradiogram of autophosphorylation reactions above a calmodulin overlay blot of the same samples. (A) Inactive (a-CaMK’) and tagged (a-CaMK*) subunits were used to assay for intersubunit autophosphorylation. Cytosols (IO ug each) containing a-CaMK’ (lane I), a-CaMK* (lane 2), a mixture of the two separately transfected (lane 3), and a mixture of the two cotransfected (lane 4) were subjected to 30 s of autophosphorylation in the presence of Ca2+/calmodulin at 4OC, and the resulting phosphoproteins were resolved on a 9% SDS-polyacrylamide gel. The positions of a-CaMK’ (54 kDa) and a-CaMK* (56 kDa) are indicated by arrows. In the lower panel, a calmodulin overlay blot of the same samples is shown with arrows indicating the position of a-CaMK* and a-CaMK’. Similar results were seen using a preparation of purified CaM kinase holoenzyme containing both subunits. (B) The series shown in (A) is repeated, except that a-CaMK’ also contains a ThrWla mutation. Samples are a-CaMKIThrZBM1’ (lane I), a-CaMK* (lane 2), a mixture of these two separately transfected (lane 3), and a mixture of these two cotransfected (lane 4). Reactions were again for 30 s at 4OC. (C) The series in (A) is again repeated, except here a-CaMK* contains a Thr28M’a mutation. Samples are cytosol with a-CaMK’ (lane I), a-CaMK*ThrzW1’ (lane 2), a mixture of these two separately transfected (lane 3), and a mixture of these two cotransfected (lane 4). Reactions were for 30 s at 4X

phorylation-induced trapping can potentiate the response of the kinase to repetitive Ca2+ transients and enable it to serve as a frequency detector.

Results Mechanism of ThP6 Autophosphorylation in a-CaM Kinase Holoenzymes Autophosphorylation of the critical autonomy site of a-CaM kinase is an intraholoenzyme reaction (Kuret and Schulman, 1985; Lai et al., 1986), which may be either an intrasubunit or an intersubunit reaction in the holoenzyme. An intersubunit mechanism would take advantage of the multimeric structure of this kinase and would have the potential of involving cooperative interactions between subunits and with calmodulin. We engineered a-CaM kinase to allow us todistinguish subunitsservingas the”substrate”from those acting as the “kinase” in autophosphorylation reactions.Active”kinase”subunitsweremadedistinct by inserting a unique immunological tag that increased their size by2 kDa but had noeffect on activity (Figures IA and IB). These tagged subunits (designated a-CaMK*) were recognized by a tag-specific monoclonal antibody (12CA5) (Wilson et al., 1984; Field et al., 1988) and by an a-CaM kinase-specific

monoclonal antibody (CB-a-2) (Figures IC and ID). 0bligate”substrate”subunitsweregenerated byinactivating the catalytic function of the kinase through replacement of Lys@, a conserved residue near the ATP-binding sites of all kinases, with Met or Arg (Taylor et al., 1990). Subunits containing Lys42Metor Lys42Ars (designated a-CaMK’) were expressed in COS cells at levels comparable to wild-type a-CaM kinase, but were inactive in both autophosphorylation and substrate phosphorylation reactions (data not shown; but see Figure 2). Holoenzymes composed of both”kinase”and “substrate” subunits were produced in COS cells by transfecting with mixtures of the corresponding DNAs (Figures IE and IF). Both a-CaMK’ (54 kDa) and a-CaMK* (56 kDa) subunits could be readily detected on calmodulin overlay blots of transfected cell cytosols (Figure IE). The tag-specific antibody immunoprecipitated both tagged a-CaMK* and untagged a-CaMK’ subunits at a ratio similar to that found in the transfected cytosol (compare Figures IF and IE). It is therefore likely that most of the expressed subunits are assembled as heteromultimers of a-CaMK* and a-CaMK’. Is CaVcalmodulin-stimulated autophosphorylation an intersubunit or an intrasubunit reaction in

Neuron 946

these holoenzymes? Both a-CaMK* (Figure 2A, lane 4, upper band) and a-CaMKr (Figure 2A, lane 4, lower band)were rapidlyautophosphorylated in heteromultimers produced as above. Whereas autophosphorylation of a-CaMK* mayoccur byeitheran intersubunit or an intrasubunit reaction, autophosphorylation of the inactive a-CaMKr can only occur by an intersubunit reaction. Homomultimers of a-CaMKr did not autophosphorylate (Figure 2A, lane I), nor were they autophosphorylated when presented to active homomultimersofa-CaMK* (Figure2A, lane3)“Kinase”and “substrate” subunits must therefore be in the same holoenzyme for significant autophosphorylation of “substrate”subunitstooccur.Thislackof interholoenzyme autophosphorylation is consistent with earlier observations that autophosphorylation of the rat brain enzyme is an intraholoenzyme reaction (Kuret and Schulman, 1985; Lai et al., 1986). By contrast, the autophosphorylation of inactive “substrate” subunits by coassembled “kinase” subunits (Figure 2A, lane 4) suggests that autophosphorylation can proceed via an intersubunit mechanism within individual holoenzymes. Similar results were obtained using a preparation of purified CaM kinase holoenzyme containing a-CaMK’ and a-CaMK* subunits (data not shown). We used site-directed mutagenesis to ask whether intersubunitautophosphorylation involvestheautonomy site or other sites of autophosphorylation. We replaced Thr286 in a-CaMK’with Ala to prevent its phosphorylation on the “substrate” subunits and found by that a-CaMKr(Thr286Ara) was not autophosphorylated coassembled a-CaMK* subunits (Figure 28, lane 4). The intersubunit phosphorylation of native a-CaMKr subunits shown above (Figure 2A, lane 4) therefore appears to target Thr2B6. With a Thr2*cAra substitution in the a-CaMK* subunits, we asked whether autophosphorylation of the autonomy site on active “kinase” subunits was a prerequisite for their ability to phosphorylate neighboring “substrate” subunits (Figure 2C). In spite of this mutation, a-CaMK*(Thr286Ara) readily phosphorylated a-CaMKr (Figure 2C, lane 4), suggesting that autophosphorylation of “kinase” subunits is not a prerequisite for their ability to phosphorylate “substrate” subunits within the holoenzyme. Autophosphorylation in Monomeric a-CaM Kinase Subunits A more quantitative and complete analysis of intersubunit autophosphorylation can be carried out with monomeric forms of CaM kinase. Since the holoenzyme does not readily dissociate into subunits, we utilized a recombinant a-CaM kinase made monomeric by truncating after amino acid 326 to eliminate the association domain of the enzyme. We first established that the physical, kinetic, and regulatory properties of the native enzyme were retained in the truncated construct. We purified a-CaMK(l-326) to near homogeneity (Figure 3A) and found that it had the biophysical properties of a monomeric protein of approximately 35 kDa, based on sedimentation in su-

A. origin

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l-326)

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dye fro”,!, =

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duces a Monomeric Kinase (A) a-CaM kinase truncated by repiacement of VaW’with a nonsense codon !a-CaMK[l-3261) was expressed in CQS-7 ceils and purified from cytosols to approximately 90% homogeneity (see Experimental Procedures). A 10% SDS-polyacryiamide gel stained with Coomassie brilliant blue shows proteins present in the cytosol, DE-52 eluate, phosphocellulose pool, and calmodulin-Sepharose pool. The apparent molecular weight of a-CaMK(l-326) is 35 kDa, consistentwith its calculated molecular weight of 36.7 kDa. lMolecular weight standards shown include myosin (200 kDa), B-galactosidase (116 kDa), phosphorylase b (97 kDa), BSA (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), and soybean trypsin inhibitor (21 kDa). (B) The sedimentation of wild-type a-CaM kinase and a-CaMKtl326) on 5%-20% sucrose density gradients is shown. COS cell cytosol (100 1-11;100 ug of total protein) containing transfected wild-type a-CaM kinase or a-CaMK(l-326) was subjected to sucrose density gradient centrifugation, and fractions collected from thetop of the gradient were assayed for CaM kinase activity as described (Experimental Procedures). The positions of BSA (4.6S), bovinecatalase (Cat; 11.3S), and bovine thyroglobulin (TG; 19.4s) run as standards in the same gradients are indicated. The peak of a-CaMKfl-326) activity corresponds to 2.55, whereas the peak of wild-type a-CaM kinase activity corresponds to 165.

