Calmodulin-Dependent Protein Kinase II–Protein Phosphatase 1 Switch Facilitates Specificity in Postsynaptic Calcium Signaling

An ultrasensitive Ca2؉͞calmodulin-dependent protein kinase II–protein phosphatase 1 switch facilitates specificity in postsynaptic calcium signaling J. Michael Bradshaw*†‡, Yoshi Kubota*, Tobias Meyer†, and Howard Schulman* Departments of *Neurobiology and †Molecular Pharmacology, Stanford University School of Medicine, Stanford, CA 94305 Edited by Roger A. Nicoll, University of California, San Francisco, CA, and approved July 21, 2003 (received for review May 7, 2003) The strength of hippocampal synapses can be persistently in- hence would provide a distinct Ca2ϩ activation region for creased by signals that activate Ca2؉͞calmodulin-dependent pro- CaMKII compared with other Ca2ϩ-activated enzymes at the tein kinase II (CaMKII). This CaMKII-dependent long-term potenti- synapse. In fact, it has been hypothesized that the signaling ation is important for hippocampal learning and memory. In this network controlling CaMKII autophosphorylation is a bistable work we show that CaMKII exhibits an intriguing switch-like type of switch that allows CaMKII to remain autophosphory- activation that likely is important for changes in synaptic strength. lated long after Ca2ϩ returns to a basal level (16). We found that autophosphorylation of CaMKII by itself showed a In this work we demonstrate experimentally that CaMKII 2؉ Ϸ ϩ steep dependence on Ca concentration [Hill coefficient (nH) 5]. responds in a switch-like fashion to Ca2 : CaMKII transitions 2؉ Ϸ However, an even steeper Ca dependence (nH 8) was observed rapidly from little to near-total autophosphorylation over a when autophosphorylation is balanced by the dephosphorylation narrow range of Ca2ϩ. Interestingly, this switch-like response was activity of protein phosphatase 1 (PP1). This autophosphorylation– enhanced by the presence of protein phosphatase 1 (PP1). dephosphorylation switch was found to be reversible because PP1 Together, CaMKII and PP1 allow CaMKII to remain dephos- ؉ dephosphorylates CaMKII when Ca2 is lowered to a basal level. phorylated in response to subthreshold Ca2ϩ signals (such as The switch-like response of a CaMKII-PP1 system suggests that those that cause LTD) but fully activate in response to strong CaMKII and PP1 may function together as a simple molecular device signals (such as those that cause LTP). This provides a mecha- 2؉ that specifically translates only strong Ca signals into all-or-none nism to help explain how modestly different Ca2ϩ levels can potentiation of individual hippocampal synapses. selectively activate different signaling pathways at the synapse. Furthermore, a CaMKII-PP1 molecular switch may provide a ϩ n the CA1 region of the hippocampus, postsynaptic Ca2 influx biochemical basis for the observed all-or-none potentiation of Ican elicit different effects on synaptic plasticity including individual hippocampal synapses. long-term potentiation (LTP), long-term depression (LTD), or no change in synaptic strength (1). Different biochemical sig- Materials and Methods naling pathways are responsible for these diverse changes, with Protein Purification. ␣CaMKII and CaM were purified as de- ϩ the magnitude of Ca2 stimulus largely determining the pathway scribed (17, 18). Purified, unstimulated CaMKII shows no that becomes activated (2). However, the dynamic range of evidence of basal Thr-286 autophosphorylation as evaluated by ϩ [Ca2 ] within dendritic spines is modest compared with other both a lack of autonomous activity and a failure to be recognized systems, varying from Ϸ100 nM under basal conditions to Ϸ10 by a Thr-286 phospho-specific antibody. ␮M on strong stimulation (3). How different pathways are ␣ ϩ The His-tagged PP1 catalytic subunit in the vector pDR540 activated with specificity over this relatively small range of Ca2 (Pharmacia) was a gift of Angus Nairn (Yale University, New is not well understood. ϩ Haven, CT). Escherichia coli cells were grown at room temper- The primary effector of Ca2 signals that elicit LTP is ature in 0.5 liters of LB with ampicillin (50 ␮g͞ml) and 100 ␮M 2ϩ͞ Ca calmodulin (CaM)-dependent protein kinase II (CaMKII) CoCl until OD ϭ 0.4 nm, induced overnight with 0.5 mM ͞ 2 (4–6). CaMKII is a multifunctional serine threonine kinase that isopropyl ␤-D-thiogalactoside, and harvested. Cells were resus- is highly abundant in neurons, particularly dendritic spines (7, 8). pended in 10 mM Tris⅐HCl, pH 8.0͞30 mM imidazole͞10% 2ϩ͞ Binding of Ca CaM both activates CaMKII toward down- glycerol͞300 mM NaCl͞1 mM CoCl (resuspension buffer), ␣ 2 stream effectors such as the -amino-3-hydroxy-5-methyl-4- disintegrated by French press, and centrifuged at 30,000 ϫ g for isoxazolepropionate receptor (9) and recruits it to the synapse 30 min. The supernatant was loaded onto a 5-ml nickel- (10). In addition, CaM binding facilitates autophosphorylation nitrilotriacetic acid agarose column (Qiagen, Valencia, CA) ␣ of CaMKII at Thr-286 (in CaMKII). This autophosphorylation equilibrated in