Interaction with the NMDA receptor locks CaMKII in an ... - CiteSeerX

Interaction with the NMDA receptor locks CaMKII in an active conformation

K.-Ulrich Bayer, Paul De Koninck, A. Soren Leonard, Johannes W. Hell & Howard Schulman

Group 9: Gomez, Arturo Gutschow, Patrick William Hanna, Michael Wageih Harp, Lindsey Faye Hayashi, Hisami Sarah Herrera, Nidfel N Hidalgo, Zander A Huang, Tracy

2 Ca2+/CaM–dependent protein kinase II (CaMKII) plays a key role in synaptic plasticity. In a brief overview of the potentiation of synaptic strength, CaMKII first becomes activated by the influx of Ca2+ through the NMDA receptors. The CaMKII then assists in the insertion of AMPA receptors at the synapse1,2 and assists in the increase of the channel conductance of AMPA receptors3. By doing so, CaMKII plays a major role in LTP expression. The proposed experiments by the authors are to prove that interaction of CaMKII with NMDA receptors will lead to an increase in the activity of CaMKII. They found 2 specific sites on the NR2B subunit of the NMDA receptor which interacted with CaMKII to allow translocation of the kinase to the synapse. The experimenters then tested for CaMKII activity in the absence of Ca2+/CaM and in the unphosphorylated T286 state. In order to understand the effects of NMDA receptor on CaMKII, the experimenters tested for the various effects of this interaction. Experiments were done to test for CaM trapping, suppression of the inhibitory autophosphorylation of CaMKII, and facilitated CaMKII response to Ca2+. The results of the various experiments only further confirm the importance of the role of CaMKII in synaptic plasticity, which lays the fundamental basis for learning and memory. NR2B is one of four subunits that make up the NMDA receptor. On the intracellular side of NR2B lays 2 sites which regulate CaMKII binding. The NR2B-P site is located at residues 839-1120 and the NR2B-C site at residues 1120-1482. CaMKII is autophosphorylated at residue T286. Autophosphorylation requires binding of 2 Ca2+/CaM molecules to the holoenzyme on CaMKII4. Previous studies have reported that the binding of CaMKII to NMDA receptors required phosphorylation while at the

3 same time NMDA-mediated translocation of the kinase to the synapse in hippocampal neurons did not5. The first experiment presented tested for the validity of this claim: autophosphorylation is required for CaMKII to bind to NMDA receptors. This was tested using immobilized fusion proteins of Glutathione S-transferase (GST) with NR2B-P and NR2B-C to visualize CaMKII binding to these two sequences. The kinase was initially autophosphorylated or not autophosphorylated in the presence or absence of Ca2+/CaM. The CaMKII bound to NR2B was seen by immunoblotting using a specific antibody. To test the hypothesis, GST-NR2B were expressed in bacteria and plated against anti-GST antibodies. The plates were washed with EGTA to buffer Ca2+ out, and kinase activity was immobilized. Then, the complex proteins were phosphorylated for 2 minutes on ice. To analyze CaMKII binding, the protein was eluted for 12 minutes in boiling SDS loading buffer, and an immunoblot using specific antibodies against the CaMKII, T286, or CAM was used. The results show that CaMKII binds to NMDA receptors with and without autophosphorylation. Autophosphorylated CaMKII wild-type (WT) binds to the NR2BP. Unphosphorylated CaMKII binds to NR2B-C only when Ca2+/CaM is present. Autophosphorylated CaMKII binds to both NR2B-P and NR2B-C sites even in the absence of Ca2+/CaM. Through this experiment, the hypothesis was proven wrong. Ca2+/CaM alone allows CaMKII to bind to the NMDA receptor, via the NR2B subunit, despite the fact that it is unphosphorylated. When WT CaMKII was autophosphorylated, CaMKII can