crose density gradients (Figure 3B) and chromatographic behavior on gel filtration through G-75 (data not shown). Kinetic analyses revealed that a-CaMK(l326) remained highly dependent on Ca*+/calmodulin

Decoding 947

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Figure 4. Regulatory of a-CaMKfl-326)

origin 200

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and was similar to the full-length enzyme in its K, for the peptide substrate autocamtide-3 (0.23 f .08 PM) and for ATP (13.6 f 1.0 vM). a-CaMK(l-326) phosphorylated autocamtide-3 with a V,,, of 13 f.rmol/min/mg; activity in the absence of Ca*+ was 200-fold lower. Before studying the molecular basis of autophosphorylation, we examined whether the monomeric kinase exhibits the two phases of autophosphorylation that are characteristic of the holoenzyme. Indeed, a-CaMK(l-326) autophosphorylated in the presence of Ca*+/calmodulin and was then able to undergo a characteristic “burst” of additional Ca*+-independent autophosphorylation leading to reduced mobility on SDS gels (Figure 4A) (Hanson et al., 1989; Hanson and Schulman, 1992a). Furthermore, the enzyme was converted by Ca2+-dependent autophosphorylation to a species (CaMKP) that was 38% autonomous (at 2 f.rM autocamtide3) and by subsequent Ca*+-independent autophosphorylation to a species (CaMKP’P) whose activity was capped at the autonomous level (Figure 48). Is the Ca2+/calmodulin-stimulated autophosphorylation of a-CaMK(l-326) an inter-or an intramolecular reaction? These two possibilities can readily be distinguished since the rate of an intermolecular reaction will depend on the concentration of monomeric kinase in the reaction, whereas an intramolecular reaction will proceed at the same rate regardless of the kinase concentration. As shown in Figure 5A, the extent of phosphorylation of a-CaMK(l-326) in a 30 s

Autophosphorylation

(A) Purified a-CaMK(l-326) was autophosphorylated with 250 gM [yJ2P]ATP in the presence of Ca~lcalmodulin and/or ECTA as indicated below the gel. Lanes 2 and 4 show 45 sand 2 min of Ca*+-stimulated autophosphorylation. Ca*+independent autophosphorylation was assayed by addition of EGTA (3.3 m M final concentration) priortoanyCaz+-stimulatedautophosphorylation (lane l)orafter45 s of Ca2+-stimulated autophosphorylation (lane 3). All reactions were stopped with EDTA (16.7 m M final concentration; at 4°C) followed by SDSstop buffer. Samples were resolved on a 10% SDS gel, and the resulting autoradiogram is shown. The functional state resulting from each autophosphorylation is labeled above the gel. Each reaction contained 200 nM a-CaMK(l-326). (B) The functional effect of autophosphorylation was determined by assaying aliquots (5 ul) from the reactions described in (A) for autocamtide-3 (2 PM) phosphorylation in the presence (solid bars) or absence (hatched bars) of Caz+/calmodulin for 20 s as described (Experimental Procedures). CaMKO had 100% activity + Ca*+ and 0.6% activity - Ca*+; CaMKP had 100% activity + Caz+ and 38.3% activity - CaZ+; CaMKPtP had 46% activity + Caz’ and 36% activity - Ca2+. Each value is the mean of triplicates; error bars show the SD.

reaction, normalized for kinase concentration, depends on the concentration of kinase in the reaction (12-250 nM). These data are clearly inconsistent with an intramolecular reaction and can be fit well to an equation describing intermolecular autophosphorylation, Y = N(l - emaxt) (Figure 5A, solid line; Experimental Procedures). The increased rate of autophosphorylation seen with increasing kinase concentration is not due to a concentration-dependent stabilization of the enzyme’s activity, since the specific activity of Ca*+-stimulated peptide phosphorylation by the kinase did not vary over the same concentration range (see Figure 5B). Most of the autophosphorylation of a-CaMK(l-326) is therefore the result of an intermolecular reaction; however, asmall contribution from an intramolecular reaction cannot be excluded. The rate of autophosphorylation observed at the lowest concentration of kinase used sets an upper limit on the rate of such an intramolecular reaction at approximately 10% of the intermolecular rate. It was important to examine specifically whether the intermolecular phosphorylation of a-CaMK(l326) was associated with the transition from the Ca*+dependent (CaMKO) to the Ca2+-independent state (CaMKP), since some of the phosphorylation measured in Figure 5Acould be unrelated to phosphorylation of the autonomy site (Thr%). The extent of Ca*+independent kinase activity can serve as a quantitative measure of the fraction of monomers which are in

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Figure 5. Intermolecular CaM Kinase

Autophosphorylation

the CaMKP state. The proportion of Ca*+-independent activity generated by a 30 s autophosphorylation of a-CaMK(l-326) at concentrations ranging from 5 to 200 nM is shown in Figure 5B (open circles). There is a concentration-dependent increase in the Ca*+independent activity generated by autophosphorylation. This increase closely parallels the concentrationdependent increase in autophosphorylation shown above (Figure 5A) and can be well fit to the same equation (Figure 5B, solid line). Autophosphorylation of ThP6 is therefore predominantly an intermolecular reaction between recombinant CaM kinase monomers. For comparison, maximal Ca2+-stimulated activity is also shown (Figure 58, closed circles) and exhibits no concentration-dependent changes.

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(A) Autophosphorylation of a-CaMK(l-326) as a function of its concentration is shown. Purified a-CaMK(l-326) (final concentrations of 12-250 nM) was autophosphorylated for 30 sat 30°C in a reaction containing 250 PM [yJ*P]ATP (Experimental Procedures). The phosphate incorporated, normalized to the concentration of kinase in the reaction, is shown as percent maximal phosphorylation (closed circles). The data points were fitted by nonlinear regression to an equation describing intermolecular autophosphorylation, Y = N(1 - e-““r), in which x is the concentration of a-CaMK(l-326), t is the reaction time in seconds, a is the ratio of the autophosphorylation rate and an equilibrium constant for the binding of monomers to each other, and N is a normalization constant (see Experimental Procedures for derivation). The data shown for a 30 s reaction were best fitted with a = 4.0 x IO-YnMls and N = 1.07 (solid line). Based on the fit to the equation above, the equilibrium constant (0 for intersubunit autophosphorylation is approximately 1.3 PM if we assume a phosphorylation rate of 0.5 s-’ (Miller and Kennedy, 1985). (B) Monomeric CaM kinase becomes Cazf independent in a concentration-dependent reaction. a-CaMK(l-326) was autophosphorylated in a 30 s preincubation (250 uM unlabeled ATP; 30°C) at final concentrations ranging from 6 to 250 nM. Autophosphorylation was terminated by addition of cold EDTA and ECTA (final concentrations of 20 and 4 mM, respectively). Autophosphorylated kinase was immediately assayed for its ability to phosphorylate autocamtide-3 (20 PM) in either the presence or the absence of Ca2+/calmodulin. The percent maximal activity is normalized for the concentration of enzyme present in each reac-