resuspension buffer. The column was washed in has the important consequence of allowing CaMKII to retain 20 mM Hepes, pH 7.4͞200 mM KCl͞2mMMg2ϩ͞10% glycerol enzymatic activity in the absence of Ca2ϩ (autonomy), thus 2ϩ (wash buffer) and eluted with wash buffer plus 400 mM imida- sustaining CaMKII activity after diminution of the initial Ca zole. Fractions were pooled to 95% purity at 16.6 ␮M PP1 and signal (11). The biological significance of CaMKII autophos- immediately snap-frozen. phorylation at Thr-286 has been demonstrated by using a PP1 catalytic subunit purified from E. coli has been shown to CaMKII ‘‘knock-in’’ mouse that cannot autophosphorylate (12). have properties that differ from PP1 purified from tissue (19). This mouse does not display hippocampal LTP and is deficient However, the PP1 purified here in CoCl resulted in decreased in spatial learning. 2 2ϩ p-nitrophenyl phosphate activity compared with PP1 purified in In contrast to LTP, Ca influx elicited by LTD-inducing the absence of CoCl , suggesting that CoCl -purified PP1 is stimuli seems not to activate CaMKII but rather the Ca2ϩ- 2 2 sensitive phosphatase calcineurin (13). How might multiple 2ϩ pathways at the synapse share Ca as their activator yet achieve This paper was submitted directly (Track II) to the PNAS office. nearly opposite responses? One mechanism for amplitude-based Abbreviations: LTP, long-term potentiation; LTD, long-term depression; CaM, calmodulin; response specificity is for CaMKII to respond in a switch-like CaMKII, Ca2ϩ͞CaM-dependent protein kinase II; PP1, protein phosphatase 1; PSD, postsyn- ϩ fashion to Ca2 signals (14, 15). This type of response would aptic density. ϩ narrow the range of Ca2 where CaMKII becomes activated and ‡To whom correspondence should be addressed. E-mail: [email protected]. 10512–10517 ͉ PNAS ͉ September 2, 2003 ͉ vol. 100 ͉ no. 18 www.pnas.org͞cgi͞doi͞10.1073͞pnas.1932759100 Downloaded by guest on September 24, 2021 enzymatically more similar to tissue-purified PP1 than PP1 purified from E. coli in the absence of CoCl2. CaMKII Autophosphorylation and the CaMKII-PP1 Signaling System. CaMKII phosphorylation experiments were performed in 40 ␮l of wash buffer plus 50 ␮M CaM, 2 mM ATP, and varying Ca2ϩ at 0°C. Eight microliters of a 5ϫ solution of CaM, Mg2ϩ, and ATP was first added to 4 ␮lofa10ϫ solution of Ca2ϩ stock buffer. Solutions of wash buffer and, if applicable, PP1 then were added to 20 ␮l. Twenty microliters of 2ϫ CaMKII solution was added to initiate the reaction. Experiments were performed at 0°C to avoid the time-dependent decrease in enzyme activity during autophosphorylation that CaMKII exhibits at higher Fig. 1. CaMKII autophosphorylation is cooperative to Ca2ϩ. Plotted is the temperatures. Performing experiments at 0°C prevents auto- CaMKII autophosphorylation level (indicated by the maximum CaMKII auton- 2ϩ Ϯ phosphorylation from occurring at the inhibitory sites on omy) after 5 min versus [Ca ] [error bars are SD; the number of autonomous activity values determined (n) was 2]. The solid line is the best fit to the Hill CaMKII (Thr-305 and Thr-306); this simplifies the CaMKII equation (Eq. 1). Here, [CaMKII] ϭ 0.2 ␮M. All shown Ca2ϩ titration data are activation process but does not explore the role of Thr-305 and representative data obtained from at least two, and often three or more, data 2ϩ Thr-306 in the Ca -dependent activation of CaMKII. sets collected at each condition. For pairs of experiments where CaMKII was initially either dephosphorylated or phosphorylated, two stock Ca2ϩ buffers (‘‘high’’ and ‘‘low’’) were used. It was calculated that the alone or along with 5 mM EGTA. The autonomy of CaMKII was combination of these two Ca2ϩ solutions would give the final assessed at 2, 5, 15, and 30 min. desired [Ca2ϩ]. For experiments where CaMKII was initially ϩ -dephosphorylated, 2 ␮l of both Ca2ϩ stocks was added to 8 ␮lof Ca2؉͞EGTA Buffer. In all experiments [Ca2 ] was controlled pre ϫ ͞ ͞ 2ϩ͞ the 5 solution of ATP MgCl2 CaM; the additional solution cisely by using a 10-fold concentrated system of Ca EGTA ϩ components then were added to initiate the reaction. However, buffer (Molecular Probes). The volume of K ͞EGTA and ϩ for experiments where CaMKII was initially phosphorylated, Ca2 ͞EGTA stock solutions to be combined was calculated ϩ only the 2-␮l high Ca2ϩ stock was initially added to 8 ␮lof based on the determined dissociation constant of Ca2 for ͞ ͞ ATP MgCl2 CaM. Twenty microliters of CaMKII then was EGTA of 60 nM under the used experimental conditions (20). ϩ added to allow CaMKII autophosphorylation. Subsequently, the To confirm the [Ca2 ], a fluorescence titration with Fluo-4 2-␮l low Ca2ϩ stock, the buffer, and PP1 were added to bring the (Molecular Probes) was performed (Fig. 6, which is published as solution to the final condition. supporting information on the PNAS web site, www.pnas.org). Autophosphorylation reactions were stopped by dilution of CaMKII to 100 nM into 20 mM Hepes, pH 7.4͞200 mM KCl͞50 Computational Models.

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