4 bind to either NR2B-P or NR2B-C sites. As long as Ca2+/CaM is available, CAMKII will at least bind to the NR2B-C site of the NR2B subunit. The experimenters then tested to see whether or not the same autophosphorylation-independent translocation of CaMKII in hippocampal neurons6,7 could be replicated in HEK cells. Translocation of CaMKII in stimulated HEK cells and hippocampal neurons was observed using a Green Fluorescent Protein labeled kinase (CaMKII-GFP) and a labeled NR2B (red-immunostain). Wild type, mutant T286A, and mutant I205K CaMKII were observed under conditions where the cell was stimulated in the presence and absence of NR2B in order to observe translocation to the synapse. The T286A mutant is unable to become phosphorylated due to the alanine residue. The I205K mutant is completely unable to bind to NR2B in vitro. However, I205K does not disrupt CaM affinity and allows normal activation of CaMKII by Ca2+/CaM8. In HEK cells, CaMKII was evenly distributed throughout the cytoplasm. Wild type CaMKII has its T286 site available to become autophosphorylated, thereby leading to activation of the kinase. HEK cells use Ca2+ to stimulate translocation (localization), while hippocampal cells use glutamate as their stimulus for translocation to the synapse. It was previously suggested that NMDA stimulated translocation of CaMKII in hippocampal neurons to their synapses did not require autophosphorylation. To determine if this was also true in HEK cells, CaMKII-GFP T286A or I205K mutants were expressed in HEK cells in the presence or absence of NR2B. HEK cells were transfected using Lipofectamine Plus. Cells were then perfused using Hank’s Balanced Salt Solution containing CaCl2, MgCl2, and HEPPES at room temperature. After this, the cells were stimulated, thereby inducing a Ca2+ influx. This was done by

5 first treating the HEK cells with ionomycin/EGTA and then perfused with CaCl2. After stimulation, they were visualized under a Nikon inverted microscope revealing that translocation of CaMKII, whether phosphorylated or not, when bound to NR2B did occur. I205K prevented NR2B from binding to CAMKII and, therefore, prevented translocation. They, then, showed this translocation in hippocampal cells derived from rats. These cells were supplemented with antibacterial solution, and after incubation of 5 days, they were transfected in the presence of CNQX (an AMPA receptor antagonist) and AP5 (blocks NMDA receptors). A 5µl of expression vector was injected into a custom made microporator before applying two electrical pulses at 190V. Neurons were maintained in the presence of AP5 before imaging. To see the translocation of CaMKII in rat hippocampal neurons, they were stimulated with glutamate in the presence of T286A and I205K CaMKII. The results of the experiments show that after stimulation of HEK cells wild type and T286A, CaMKII translocate to the synapse but only when coexpressed with NR2B. The kinase and peptide are visualized and localized together, and in the image, the images of the kinase and the peptide overlapped. I205K does not show translocation of CaMKII even in the presence of NR2B. The I205K-GFP image did not overlap with that of the NR2B peptide confirming their inability to localize. Lastly, in stimulated hippocampal neurons, I205K also fails to translocate where wild type and T286A did. From these experiments, the hypothesis was shown to be accurate in that phosphorylation is not needed for translocation and that NR2B binding is required. In

6 HEK cells, it is apparent that NR2B interacts with CaMKII as long as it has an affinity for the kinase. This binding of CaMKII to NR2B in hippocampal neurons also shows translocation of CaMKII to the postsynaptic site. After observing from the previous two experiments that CaMKII and NR2B were interacting and becoming co-localized upon stimulation, the authors wanted to find the specific NR2B sequence responsible for this interaction and its mechanism of CaMKII activity regulation. To test for the affect of NR2B-C binding on the affinity of CaMKII for CaM, NR2B-C-GST proteins were made and immobilized on anti-GST plates. They were then incubated with CaMKII and Ca2+/CaM, to allow peptide binding, and then washed under different conditions. When Ca2+/CaM was present during the binding steps, CaMKII always bound to the fusion protein. If Ca2+/CaM or Ca2+ was present in the washing buffer, CaM was also found bound to the CaMKII/NR2B-C complex. CaM was not found with the complex if the washing buffer contained EGTA. So, CaM trapping was observed in the presence of NR2B-C, similar to what is seen with autophosphorylated kinase. To find the precise region responsible for this binding, an assay was performed with different peptide fragments derived from NR2B. The fragments were immobilized then incubated with CaMKII, and IAEDANS-labeled CaM (IAEDANS is a flourophore). After incubation, unlabelled CaM was added to observe for competition and dissociation. A fragment N2B-s, corresponding to residues 1,095-1,119 of NR2b-C was found to reduce the dissociation, i.e. increase the affinity of CaMKII for CaM. Upon sequence analysis, the N2B-s fragment was found to have homology to the sequence surrounding T286 in CaMKII. The T286 region in a normal kinase interacts with two sites; the T-site