Calmodulin Must Be Bound to the Substrate Subunit for Effective lntersubunit Phosphorylation of Thr286 It is important to determine whether Ca2+/calmodulin must be bound to the subunit serving as “substrate” in an autophosphorylation reaction. Finding that an autonomous “kinase” subunit could phosphorylate a substrate subunit in the absence of Ca2+/calmodulin ‘would provide support for the memory model of CaM lkinase (Lisman, 1985; Miller and Kennedy, 1986; Lislman and Coldring, 1988). A mechanism requiring binding of Ca2+/calmodulin to both the “kinase” and “substrate” subunits predicts a cooperative role for calmoduiin that would be important in situ when only a few subunits per holoenzyme are likely to bind Cai”/ calmodulin because of limitations in both Ca*+ and calmodulin (Estep et al., 1989; Skene, 1990; Klee, 1991; Fukunaga et al., 1989; MacNicol et al., 1990; MacNicol and Schulman, 1992a, 1992b). We performed two experiments using monomeric CaM kinase to address this question. In the first, we generated the autonomous form of CaM kinase, CaMKP, using a-CaMK(l-326) and compared its ability to phosphorylate dephospho-a-CaMK(l-326) (CaMKO) in the absence and presence of Ca*+/calmodulin (Figure 6A). Addition of two arbitrary units of CaMKO to one unit of autonomous CaMKP in the absence of Ca*” did not lead to any more autophosphorylation than is obtained by the Ca*+-independent autophosphorylation of one unit of CaMKP alone. The autonomous kinase is active in the absence of Ca*+/calmodulin, as seen by its continued autophosphorylation to produce CaMKPjP as well as its ability to phosphorylate other substrates (see Figure 4B), but it does not phos-

tion. Activity in the presence (closed circles; dashed line) or adsence (open circles) of Caz’/calmodulin is shown. Maximal Ca2+-independent activity in this set of reactions was 80% of Ca2+-stimulated activity and was normalized to 100%. The Ca2*independent data were fitted (solid line) by minimizing the chisquared function for the parameterized model; in this case, the best fit parameters were a = 4.9 x IO-YnMls and IV = 0.991 (Experimental Procedures).

Decoding 949

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Figure 6. Calmodulin Effective Intersubunit

a-CaMKI

+ a-CaMK1(l-326)

(l-326)

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Must Be Bound to a Substrate Phosphorylation

Subunit

for

(A) Purified a-CaMK(l-326) (325 nM) was autophosphorylated in 15 ul reactions containing 250 PM ATP and standard reaction components (Experimental Procedures) to generate CaMKP. Ca2+-stimulated reactions contained 800 uM CaCb, 400 uM ECTA, and 600 nM calmodulin; where indicated, ECTA was added to a final concentration of 3.5 mM. Fresh a-CaMK(l-326) was added asindicated(columns3-5)in30uIof reaction mixture(+or-Ca*+ as shown), keeping the kinase concentration constant (325 nM). All reactions were stopped at 2 min by addition of SDS-stop buffer. Samples were resolved on SDS gels and quantified by excision of ‘*P-labeled bands and counting Cerenkov radiation. Reactions 1 and 2 contained 4.9 pmol of kinase, and reactions 3-5 contained 14.7 pmol of kinase. Phosphate (2.2 pmol) were incorporated into lx CaMKP (column I), 8.5 pmol into lx CaMKPfP (column 2), 9.7 into 1 x CaMKPfP + 2x CaMK” (column 3), 7.6 into 3x CaMKr (column 4), and 30.1 into 3x CaMKPfP (column 5). (B) Phosphorylation of inactive monomer (a-CaMKr[l-3261) and peptide substrate (autocamtide3) was carried out using a prephosphorylated CaMKP’P species of monomeric CaM kinase. This species was prepared by prephosphorylation of a-CaMK(l-326) through the two phases of autophosphorylation described earlier (see Figure 4) in the presence of unlabeled 250 PM ATP. The substrate phosphorylation reactions shown in this panel were started by addition of 3 ul of CaMKr’r (final concentration of 3.6

phorylate the added CaMKO in the absence of Ca2+1 calmodulin (Figure 6A). The added CaMKO only becomes phosphorylated when Ca2+/calmodulin is present. This finding suggests a need for Ca2+/calmodulin on the subunit that is to be phosphorylated. To monitor the requirement for calmodulin on the target of autophosphorylation directly, we generated a “kinase” and “substrate” pair in which only the “substrate”could bind Ca2+/calmodulin.Theobligate”substrate” subunit in the reaction, a-CaMK’(l-326), is the a-CaMK(l-326) monomer containing the inactivating LYS42Metmutation. The calmodulin-insensitive”kinase” subunit is monomeric a-CaMK(l-326) maximally prephosphorylated with unlabeled ATP to the CaMKPIP state. This species of kinase is active but does not bind calmodulin (see Figure 4B) (Hanson and Schulman, 1992a). Indeed, phosphorylation of the inactive substrate subunit, but not of other substrates, was calmodulin dependent (Figure 6B). Since both the autonomy and inhibitory sites of autophosphorylation were fully prephosphorylated with unlabeled ATP, the active kinase (CaMKpjP) exhibited no further autophosphorylation when [Y-~~P]ATPwas added in either the absence or presence of Ca2+/calmodulin (Figure 6B, columns 1 and 2). It was also unable to phosphorylate purified a-CaMKr(l-326) in the absence of Ca2+/ calmodulin (Figure 6B, lane 3) despite the fact that it phosphorylated autocamtide-3 equally well in either the absence or the presence of Ca2+ (Figure 68). Phosphorylation of the obligate “substrate” subunit (a-CaMK[l-326])onlyoccurred when Ca2+/calmodulin was added (Figure 6B, lane 4). Since the only calmodulin-binding protein in this reaction was the “substrate” subunit,therequirementforcalmodulinmustinvolve binding of calmodulin to the substrate in the reaction. Calmodulin may therefore have dual roles in autophosphorylation-to make one subunit catalytically active and to make the other an effective substrate. This suggests further that Thr2% may only be available for intermolecular autophosphorylation when exposed or “presented” bythe binding of Ca2+/calmodulin. A dual requirement for calmodulin on kinase and substrate subunits predicts cooperativity in generation of the autonomous and trapped states of the kinase by calmodulin. Trapping of Calmodulin Is Cooperative and Requires Binding of At least Two Molecules of Calmodulin to the Holoenzyme Is trapping of calmodulin by autophosphorylation of

nM) to a 30 ul substrate phosphorylation reaction containing no substrate (columns 1 and 2), a-CaMK’(l-326) (approximately 200 nM; columns 3 and 4), and autocamtide-3 (20 PM; columns 5 and 6). Each pair of reactions contained either EGTA (-) or Ca2+/ calmodulin (+). The activity is expressed as percentage of maximal Caz+-stimulated activity toward a-CaMK’(l-326) (columns 1-4)orautocamtide-3(columns5and6).Theinsetabovecolumns l-4 shows the kinase band seen at 35 kDa on the autoradiogram of the phosphorylation reactions.

Neuron 950

Time of incubation

rate of trapping was strongly dependent on the degree of saturation of CaM kinase-binding sites by CaMF. Whereas trapping half of bound calmodulin took 4 s at 40% occupancy, it took 20 s at 15% occupancy. The rate at which calmodulin was trapped over a range of occupancy levels, determined in similar experiments, is shown in Figure 78 (closed circles). The dashed line indicates the data expected of a noncooperative process. in fact, there is a marked increase in the rate of trapping as the number of subunits in the multimeric enzyme which are occupied by CaMF increases. Hence, trapping of calmodulin is a cooperative process that would be much slower at low levels of free cytoplasmic calmodulin than at high ones. There was no detectable Lag between autophosphorylation and trapping, indicating that cooperativity is not due to a concentration-dependent delay time between autophosphorylation and trapping (data not shown). This cooperativitycould be explained if CaMF must be bound to the target subunit for its autonomy site to be phosphorylated. These data are therefore consistent with the findings above (Figure 6) indicating that one calmodulin molecule must be bound to the “kinase” subunit and another calmodulin mo!ecule to the “substrate” subunit before intersubunit phosphorylation of the autonomy site and trapping can occur. This dual calmodulin requirement is likely to be the basis for the observed cooperativity.

(see)

I3 I

0.8 s

&j c

. 0.6

.

