Biophysical Journal Volume 70 June 1996 2493-2501 2493 A Mechanism for Synaptic Frequency Detection through Autophosphorylation of CaM Kinase 11 A. Dosemeci,* and R. W. Alberst *Laboratory of Neurobiology and *Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892 ABSTRACT A model for the regulation of CaM kinase 11 is presented based on the following reported properties of the molecule: 1) the holoenzyme is composed of 8-12 subunits, each with the same set of autophosphorylation sites; 2) autophosphorylation at one group of sites (A sites) requires the presence of Ca2+ and causes a subunit to remain active following the removal of Ca2+; 3) autophosphorylation at another group of sites (B sites) occurs only after the removal of Ca2+ but requires prior phosphorylation of a threshold number of A sites within the holoenzyme. Because B-site phosphorylation inhibits Ca2+/calmodulin binding, we propose that, for a given subunit, phosphorylation of a B site before an A site prevents subsequent phosphorylation at the A site and thereby locks that subunit in an inactive state. The model predicts that a threshold activation by Ca2+ will initiate an "autophosphorylation phase." Once started, intra-holoenzyme autophosphory- lation will proceed, on A sites during periods of high [Ca2+] and on B sites during periods of low [Ca2+]. At "saturation," that is when every subunit has been phosphorylated on a B site, the number of phosphorylated A sites and, therefore, the kinase activity will reflect the relative durations of periods of high [Ca2+] to periods of low [Ca2+] that occurred during the autophosphorylation phase. Using a computer program designed to simulate the above mechanism, we show that the ultimate state of phosphorylation of an array of CaM kinase 11 molecules could be sensitive to the temporal pattern of Ca2+ pulses. We speculate that such a mechanism may allow arrays of CaM kinase 11 molecules in postsynaptic densities to act as synaptic frequency detectors involved in setting the direction and level of synaptic modification. INTRODUCTION The temporal pattern of synaptic activity can be an impor- levels in the brain, particularly in the hippocampus (Ouimet tant determinant in long term modification of synaptic ef- et al., 1984; Erondu and Kennedy, 1985; Fukunaga et al., ficacy. For example, in the CAl region of the hippocampus, 1988). The enzyme occurs in soluble form throughout neu- Dudek and Bear (1992) established that afferent stimulation rons and is a major component of postsynaptic densities consisting of a few hundred pulses delivered at 1-3 pulses (Kennedy et al., 1983; Kelly et al., 1984; Dosemeci and per second induces long term depression (LTD), whereas Reese, 1993). Numerous studies indicate that the induction the same number of pulses delivered at 50 pulses per second of LTP requires CaM kinase II activation (Malenka et al., induces long term potentiation (LTP). It is proposed that 1989; Malinow et al., 1989; Ito et al., 1991; Fukunaga et al., both phenomena require a postsynaptic increase of [Ca2+] 1993; Pettit et al., 1994). In addition, gene knockout studies via NMDA receptors (For reviews see Malenka and Nicoll, show that both LTP and LTD are either absent or attenuated 1993; Bear and Malenka, 1994). Again, in the same region in mice lacking the gene for a-CaM kinase II (Silva et al., of the hippocampus, prior synaptic activity of a certain 1992; Stevens et al., 1994). pattern can inhibit induction of LTP (Huang et al., 1992). A hypothesis to explain the bidirectional effect of Ca21 in These observations indicate the presence of a molecular inducing both potentiation and depression is based on a system for the detection of temporal patterns. model by Lisman (1989) that proposes that high levels of A mechanism by which Ca2+ transients induce long term Ca2+ will result in increased autophosphorylation of CaM changes in the nervous system may be through the regula- kinase II, whereas intermediate levels of Ca2+ will cause its tion of Ca2+/calmodulin-dependent protein kinase II (CaM dephosphorylation by selective activation of phosphatases. kinase II) activity. CaM kinase II is a en- Ca2+-activated The hypothesis assumes that low frequency and high fre- zyme that becomes Ca2+-independent upon autophosphor- stimulation will ylation (Miller and Kennedy, 1986). It is expressed at high quency patterns produce intermediate and high levels of Ca2+, respectively. Although this hypothesis is consistent with numerous findings, Malenka (1994) has Received for publication 4 December 1995 and in final form 19 March pointed out 1) that experimental manipulations expected to 1996. give rise to intermediate Ca21 levels do not necessarily Abbreviations: Long Term