7 which binds to ‘target’ proteins when unoccupied and the S-site which binds to substrate when unoccupied. Combining the sequence homology data with the results from the CaM trapping and dissociation experiments, the authors speculated that the N2B-s fragment would interact with the kinase domain of CaMKII in a similar fashion to how its own autoinhibitory region interacts with it. This has an affect similar to that seen with CaM independent responses. From the results of these experiments the authors were able to formulate a hypothesis as to the mechanism of NR2B interaction and modulation of CaMKII activity. Their proposed theory was that upon CaM binding the auto-inhibitory region of the kinase is displaced and the T-site and S-site are open for interactions. If NR2B is bound to the T-site it prevents the auto-inhibitory segment from rebinding even if T286 is unphosphorylated. However, if it binds to the S-site it would inhibit the kinase by preventing substrate binding. In the previous experiments, we saw that NR2B-C is the binding site on the NR2B region for Ca2+/CaM and that CaM trapping is a result of the interaction between NR2B and CaMKII. The authors wanted to further experiment on the activity of CaMKII. In the fourth experiment, the experimenters were curious to see whether or not CaMKII activity would occur in the absence of Ca2+/CaM and in the absence of T286 phosphorylation. First, the experimenters worked with wild type CaMKII and T286A mutant CaMKII. They allowed some of the wild type CaMKII and mutant T286A to bind to immobilized NR2B-C by supplying Ca2+/CaM. With a batch of these wild type and

8 mutant CaMKII bound to NR2B-C, they further stimulated the CaMKII by supplying more Ca2+/CaM. With a separate batch of NR2B-C bound CaMKIIs (both mutant and wild type), the Ca2+/CaM was removed by the addition of EGTA. After this, the CaMKII activity was observed and recorded. The experimenters also used control groups of wild type CaMKII and T286A mutant CaMKII not bound to NR2B-C. The control groups underwent the same procedures and were either stimulated with Ca2+/CaM or washed with EGTA for Ca2+/CaM removal. The experimenters graphed stimulated wild type and T286A mutant pre-incubated with Ca2+, thus NR2B bound, and without pre-incubation, thus not bound to NR2B. This was also done with autonomous wild type and mutant. It was found that NR2B-Cimmobilized kinase still retained 70% of Ca2+/CaM stimulated activity. In addition, it was observed that though Ca2+/CaM was washed away with EGTA, there was still an autonomous activity of 19% of the maximal Ca2+/CaM stimulated activity with NR2B-C bound kinase. This type of autonomous activity had been previously observed after phosphorylation at T286 of CaMKII with a maximal autonomy of about 20-80% in another paper9. It was also observed that CaMKII T286A mutant showed autonomous activity when bound to NR2B-C which showed that Ca2+ pre-incubated mutant T286A washed free of Ca2+/ CaM is capable of producing activity while bound to NR2B-C. The observed autonomous activity after NR2B-C binding is thus created by a T286 phosphorylation-independent mechanism that is also not due to the trapping of CaM. A proposal made by the experimenters was that once NR2B-C binds and occupies the T-site of the kinase, it prevents the inhibition of the T286 segment of CaMKII so that the enzyme remains active even after Ca2+/CaM dissociation.