0 i 0

02

0.6

0.4

Fraction oEcaIniodulin-bindIng Figure 7. The Rate of Trapping Calmodulin Occupancy

by a-CaM

08

I0

citei occupied Kinase Increases

with

(A) Time course of trapping at different calmodulin occupancy. Fluorescence anisotropy was used to assess the fraction of bound CaMF that is trapped, i.e., with a decreased dissociation rate as described (Experimental Procedures). Values are normalized so that 1.0 corresponds to complete trapping of all bound CaM’. The two concentrations shown correspond to 15% (circle) or 40% (triangle) occupancy of calmodulin-binding sites on CaM kinase. These occupancies were achieved by adding 4 nM CaM kinase and IO nM or 2.5 nM CaMF. The solid lines are fits using rate constants of 0.25 s-: (40% occupancy) and ,045 5-l (15% occu-

pancy). (B) Dependence of the rate of trapping on the fraction of occupancy of CaM kinase sites by CaM’. Experiments similar to those in (A) were carried out over a range of occupancies. The plotted rate of trapping is defined as the inverse of the time required for 63% of calmodulin to be trapped.

ThP6 in multimeric CaM kinase a cooperative process? We incubated a-CaM kinase with concentrations of dansylated calmodulin (CaMF) that resulted in either 15% or 40% occupancy of the calmodulinbinding sites and used fluorescence anisotropy to determine the rate at which bound calmodulin became trapped on the kinase (Figure 7A). We found that the

Molecular Potentiation and Frequency Detection Based on the requirement for the simultaneous presence of Ca2’/calmodulin on proximate neighbors for autophosphorylation, we propose that a new consequence of autophosphorylation at limiting concentra tions of Ca2’/calmodulin is the ability of CaM kinase to decode the frequency of Ca2+ signals. Computer simulation of the response of CaM kinase to a series of repetitive Ca*+ spikes of equal amplitude at two different frequencies suggests a frequency-dependent response (Figure 8A). Let us assume that only 5% of the kinase sites contain bound Ca2+/calmodulin and are active at the peak of the first spike. Of these, only a small percentage will trap calmodulin because the probability of two calmodulin molecules bound to proximate neighbors on the same holoenzyme is relatively low at 5% occupancy. If the CaZi spikes are infrequent (say every 20 s), potentiation of the Ca2’ signal will be minimal since phosphatase action is likely to prevent the buildup of autophosphorylated enzyme with trapped calmodulin during successive spikes. In contrast, if Ca2+ spikes occur frequently (say every 2 s), autophosphorylation will outpace dephosphorylation and lead to increased trapping with time. Trapping will enhance the response of the kinase to successive CaZi pulses, because increasing amounts of calmodulin will be recruited from other calmodulinbinding proteins to the kinase with each spike. Since trapping will increase the probability that a newly bound calmodulin will have an active subunit as its proximate neighbor, it will increase the yield of

Decoding 951

Calcium

Signals

20

0

40

60

feedforward loop for trapping and establishes a threshold frequency for Ca2+ spikes above which the kinase becomes highly activated (Figure 8B). This capacity to detect only rapid Ca*+ spiking is dependent on a cooperative autophosphorylation and submaximal calmodulin occupancy during individual Ca*+ spikes. An additional consequence of trapping arises if CaM kinase and other calmodulin-binding proteins are far apart (>I pm), since acondition for recruitment of calmodulin by CaM kinase is the fast spatial equilibration of free calmodulin by diffusion. Diffusion of calmodulin could be much slower at high than at low Ca*+ levels because Ca*+/calmodulin is then mostly bound to target proteins (Cough and Taylor, 1993). If Ca2+ levels stay high, calmodulin bound to other proteins cannot diffuse to CaM kinase. Thus, little trapping by CaM kinase would occur at persistently high Ca*+ levels because calmodulin diffusion would be too slow. Experiments are needed to determine whether calmodulin binding in vivo is limiting and whether its diffusion is dependent on the cytosolic Ca2+ level. It will be interesting to learn whether CaM kinase has a frequency threshold in vivo and whether this inferred capability is exploited in the regulation of synapses.

80

Time (see)

B

Discussion

0

0.01 0.10 Frequency of calcium spikes (spikedsec)

Figure 8. Simulation Spike Frequency

of the Dependence

I.0

of Kinase Activity

on

(A) When spikes are infrequent (20 s interval; lower trace) and Ca2+/calmodulin is limiting (5% occupancy at each Ca2+ spike), the peak activity is low and remains relatively constant through successive spikes. In contrast, when spikes are frequent (2 s interval; upper trace), the peak activity increases with successive spikes and approaches maximal levels. Successive spikes lead to the recruitment of calmodulin, phosphorylation of the autonomy site, and trapping. (B)The peak kinaseactivityafter 30 spikes is plotted against spike frequency. The same parameters were used for this simulation as those in (A).

trapped kinase subunits during successive spikes in a feedforward manner. The requirement for the binding of two molecules of calmodulin to CaM kinase generates a cooperative

Multifunctional CaM kinase is an oligomeric enzyme whose activation is inherent in each of its individual subunits but whose molecular potentiation is propertyof its multimeric structure. The active site of each subunit is individually controlled byCa2+/calmodulin, which binds and perturbs the autoinhibitory domain residing on the same subunit (Yamauchi et al., 1989; Cruzalegui et al., 1992). We have shown here that potentiation of kinase activity by autophosphorylation at the autonomy site (Thr*&) depends on interactions between subunits and is therefore a property of the multimeric holoenzyme. This modulation occurs when one CaM kinase subunit phosphorylates the autonomy site of a proximate neighbor subunit in an intersubunit reaction. This reaction has a requirement for Ca*+/calmodulin on both the”kinase”and the”substrate”subunits. The multimeric design of CaM kinase and its regulation at the level of individual holoenzymes ensure that modulation of kinase activity will be independent of kinase concentration. These regulatory properties may enable CaM kinase to respond to cellular Ca2+ oscillations and modify our view of how CaM kinase may serve as a “memory molecule.” Binding of Ca*+/calmodulin to the regulatory domain of CaM kinase has three consequences. First, since the calmodulin-binding site overlaps with the autoinhibitory segment of the subunit, binding of Caz+/calmodulin disables the autoinhibitory domain and allows binding of ATP and peptide substrates to the active site. Calmodulin has been shown to wrap around its peptide-binding site, and it is likely that

theautoinhibitorydomain isdisplaced bythe binding of Ca2+/calmodulin (Meador et al., 1992; lkura et al., 1992). Second, binding of Ca2+/calmodulin exposes Thr2a6 so that it can be phosphorylated by an adjacent subunit. Monomeric kinase was only able to phoswas bound to phorylate Thr 286when Ca*+/calmodulin the target of the intermolecular reaction (Figure 6). The ability to propagate Thrza6 phosphorylation in the absence of Ca*+/calmodulin has been a key feature of previous memory models of CaM kinase (Lisman, 1985; Miller and Kennedy, 1986; Lisman and Goldring, 1988) but does not appear to occur in vitro. Displacement of the autoinhibitory segment byCa2+/calmodulin extends distally, from the calmodulin-binding site to the autonomy site, since binding of Ca2+/calmodulin has been shown to increase the accessibility of Hiszg to solvent (Smith et al., 1992). Thus, Thr2@ may only be accessible for autophosphorylation when it is exposed by the binding of Ca*+/calmodulin nearby. Electron microscopic studies suggest that the kinase may consist of octamers or decamers in which catalytic/regulatory domains are arranged in a circle around a central globular structure made up of the association domain of all subunits (Kanaseki et al., 1991). In such a holoenzyme, one subunit binding calmodulin would function as a “kinase” for a second proximatecalmodulin-bound subunit servingas”substrate.“The activated subunit can only phosphorylate when calmodulin “presents” it with Thr286 on a proximate subunit. Third, the inhibitory sites (Thr305 and Thr3ffi) which are within the calmodulin-binding domain are protected from autophosphorylation, whereas calmodulin is bound and only becomes autophosphorylated when calmodulin dissociates from an autonomous subunit (Patton et al., 1990; Hanson and Schulman,1992a).Thus,calmodulinregulatestheactivation of CaM kinase and determines which sites are to be autophosphorylated, facilitating phosphorylation of the autonomy site and blocking phosphorylation of the inhibitory sites. The intersubunit autophosphorylation may explain whytheautonomy site is within a prototypical consensus autophosphorylation site of Arg-Cln-Clu-Thr (Basic-X-X-Thr). Synthetic peptides based on the sequence surrounding Thr286 make excellent CaM kinase substrates, demonstrating that this site is well suited to be a substrate (Hanson et al., 1989; Ocorr and Schulman, 1991; Hanson and Schulman, 1992a). Furthermore, when freed of their association domains, recombinant kinase subunits are monomeric and phosphorylate each other on Thr*@j (Figure 5). By contrast Thr305 and Thr3%, sites of Ca2+-independent autophosphorylation, do not conform to consensus sites, do not make good substrates as peptides, and appear to be autophosphorylated by an intramolecular reaction (P. I. H. and H. S., unpublished data). An important consequence of the requirement for two molecules of calmodulin in generating the autonomous and trapped states is that activation of a single subunit will not necessarily lead to autophosphoryla-