Potentiation (LTP); Long Term Depression produce LTD, and 2) that this model does not explain the (LTD); Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) need for "prolonged and patterned afferent stimulation" to Address reprint requests to Dr. Ayse Dosemeci, Laboratory of Neurobiol- ogy, Building 36, 2A21, National Institutes of Health, Bethesda, MD achieve homosynaptic LTD. 20892. Tel.: 301-496-1296; Fax: 301-480-1485; E-mail: The forgoing hypothesis is based on the assumption that [email protected]. the frequencies of presynaptic firing patterns determine the C 1996 by the Biophysical Society levels of postsynaptic Ca2 . It is also likely that the pre- 0006-3495/96/06/2493/09 $2.00 synaptic firing pattern is conveyed postsynaptically as a 2494 Biophysical Joumal Volume 70 June 1996 pattern or "frequency" of postsynaptic Ca2+ transients; that kinase II subunits; in neurons the main types are a and P3. Because the a is, the presynaptic firing frequency may influence not only form appears to predominate in the forebrain (Kanaseki et al., 1991), we the amplitude but also the frequency of the postsynaptic will discuss our model in terms of the a-isoform. However, it should be noted that the properties of the ,3-isoform are also compatible with the Ca2+ signal. In this case, a mechanism for the detection of requirements of the model. postsynaptic Ca2+-pulse frequency is needed to translate the A particular threonine residue (Thr-286) is phosphorylated in the pres- temporal pattern of activity into synaptic modification. The ence of Ca2+. This process of phosphorylation is intraholoenzyme and "calmodulin trapping" model proposed by Meyer et al. intersubunit (Hanson et al., 1994; Mukherji and Soderling, 1994). We have (1992) suggests that CaM kinase II may discriminate tem- designated these residues as A sites in this paper. Experiments using various autonomous forms of the enzyme indicate that, even when Ca21/ poral patterns of Ca2+ under certain conditions. calmodulin is not needed for enzymatic activity, A-site phosphorylation The calmodulin trapping model for the detection of Ca2+- cannot occur unless Ca2+/calmodulin binds to the regulatory portion of the pulse frequency by CaM kinase II (Meyer et al., 1992; subunit, presumably to "expose" the site (Hanson et al., 1994; Brickey et Hanson et. al., 1994) is based on the observation that al., 1994; Colbran et al., 1988). Thus, A-site phosphorylation cannot autophosphorylation on Thr-286 increases the happen in the absence of Ca2 . Phosphorylation of an A site generates enzyme's Ca2+-independent activity, which is, however, less than the maximal affinity for calmodulin. Because of this, when calmodulin activity in the presence of Ca2' (Miller and Kennedy, 1986; Hanson et al., concentration is limiting, repetitive Ca2+ pulses will have a 1989). According to a recent study, this Ca2+-independent kinase activity cooperative effect in promoting autophosphorylation and toward exogenous substrates increases with the number of A sites being consequent kinase activity. However, because the dissocia- phosphorylated (Ikeda et al., 1991). a of subunits tion time of calmodulin from a subunit phosphorylated on Following the A-site phosphorylation of threshold number per holoenzyme, another set of sites becomes subject to autophosphoryla- Thr-286 is on the order of seconds, trains of Ca2+ pulses tion. Phosphorylation of particular residues located within the calmodulin- with intervals <1 s could not be differentiated by this binding domain (Thr-305/306) occurs only after calmodulin dissociates mechanism. More importantly, according to this model, from the enzyme upon removal of Ca2' (Patton et al, 1990). We have prolonged exposure of the molecule to Ca2+ pulses, even at designate these residues as B sites. Phosphorylation of B sites inhibits and thus blocks Ca21 low frequency, will eventually lead to maximal Ca2+-de- binding of Ca2+/calmodulin -dependent activity (Lickteig et al., 1988; Patton et al., 1990; Hanson and Schulman, 1992). pendent autophosphorylation and thus to maximal autono- Both intra- and intersubunit phosphorylation of B sites appears to occur. mous activity. That is, prolonged low frequency and high Hanson et. al. (1994) presented data indicating that Ca2'-independent frequency Ca2+ pulses will have identical effects. phosphorylation can occur by an intrasubunit reaction. Mukherji and In this article we propose a different model which takes Soderling (1994) observed that although basal autophosphorylation at a an intersubunit into account the experimental observations that CaM kinase Thr-306 is intrasubunit in nonautonomous enzyme, phos- phorylation of the same residue occurs when the enzyme is first made II has at