9 Next, the experimenters were interested in narrowing down the location on the NR2B subunit of CaMKII interaction. They tested different derivatives of the NR2B peptide to observe the activity it would generate if the CaMKII was pre-incubated with Ca2+ and the NR2B peptide. The experimenters worked with four different NR2Bderived peptides: N2B-con (residues 1,095-1,119), N2B-l (residues 1,259-1,310), N2B-s (residues 1,289-1,310), and N2B-a (a NR2B mutant that cannot be phosphorylated due to the mutant S1,303A). Half of these NR2B-derived peptides were pre-incubated with Ca2+ in the presence of CaMKII (thereby allowing for the binding of the NR2B peptide with CaMKII) while the other half were not pre-incubated with Ca2+ (thereby did not associate with CaMKII). Batches of these were further stimulated with Ca2+/CaM while other batches were washed with EGTA to remove the Ca2+/CaM. The kinase activity of the different batches was observed. The experimenters found significant autonomous activity of CaMKII when CaMKII was pre-incubated with Ca2+ and thereby bound to N2B-l and N2B-s. The relative kinase activity was 84% and 78% for N2B-l bound CaMKII and N2B-s bound CaMKII respectively. In batches where the CaMKII and NR2B peptides were not preincubated with Ca2+ and were later washed with EGTA, there was no observed kinase activity. To further study the interaction between the N2B-l peptide and CaMKII, the experimenters worked with varying concentrations of N2B-l peptide. When CaMKII was pre-incubated with Ca2+, the experimenters added different concentrations of N2B-l peptide. Then, with certain batches, further Ca2+ was added for stimulation. With the

10 remaining batches, EGTA was added to remove Ca2+/CaM. The relative kinase activity was then observed and recorded. From this, it was found that with increasing concentrations of the N2B-l derivative, autonomous kinase activity attained about 60% of its initial maximal activity. This observation verified a separate paper’s claim that N2B-l binds to and directly activates the CaMKII subunits10. It was also found that there was some reduction in the maximal stimulated kinase activity at very high concentrations of the N2B-l peptide. Though the reasons for this are still uncertain, the experimenters believe this to be the result of the secondary binding of NR2-l peptides at the S-site of CaMKII. Overall, experiment 4 showed that phosphorylation at T286 of CaMKII and CaM trapping are not the only factors in the generation of CaMKII activity. Interaction of CaMKII with NR2B (or more specifically N2B-l) allows for the increase in CaMKII activity. While the N2B-l peptide is a longer version of the N2B-s peptide (with an additional 30 residues), the N2B-l peptide will interact with greater preference to the Tsite of the CaMKII. This will result in the activity of CaMKII. Knowing that phosphorylation of T286 and the presence of Ca2+/CaM are other factors that generate kinase activity, the interaction of CaMKII with NR2B will further increase CaMKII activity and assist in the potentiation of the synaptic strength. Provided that CaMKII is bound to the NR2B-C region of a NMDA receptor, it can be assumed that changes in structure might result in a change in affinity for both calcium and calmodulin. Calcium and calmodulin affect the activity of the kinase, so the implications of changing the affinity for these substrates include changing the efficacy by which long-term potentiation is achieved. In experiment 5, there were three tests done in

11 order to observe the difference in phosphorylation when the sample was stimulated with calcium and when it was autonomous which referred to the usage of EGTA. In the first part of the experiment, selective T286 phosphate antibodies were used. Other methods used to trace phosphate included autoradiograph and immunoblotting with a phosphorylated T286 specific antibody or CaM overlay. The samples were prepared as follows: GST-NR2B fusion proteins were expressed in bacteria and immobilized on antiGST-antibody-coated well plates. This was done in preparation for immunoblotting to occur, as indicated by the authors. Once the wells were prepared, a 50 microliter of a binding mix was added. This mix consisted of kinase, NaCl, calcium chloride, and CaM or EGTA, and overlaid lasted for an hour at 4 degrees Celsius. Then, for 7-8 times, the wells were washed with 0.5-1 mM of EGTA or with 300nM of CaM for 20 minutes. Afterwards, immobilized kinase activity was measured for 1 minute at 30 degrees Celsius. A mix was then applied with radiated ATP (32P). Immunoblotting was done by using antibodies against _-CaMKII, phosphorylated T286, or CaM, and the protein was boiled with SDS loading buffer for 12 minutes. When looking for a trace of phospho-T286, the wild type, T286, when stimulated, contained traces of phosphate. However, on the defected type, T286A, after CaMKII was stimulated by Ca2+, no trace of phosphate was found.