tion of Thr286, the autonomy site. Activation and autophosphorylation (leading to molecular potentiation) need not always occur in tandem. Ca*+ signals leading to a kinase with a low calmodulin occupancy would activate individual calmodulin-bound subunits but would not necessarily potentiate each of these subunits (by autophosphorylation and calmodulin trapping) because the probability of proximate subunits with bound calmodulin would be low (Figure 7). Ca2+ signals leading to a higher level of calmodulin occupancy would enable autophosphorylation and resultant potentiation to occur; this level of Ca*+/calmodulin is the threshold for potentiation of CaM kinase activity. A threshold for autophosphorylation is important since autophosphorylation modulates the effective duration of a Ca*+ signal. Such a threshold decreases the probability of spurious autophosphorylation and potentiation. Low frequency Ca2+ signals may produce activation without potentiation, whereas high frequency Ca2’ signals would produce both a higher maximal activity (calmodulin-bound subunits) and a higher level of potentiation (autophosphorylated subunits). Discriminating between low and high Ca*+ signals may be important in vivo. For example, calcineurin, a Ca2+/calmodulin-dependent phosphatase which is sensitive to low levels of Ca”+/calmodulin, may be fully activated by low Ca2’ signals that result in long-term depression, whereas CaM kinase would only be activated by high Ca2’ signals that produce long-term potentiation (Lisman, 1989; Mulkey et al., 1993; Fukunaga et al., 1993). A distinctive feature of cellular Ca2+ signaling is the occurrence of oscillations or repetitive spikes in cellular Ca2+ levels (Berridge, 1990; Meyer and Stryer, 1991). Intracellular mechanisms for detecting and responding to these spikes are not well understood, and we have been interested in understanding how proteins may decode some of the dynamics peculiar to Ca*+ signals. Properties that would theoretically enable an enzyme such as CaM kinase to sense and respond to Ca2+ spikes were outlined in the introduction and include the presence of multiple activity states in each detector unit which enable it to register the cumulative effect of repeated submaximal spikes, a slow rate of deactivation of the detectorsto permit repetitive stimuli to produce additive effects, and cooperativity in generating the active state to produce a threshold for activation. Based on prior work as well as the results of the present study, we propose that CaM kinase is well suited to this role. As a multimer of individually active subunits, CaM kinase has multiple activity states; furthermore, it is likely that individual Ca*+ signals in vivo are submaximal for activation of this multimer owing to limitations in either Ca*+ or calmodulin (Meyer and Stryer, 1990; Stump0 et al., 1989; DuPont and Goldbeter, 1992). Because of its relatively low affinity for calmodulin, the kinase exhibits a fast deactivation (
Decoding 953

Calcium

Signals

reduce the rate of deactivation to several seconds or longer (Meyer et al., 1992). Whereas autophosphorylated subunits retain trapped calmodulin during the interspike interval, other calmodulin-binding proteins with rapid deactivation propertieswould replenish the “free” calmodulin so that additional calmodulin can bind to CaM kinase when the next Ca*+ spike arrives. Recruitment of calmodulin by repetitive submaximal Ca*+ spikes (Figure 8A) could lead to a progressive increase in the number of active subunits per CaM kinase holoenzyme. In essence, such a kinase would be a coincidence detector whose activity level increases incrementally when it is exposed to a Ca*+ spike while still retaining active subunits from a previous, closely spaced, stimulus. Furthermore, recruitment of calmodulin becomes increasingly more efficient since trapping becomes more efficient with increasing calmodulin occupancy (Figure 7). A CaM kinase multimer may therefore register the cumulative effect of repeated spikes with a steep threshold. In addition, it is conceivable that CaM kinase is tuned to a narrow range of oscillation frequencies because, at high frequencies, the interspike interval is short and may be insufficient to allow full equilibration of the free calmodulin pool and recruitment of calmodulin to the kinase. The properties of CaM kinase create a high threshold for significant activation and autophosphorylation but may enable subsequent maintenance of the activated state at lower frequencies of stimulation. The efficiency of converting a calmodulin-binding event to autophosphorylation and trapping increases as the number of autophosphorylated subunits per holoenzyme increases. Maintenance of the “on” state could occur with a lower stimulation frequency than the high threshold frequency needed to produce significant autophosphorylation. Maintenance of the on state may even be aided by basal Ca*+. Whereas spontaneous binding of calmodulin at basal Ca2+ would occur with a low probability, the efficiency of convertingsuch bindingtotrappingwould behigh.These properties may underlie the high degree of autonomous activity (8%-20%) found in hippocampal tissue, under apparent basal conditions (Molloy and Kennedy, 1991; Ocorr and Schulman, 1991). The readout of a CaM kinase molecule in the on state may be its abilityto phosphorylate substrates at basal Ca2+ for an extended period of time. Because of the abundance of this enzyme in brain, additional readouts may include noncatalytic interactions that are altered by its state of autophosphorylation and calmodulin binding or trapping and recruitment of significant amounts of calmodulin away from other calmodulin-dependent enzymes. Experimental

Procedures

Materials and Chemicals Except where indicated below, all materials and chemicals were those previously described (Lou and Schulman, 1989; Hanson and Schulman, 1992a). Dansylated calmodulin was synthesized

as described (Meyer et al., 1992). Porcine brain calmodulin was purchased from Ocean Biologics (Edmonds, WA). Ultra-pure sucrose was from Schwarz/Mann Biotechnology (Cleveland, OH). The CaM kinase substrate peptide autocamtide-3 has the sequence Lys-Lys-Ala-Leu-His-Arg-Gln-Glu-Thr-Val-Asp-AlaLeu and was synthesized and purified (>98%) by David King (University of California, Berkeley). The monoclonal antibody 12CA5 (mouse monoclonal, IgC2b), which recognizes the 9 amino acid epitope YPYDVPDYA from hemagglutinin HA1 (Wilson et al., 1984; Field et al., 1988), was kindly provided by I. Wilson and is now available commercially through Berkeley Antibody Company (Richmond, CA). Ascites containing monoclonal antibody recognizing the CaM kinase a subunit (CB-a-2) were prepared as previously described (Scholz et al., 1988). Immobilized protein A (coupled to Sepharose beads) was from Repligen Corporation (Cambridge, MA). All other chemicals were of reagent grade or better and were obtained from commercial sources. Site-Directed Mutagenesis and Construction of Expression Vectors Site-directed mutagenesis of a-CaM kinase (as M13-CaM kinase) was performed as described previously (Hanson and Schulman, 1992a; Waldmann et al., 1990). To truncate the kinase, a nonsense codon was introduced in place of VaP2’, thereby creating a-CaMK(l-326). Gly326 was also replaced with Val, so that the truncated kinase ended with Val. The oligonucleotide used for this mutagenesis was 5’-GCAnCCTTCTAGACATCAlTClTC-3 and introduced a new Xbal restriction site. To generate CaMK’, LYS~~was replaced with Met or Arg using the oligonucleotide 5’-ClTGATAATC[A,C]TGCCAGCATAC3’. Thr2% was replaced with Ala using 5’-GGCACTCCACGGCCTCCTCTCTCTG-3’. Sitedirected mutagenesis was also used to “loop-in” a sequence coding for the 18 amino acid “tag” sequence (creating CaMK*). The 90-mer oligonucleotide used had the sequence S-CAATCCCGTGCACCTCATCACCTCACCTCCACCTCCGGCGGCGTAGTCGGGGACGTCGTAACCATAAGCAGCCCGGTAGCCA-CCCTGGCACT-3’ and introduced the amino acid sequence GAPYPYDVPDYAGPCAQL between Thr’ and Ile”. Mutations introducing changes at the amino-terminal end of the kinase (Lys”‘-‘“, Lysamrs, and the tag of CaMK*) were subcloned into SRa-BKS (SRa-296 with the Bluescript KS polylinker between Pstl and Kpnl) as full-length Pstl-Kpnl cDNAs (1.5 kb). Mutants introducing changes in the central regulatory domain of the kinase (ThrZ86A’a; stop at 327) were transferred into SRa-aCaM kinase as 1100 bp Smal-Bglll fragments. Combinations of amino-terminal and regulatory domain mutationswereconstructed bysubcloningtheappropriate Smal-Bglll fragment into a parent SRa-a-CaM kinase plasmid containing the desired amino-terminal kinase sequence. Expression and Purification of CaM Kinase Transient transfection and harvesting of COS-7 cells were performed as described previously (Hanson et al., 1989; Hanson and SchuIman,1992a).Holoenzymescontainingamixtureofa-CaMK and a-CaMK* subunits were prepared by transfecting mixtures of the two SRa expression plasmids into COS-7 cells (typically IO pg of each plasmid; total of 20 pg of DNA per IO cm plate). Wild-type a-CaM kinase was purified essentially as described (Hanson and Schulman, 1992a), and the same procedures were used to purify a-CaMK* and a-CaMK’. A two column purification (phosphocellulose followed by calmodulin-Sepharose) was found to be almost as effective as the original three column procedure and was used in later purifications. The resulting a-CaM kinases were >90% pure as judged by Coomassie blue staining of 9% SDS-polyacrylamide gels. Purification of monomeric a-CaMK(l-326) differed since this kinase did not bind DEAE-cellulose (DE-52) at pH 7 and was instead found in the flow-through (see Figure 3A). A DEAE-cellulose step was therefore used to deplete the starting material of proteins that bind DE-52. Phosphocellulose chromatography was then carried out as for thewild-type enzyme, except that the column was washed with buffer A plus 0.1 N NaCl (buffer A contains 25 m M PIPES [pH 7’j, 1 m M EGTA, and 5 m M dithiothreitol) and eluted with buffer A plus 0.5 N NaCI. Calmodulin-Sepharose chromatog-

Neuron 954

raphy was as for wild-type enzyme (Hanson and Schulman, 1992a). Pools from a typical purification are shown in Figure 3A. a-CaMK(1-326)Ly~~~~” was purified using the same procedures, except that immunoblots or calmodulin blots were used to follow recovery of the inactive kinase. Sucrose Gradients Sedimentation coefficients of wild-type a-CaM kinase and a-CaMK(l-326) were measured relative to reference proteins as previously described (Martin and Ames, 1961; Kuret and Schulman, 1984). Sedimentation was carried out through 4.6 ml of 5%-20% linear sucrose gradients in 50 m M PIPES (pH 6.9), 150 m M NaCI, 1 m M EDTA, and 5% glycerol. One hundred microliters of sample (containing enzyme and three proteins as standards) was layered on top of the gradients which were then spun at 36,000 rpm for 14 hr in a Beckman SW50.1 rotor at 4OC. Fractions (150 ~1) were collected from the top of the gradients and assayed for calmodulin-stimulated phosphorylation of the CaM kinase peptide substrate autocamtide-3 and for protein by the method of Bradford (Bradford, 1976). Substrate Phosphorylation and Kinetic Analysis Kinase activity was routinely measured using the CaM kinase selective substrate autocamtide3 as described previously (Hanson and Schulman, 1992a). Standard Ca*+-stimulated assays were performed at 30°C for 15-40 s and contained 50 m M PIPES (pH 7.0), IO m M MgCI,, 200-600 VM CaCI,, 300-600 nM calmodulin, 100 pglml bovine serum albumin (BSA), 20-250 PM [yJ2P]ATP (1 Ci/mmol), 20 pM autocamtide-3, and CaM kinase as indicated. Assays of Ca*+independent activity contained 1 m M ECTA instead of CaC12 and calmodulin, but were otherwise identical. K, (ATP) was measured in reactions containing 300-600 nM calmodulin, 20 W M autocamtide-3, and ATP ranging from 1 to 200 FM. K, (autocamtide-3) was measured with 300 nM calmodulin, 250 PM ATP, and autocamtide-3 from 0.05 to 20 PM. Half-maximal calmodulin activation (K,,) was measured with 250 pM ATP, 20 PM autocamtide-3, and calmodulin from 1 to 600 nM. The apparent V,,, values for autocamtide-3 phosphorylation ranged from 7.7to 10 pmol/min/ng for a-CaMK and from IO to 12.9 pmol/min/ ng for a-CaMK(l-326). The protein concentration was derived from a quantitative amino acid composition determination. Values for both K, and V,,, were calculated by a direct linear and maximum likelihood estimation (Hanson and Schulman, 1992a). Autophosphorylation a-CaMK(l-326) was autophosphorylated at 30°C in 15-50 pl reactions containing 50 m M PIPES (pH 7.0), IO m M MgCI,, 200-600 VM CaC&, 600 nM calmodulin, 25-100 pglml BSA, and 50-250 PM [yJ2P]ATP (2,000-12,000 cpm/pmol). The concentration of a-CaMK(l-326) was varied between IO and 300 nM as indicated. Reactions were started by the addition of enzyme (typically 15 pl into a 50 pl reaction mix or 7 pl into a 15 PI mix); enzyme was diluted as needed in buffer (25 m M PIPES, 1 m M EGTA, 300 m M NaCI, 10% glycerol, lOOpg/ml BSA). Where indicated (e.g., Figure 4A), EGTA (3.3 m M final concentration) was added to initiate Ca*+-independent autophosphorylation. Reactions were stopped at the time indicated by addition of EDTA (16.7 m M final concentration) and/or 3x SDS-stop solution (3% SDS, 8.3% glycerol, 62 m M Tris [pH 6.71, 3 m M !3-mercaptoethanol). Samples were either resolved on 10% SDS-polyacrylamide gels and subjected to autoradiography as described (Lou et al., 1986) or used for subsequent activity assays as described above. Cerenkov radiation in bands excised from SDS gels was quantified in a Beckman liquid scintillation spectrophotometer (LS3801). Background s2P in the gel was corrected for by counting and subtracting the Cerenkov radiation in comparably sized bands from blank areas of a gel lane. lmmunoprecipitation Samples (40 pg of transfected cell cytosol) were diluted in Triton immunoprecipitation buffer (20 m M Tris-HCI [pH 7.41, 5 m M EDTA, 150 m M NaCI, 1.1% Triton X-100,1 m M o-phenanthroline,

2 m M phenylmethylsulfonyl fluoride) and immunoprecipitated with 1 Kg of 12CA5 IgG or CB-a-2 (2H5) for 2 hr overnight at 4OC. Seventy microliters of a 30% Repligen bead (protein A-Sepharose) slurry was then added and incubated for an additional 60 min, after which the beads were extensively washed prior to analysis on SDS gels. Model for Intermolecular Autophosphorylation between Monomers Wecan represent the intermolecular autophosphorylation tion between two kinase monomers as

CaMK + CaMK & k,

reac-

(CaMK - CaMK) II (CaMKP - CaMK)

(1)

fast

+ CaMKP + CaMK.