In addition, no trace of

phosphorylation was found in the autonomous sample in spite of it being exposed to ATP. In the second part of this experiment, the experimenters decided to saturate CaMKII with NR2B-derived peptides, N2B-l, N2B-s, and N2B-a. This part of the experiment tested for the phosphorylation of T286. First, CaMKII was pre-incubated

12 with the peptides for 12 minutes when Ca2+/Calmodulin was present. Afterwards, 2.5mM of EGTA was added, and the samples were diluted. Two protocols were done. One was to stimulate the samples in the presence of Ca2+/CaM while the other was autonomous with an EGTA wash. In order to observe whether T286 was phosphorylated in CaMKII, immunoblotting was performed by using phospho-specific antibodies. The results obtained were consistent with the previous results. The stimulated samples of N2B-l, N2B-s, and N2B-a correlated with previous experiments done, which showed that T286 in fact was phosphorylated in the presence of the calcium-calmodulin complex. In the samples which were autonomous, with the sequestering of Ca2+ by EGTA, no autophosphorylation was seen in T286 of CaMKII. This, therefore, further proves the fact that autophosphorylation is not necessary for the binding and the activation of _-CaMKII to NMDA (specifically the NR2B portion of the receptor). The third test of experiment five tested for the effects of binding NMDA receptors to CaMKII on the burst phosphorylation at T305/306. Phosphorylation at T305 and T306 on CaMKII can be achieved when CaM dissociates from an autonomous enzyme11,12. The effectiveness of this process was analyzed with CaMKII bound to the NR2B-C region of the NMDA receptor and with soluble CaMKII. Phosphorylation of the kinase was induced and the relative effects on calmodulin binding provided another means by which NMDA binding can affect LTP. This test was conducted with soluble CaMKII and NR2B-C bound CaMKII. ATP was introduced into the medium, and then calcium and calmodulin were allowed to bind to both forms of CaMKII. The relative amount of calmodulin bound to the kinase and the phosphorylated CaMKII were quantified at this point with gel electrophoresis. Phosphorylation of CaMKII was then induced by the

13 addition of EGTA for both of the samples. Again, the relative amounts of phosphorylated CaMKII and calmodulin bound to CaMKII were quantified with gel electrophoresis. At T305/306 of CaMKII, the experimenters observed a very slight amount of phosphorylation in CaMKII apparent on the gel. After burst phosphorylation of T305/306 was induced, phosphorylated CaMKII was more abundant and provided a much heavier band on the gel. Calmodulin bound to CaMKII, however, was eliminated after the induction of phosphorylation at T305/306. For CaMKII bound to NR2B-C, the amount of phosphorylated CaMKII present before and after induced phosphorylation remained constant. Likewise, calmodulin bound to CaMKII remained constant despite phosphorylation at T305/306.

From the results obtained in this experiment, it is obvious that phosphorylation at T286 of CaMKII depends heavily on the presence of Ca2+/CaM. Only if the medium is supplied with Ca2+/CaM does T286 become phosphorylated. However, phosphorylation of NR2B-C does not rely at all on the presence of Ca2+/CaM. In addition, this experiment supports the assumption that binding CaMKII to a NMDA receptor inhibits autophosphorylation that would otherwise result in the displacement of calmodulin and calcium from the CaMKII, thereby resulting in the inactivation of the kinase. Provided that CaMKII is made tonically active when bound to a NMDA subunit, there would be no dependence on the presence of Ca2+/CaM to promote a more effective means of amplifying the necessary signals that may eventually lead to AMPA receptor inclusion at

14 the synapse. This mechanism, then, has implications for structural changes of the neuron and long-term potentiation. The authors argue that this long-term-potentiation arises from interactions between CaMKII and NMDA through four main forms: increased CaMKII activity due to an influx of Ca2+, suppression of inhibitory phosphorylation of the kinase, the formation of sustained CaM-independent kinase activity generated by a phosphorylationindependent mechanism, and sustained CaMKII activity due to CaM trapping. First, they showed that Ca2+/CaM alone could induce the binding of CaMKII to the NMDA receptors at the NR2B-C domain. This was a contradiction to previous reports that kinase-NMDA binding required phosphorylation.