If the total concentration of CaMK in the reaction is x and the concentration of (CaMK-CaMK) dimers is z, then the concentration of free CaMK monomers is x - 2z. Assuming that binding intoa(CaMK-CaMK)dimer is independentofamonomer’s phosphorylation status, the equilibrium constant K is

The (CaMK-CaMK) dimer population consists of four possible combinations of phosphorylated and unphosphorylated CaMK monomers: (CaMKO-CaMKO), (CaMKO-CaMKP), (CaMKP-CaMKO), and (CaMKP-CaMKP), ordered in parentheses as (substrate subunit, kinase subunit). Toquantitatethetimecoursefor kinasemonomerphosphorylation, we define the fraction of phosphorylated CaMK molecules (CaMKP) as y (0 Q y =G 1); the concentration of CaMKP is xy, and the concentration of CaMKO is x(1 - y). The fraction of (CaMK-CaMK) dimers which are (CaMKO-CaMKO) is therefore given by (1 - y)‘; (CaMKO-CaMKS by y(1 - y); (CaMKP-CaMKn) by y(1 - y); and (CaMKP-CaMKP) by yZ. We define a’ as the autophosphorylation reaction rate for either a (CaMKO-CaMKO) dimer going to (CaMKP-CaMKS or a (CaMKO-CaMKP) dimer going to (CaMKP-CaMKP); (CaMKPCaMKO)and (CaMK”-CaMKP)dimerscannot undergo phosphorylation. Assuming a rate p for intramolecular phosphorylation, CaMKP concentration then changes at the rate

f

[CaMKPl = x!$

= (1 - y)2 za’ + ~(1 - y)za’ +

x(1 - y)p -(back Ignoring

the back reactions

reactions).

and defining (4)

with x and z(x) constant,

this simplifies

to

Then, y(x,t) = 1 - emdrif y(t = 0) = 0. We can explicitly by solving for z in the equilibrium constant equation,

give a(x)

6) This can be Taylor

expanded

for small x, (7-I

Decoding 955

Calcium

Signals

Since the first term predominates is given by

for the present

data, and a(x)

a’Z-+p3 ; X+p, X 0

hereby marked “advertisement” in accordance tion 1734 solely to indicate this fact. Received

November

23,1993;

Berridge, 9586.

M. (1990). Calcium

revised

with 18 USC Sec-

February

23,1994.

only the ratio

Berridge, M. J. (1993). lnositol ling. Nature 367, 315-325. can be well constrained by the data. We therefore fit the data to the parameterized model function Y = N . (I- e-“n) in which N = 1 is a normalization constant. Minimizing the chi-squared function for Y yielded the best fit values for a and N given in the figure legends. Fits with the full expression for z(x) gave consistent values, but converged only if K was held fixed at a reasonable value. The fits held p = 0 fixed, since the data are consistent only with ax >> p; the rate of intrasubunit autophosphorylation does not exceed 10% of the rate of the intersubunit reaction at the lowest kinase concentration used. Calmodulin Trapping Fluorescence anisotropy measurements were carried out using a custom-made instrument as described (Meyer et al., 1992). To determine the fraction of trapped calmodulin, a Ca*-ECTA mixture was added to lower free Caz+ to approximately 200 nM. CaMF then dissociated with a fast and slow component from unphosphorylated and phosphorylated subunits, respectively. The fraction of trapped CaMF was then determined as the amplitudeofanexponential fittotheslowcomponentof CaMFdissociation, A(t) = A e(m with f,,d = A/AA. AA was the initial anisotropy change when kinase was added. The same average dissociation time of r = 16 s was used for all fits. Computer Modeling Computer simulation of the response of CaM kinase to variable frequencies of Ca* oscillations was carried out using Visual Basic. Ca2+ spikes were assumed to be 1 s long at all frequencies. The fraction of calmodulin bound but not trapped, f&d, is defined as, fbounf = f, . (1 - f,,*,) during Ca*+ spikes and fb, = 0 between spikes. The rate of trapping is given by df,,a,/dt

= R . fbaund . hmu”d + f,,)

- P . ftrlp,

(10)

in which f,,+ is the fraction of calmodulin-binding sites with trapped calmodulin; f., here assumed to be 5%, is the fractional occupancy at equilibrium of the low affinity calmodulin-binding sites (with unphosphorylated autonomy site); R is the rate of autoptiosphorylation for fully occupied kinase and is assumed to be 5 s-j; and P is the rate of dephosphorylation of Thr%, assumed to be 0.03 0. The cooperativity of the trapping process is reflected in the term f&d . (fbound+ f,,), and the slow dephosphorylation rate of the autonomy site is here assumed to be independent of calmodulin occupancy. It is assumed that trapped calmodulin does not significantly dissociate between individual spikes. Acknowledgments This study was supported by National Institutes of Health grants GM30179 to H. S. and GM24032 and MH45324 to L. 5. and PHS training grant CA09302 to P. I. H. We wish to thank Lillian Lou for early studies with monomeric CaM kinase, Geoff Rosenfeld and Michael Kapiloff for the monomeric construct, Stephen Selipsky for derivation of the equation for autophosphorylation between monomers, Samantha Brown for helpwith site-directed mutagenesis, and Paul Nghiem for helpful discussions. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be

oscillations.

J. Biol. Chem.265,9583-

trisphosphate

and calcium

signal-

Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. B&hem. 72, 248-254. Colbran, R. J., and Soderling, T. R. (1990). Calcium/calmodulindependent protein kinase II. Curr. Topics Cell. Reg. 37,181~221. Cruzalegui, F. H., Kapiloff, M. S., Morfin, J. P., Kemp, B. E., Rosenfeld, M. C., and Means, A. R. (1992). Regulation of intrasteric inhibition of the multifunctional calciumtalmodulindependent protein kinase. Proc. Natl. Acad. Sci. USA 89,12127-12131. DuPont, G., and Goldbeter, cytosolic calcium: insights 14,4x393.

A. (1992). Oscillations and waves of from theoretical models. Bioessays

Estep, R. P., Alexander, K. A., and Storm, D. R. (1989). Regulation of free calmodulin levels in neurons by neuromodulin: relationship to neuronal growth and regeneration. Curr. Topics Cell. Reg. 37,161-180. Field, J., Nikawa, J.-l., Broek, D., MacDonald, B., Rodgers, L., Wilson, I. A., Lerner, R. A., and Wigler, M. (1988). Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol. Cell. Biol. 8, 2159-2165. Fong,Y.-L.,Taylor, W. L., Means,A. R., and Soderling,T. R. (1989). Studies of the regulatory mechanism of Ca*/calmodulindependent protein kinase II. Mutation of threonine 286 to alanine and aspartate. J. Biol. Chem. 264, 16759-16763. Fukunaga, K., Rich, D. P., and Soderling, T. R. (1989). Generation of the Ca*-independent form of Ca*/calmodulindependent protein kinase II in cerebellar granule cells. J. Biol. Chem. 264, 21830-21836. Fukunaga, K., Stoppini, L., Miyamoto, E., and Muller, D. (1993). Long-term potentiation is associated with an increased activity of Caz+/calmodulindependent protein kinase. J. Biol. Chem. 268, 7863-7867. Cough, A. H., and Taylor, D. L. (1993). Fluorescence anisotropy imaging microscopy maps calmodulin binding during cellular contraction and locomotion. J. Cell Biol. 727,1095-1107. Hanson, P. I., and Schulman, H. (1992a). Inhibitory autophosphorylation of multifunctional Ca*/calmodulindependent pro tein kinaseanalyzed bysitedirected mutagenesis. J. Biol. Chem. 267,17216-17224. Hanson, P. I., and Schulman, H. (1992b). Neuronal Ca*/calmoduIindependent protein kinases. Annu. Rev. Biochem. 67,559-601. Hanson, P. I., Kapiloff, M. S., Lou, L. L., Rosenfeld, Schulman, H. (1989). Expression of a multifunctional modulindependent protein kinase and mutational its autoregulation. Neuron 3, 59-70.

M. G., and Ca%alanalysis of

Ikura, M., Clore, G. M., Fronenborn, A. M., Zhu, C., Klee, C. B., and Bax, A. (1992). Solution structureof acalmodulin-target peptide complex by multldimensional NMR. Science 256, 632638. Ip, N. Y., and Zigmond, R. E. (1984). Pattern of presynaptic nerve activity can determine the type of neurotransmitter regulating a postsynaptic event. Nature 377, 472-474. Kanaseki, T., Ikeuchi, Y., Sugiura, H., and Yamauchi, T. (1991). Structural features of Ca*/calmodulindependent protein kinase II revealed by electron microscopy. J. Cell Biol. 775,1049-1068. Klee, C. B. (1991). Concerted

regulation

of protein

phosphoryla-

Neuron 956

tion and dephosphorylation 76, 1059-1065.

by calmodulin.

Neurochem.

Res.

Kuret, J., and Schulman, H. (1984). Purification and characterization of a Ca*+/calmodulin-dependent protein kinase from rat brain. Biochemistry 23, 5495-5504.

Ocorr, K. A., and Schulman, H. (1991). Activaton of multifunctional Ca2+/calmodulin-dependent kinase in intact hippocampal slices. Neuron 6, 907-914.

Kuret, J., and Schulman, H. (1985). Mechanism of autophosphorylation of the multifunctional Ca2+lcalmodulin-dependent protein kinase. J. Biol. Chem. 260, 64276433.

Patton, B. L., Miller, S. G., and Kennedy, M. B. (1990). Activation of type II calciumicalmodulin-dependent protein kinase by Ca2+/ calmodulin is inhibited by autophosphorylation of threonine withinthecalmodulin-bindingdomain.J.Biol.Chem.265,~120411212.

Lai, Y., Nairn, A. C., and Greengard, P. (1986). Autophosphorylation reversibly regulates the Ca2+/calmodulin-dependence of Ca2+/calmodulin-dependent protein kinase II. Proc. Natl. Acad. Sci. USA 83, 4253-4257.

Scholz, W. K., Baitinger, C., Schulman, H., and Kelly, P. T. (1988). Developmental changes in Ca2+/calmodulin-dependent protein kinase II in cultures of hippocampal pyramidal neurons and astrocytes. J. Neurosci. 8, 1039-1051.

Lisman, J. E. (1985). A mechanism for memory storage insensitive to molecular turnover: a bistable autophosphorylating kinase. Proc. Natl. Acad. Sci. USA 82, 3055-3057.

Schulman, H., Hanson, P. I., and Meyer, T. (1992). Decoding calcium signals by multifunctional CaM kinase. Cell Calcium 73, 401-411.

Lisman, J. (1989). A mechanism processes underlying learning Sci. USA 86, 9574-9578.

Skene, J. H. P. (1990). GAP-43 as a’calmodulin sponge’and some implications for calcium signalling in axon terminals. Neurosci. Res. 73 (SuppI.), S112-S125.

for the Hebb and the anti-Hebb and memory. Proc. Natl. Acad.

Lisman, J. E., and Coldring, M. A. (1988). Feasibility of long-term storage of graded information by the Ca2+/calmodulin-dependent protein kinase molecules of the postsynaptic density. Proc. Natl. Acad. Sci. USA 85, 5320-5324.

Smith, M. K., Colbran, R. J., Brickey, D.A., and Soderling, T. R. (1992). Functional determinants in the autoinhibitory domain of calciumlcalmodulin-dependent protein kinase It. Role of His282 and multiple basic residues. J. Biol. Chem. 267, 1761-1768.

Lou, L. L., and Schulman, H. (1989). Distinctautophosphorylation sites sequentially produce autonomy and inhibition of the multifunctional Ca2+/calmodulin-dependent protein kinase. J. Neurosci. 9, 2020-2032.

Stumpo, D. J., Craff,J. M.,Albert, K.A., Greengard, P., and Blackshear, P. J. (1989). “80- to 87-kDa” myristoylated alanine-rich C kinase substrate: a major cellular substrate for protein kinase C. Proc. Natl. Acad. Sci. USA 86, 4012-4016.

Lou, L. L., Lloyd, S. J., and Schulman, H. (1986). Activation of the multifunctional Ca*+/calmodulin-dependent protein kinase by autophosphorylation: ATP modulates production of an autonomous enzyme. Proc. Natl. Acad. Sci. USA 83, 9497-9501.

Taylor, S. S., Buechler, J. A., and Yonemoto, W. (1990). CAMPdependent protein kinase: framework for a diverse family of regulatory enzymes. Annu. Rev. Biochem. 59, 971-1005.

MacNicol, M., and Schulman, H. (1992a). Cross-talk between protein kinase C and multifunctional Ca2+lcalmodulin-dependent protein kinase. J. Biol. Chem. 267, 12197-12201. MacNicol, pathways 237-240.

M., and Schulman, H. (199213). Multiple Ca2+ signaling converge on CaM kinase in PC12 cells. FEBS Lett. 304,

MacNicol, M., Jefferson, A. B., and Schulman, H. (1990). Ca2+/ calmodulin kinase is activated by the phosphatidylinositol signaling pathway and becomes Ca*+independent in PC12 cells. J. Biol. Chem. 265, 18055-18058. Martin, R. G., and Ames, B. N. (1961). A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J. Biol. Chem. 236, 1372-1379. Meador, W. E., Means, A. R., and Quiocho, F. A. (1992). Target enzyme recognition bycalmodulin:2.4Astructureofacalmodulin-peptide complex. Science 257, 1251-1255. Meyer, T., and Stryer, L. (1990).Transient calcium release induced by successive increments of inositol 1,4,5trisphosphate. Proc. Natl. Acad. Sci. USA 87, 3841-3845. Meyer, T., and Stryer, L. (1991). Calcium phys. Chem. 20, 153-174.

spiking.

Meyer, T., Hanson, P. I., Stryer, L., and Schulman, modulin trapping by calcium-calmodulin-dependent nase. Science 256, 1199-1201.

Annu.

Rev. Bio-

H. (1992). Calprotein ki-

Miller, S. C., and Kennedy, M. B. (1985). Distinct forebrain and cerebellar isozymes of type II Ca*+/calmodulin-dependent protein kinase associate differently with the postsynaptic density fraction. J. Biol. Chem. 260, 9039-9046. Miller, S. C., and Kennedy, M. B. (1986). Regulation of brain type II Ca2+/calmodulin-dependent protein kinase byautophosphory lation: a Ca*+-triggered molecular switch. Cell 44, 861-870. Molloy, S. S., and Kennedy, M. B. (1991). Autophosphorylation of type II Ca*+/calmodulin-dependent protein kinase in cultures of postnatal rat hippocampal slices. Proc. Natl. Acad. Sci. USA 88, 4756-4760. Mulkey, R. M., Herron, C. E.,and Malenka, R. C. (1993). An essential role for protein phosphatases in hippocampal long-term depression. Science 267, 1051-1055.

Waldmann, R., Hanson, P. I., and Schulman, H. (1990). Multifunctional Ca2+/calmodulin-dependent protein kinase made Ca2’independent for functional studies. Biochemistry29,1679-1684. Waxham, M. N., Aronowski, J., Westgate, S. A., and Kelly, P. T. (1990). Mutagenesis of Th$-285 in monomeric Ca*+/calmodulindependent protein kinase II eliminates Ca2+/calmodulin-independent activity. Proc. Natl. Acad. Sci. USA 87, 1273-1277. Wilson, I. A., Niman, H. L., Houghten, R. A., Cherenson, Connolly, M. L., and Lerner, R. A. (1984). The structure antigenic determinant in a protein. Cell 37, 767-778.

A. R., of an

Yamauchi, T., Ohsako, S., and Deguchi, T. (1989). Expression and characterization of calmodulin-dependent protein kinase II from cloned cDNAs in Chinese hamster ovary cells. J. Biol. Chem. 264, 19108-19116.