Now, the autophosphorylation-

independent translocation of the kinase to the synapse can be linked to an autophosphorylation-independent binding of CaMKII to NMDA receptors. They proceeded to show that NR2B-bound CaMKII can suppress the burst phosphorylation of T305/306 of CaMKII thereby allowing CaMKII to remain active for a longer period of time. CaMKII was also shown to possess autonomous activity, that is, activity without phosphorylation and without bound CaM. Once N2B-l binds to the unphosphorylated kinase, the CaM can be removed with EGTA, and CaMKII will continue to remain active. On top of this autonomous activity, NR2B bound to CaMKII increases CaMKII’s affinity for CaM by lowering the rate of dissociation. This trapping of CaM will allow sustained NMDA receptor activity by reducing the down-regulation of NMDA receptors.

References: 1. Rongo, C. & Kaplan, J.M. CaMKII regulates the density of central glutamatergic synapses in vivo. Nature. 402, 195-199 (1999).

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2. Hayashi, Y. et al. Driving AMPA receptors into synapses by LTP and CaMKII: requirement for GluR1 and PDZ domain interaction. Science. 287, 2262-2267 (2000). 3. Derkach, V., Barria, A. & Soderling, T.R. Ca2+/calmodulin-kinase II enhances channel conductance of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate type glutamate receptors. Proc. Natl Acad. Sci. USA. 96, 3269-3274 (1999). 4. Rich, R.C. & Schulman, H. Substrate-directed function of calmodulin in autophosphorylation of Ca2+/calmodulin-dependent protein kinase II. J. Biol.Chem. 273, 28424-28429 (1998). 5. Strack, S., McNeill, R.B. & Colbran, R.J. Mechanism and regulation of calcium/calmodulin-dependent protein kinase II targeting to the NR2B subunit of the NMDA receptor. J. Biol. Chem. 275, 23798-23806 (2000). 6. Shen, K. & Meyer, T. Dynamic control of CaMKII translocation and localization in hippocampal neurons by NMDA receptor stimulation. Science. 284, 162-166 (1999). 7. Shen, K., Teruel, M. N., Connor, J. H., Shenolikar, S. & Meyer, T. Molecular memory by reversible translocation of calcium/calmodulin-dependent protein kinase II. Nature Neurosci. 3, 881-886 (2000). 8. Yang, E. & Schulman, H. Structural examination of autoregulation of multifunctional calcium/calmodulin-dependent protein kinase II. J. Biol. Chem. 274, 26199-26208 (1999). 9. Schulman, H. & Brown, A. in Calcium as Cellular Regulator (eds. Carafoli, E. & Klee, C.) 311-343 (Oxford Univ. Press, New York, 1999). 10. Hanson, P. I., Meyer, T., Stryer, L. & Schulman, H. Dual role of calmodulin in autophosphorylation of multifunctional CaM kinase may underlie decoding of calcium signals. Neuron. 12, 943-956 (1994). 11. Colbran, R. J. & Soderling, T. R. Calcium/calmodulin-independent autophosphorylation sites of calcium/calmodulin-dependent protein kinase II. Studies on the effect of phosphorylation of threonine 305/306 and serine 314 on calmodulin binding using synthetic peptides. J. Biol. Chem. 265, 11213-11219 (1990). 12. Hanson, P. I. & Schulman, H. Inhibitory autophosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase analyzed by site-directed mutagenesis. J. Biol. Chem. 267, 17216-17224 (1992).