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PLOS ONE  2012 

A Significant but Rather Mild Contribution of T286 Autophosphorylation to Ca2+/CaM-Stimulated CaMKII Activity

DOI: 10.1371/journal.pone.0037176

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Background Autophosphorylation of the Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) at T286 generates partially Ca2+/CaM-independent “autonomous” activity, which is thought to be required for long-term potentiation (LTP), a form of synaptic plasticity thought to underlie learning and memory. A requirement for T286 autophosphorylation also for efficient Ca2+/CaM-stimulated CaMKII activity has been described, but remains controversial. Methodology/Principal Findings In order to determine the contribution of T286 autophosphorylation to Ca2+/CaM-stimulated CaMKII activity, the activity of CaMKII wild type and its phosphorylation-incompetent T286A mutant was compared. As the absolute activity can vary between individual kinase preparations, the activity was measured in six different extracts for each kinase (expressed in HEK-293 cells). Consistent with measurements on purified kinase (from a baculovirus/Sf9 cell expression system), CaMKII T286A showed a mildly but significantly reduced rate of Ca2+/CaM-stimulated phosphorylation for two different peptide substrates (to ~75–84% of wild type). Additional slower CaMKII autophosphorylation at T305/306 inhibits stimulation by Ca2+/CaM, but occurs only minimally for CaMKII wild type during CaM-stimulated activity assays. Thus, we tested if the T286A mutant may show more extensive inhibitory autophosphorylation, which could explain its reduced stimulated activity. By contrast, inhibitory autophosphorylation was instead found to be even further reduced for the T286A mutant under our assay conditions. On a side note, the phospho-T305 antibody showed some basal background immuno-reactivity also with non-phosphorylated CaMKII, as indicated by T305/306A mutants. Conclusions/Significance These results indicate that Ca2+/CaM-stimulated CaMKII activity is mildly (~1.2–1.3fold) further increased by additional T286 autophosphorylation, but that this autophosphorylation is not required for the major part of the stimulated activity. This indicates that the phenotype of CaMKII T286A mutant mice is indeed due to the lack of autonomous activity, as the T286A mutant showed no dramatic reduction in stimulated activity.


[1]  Erondu NE, Kennedy MB (1985) Regional distribution of type II Ca2+/calmodulin-dependent protein kinase in rat brain. J Neurosci 5: 3270–3277.
[2]  Tobimatsu T, Fujisawa H (1989) Tissue-specific expression of four types of rat calmodulin-dependent protein kinase II mRNAs. J Biol Chem 264: 17907–17912.
[3]  Bayer KU, Lohler J, Schulman H, Harbers K (1999) Developmental expression of the CaM kinase II isoforms: ubiquitous gamma- and delta-CaM kinase II are the early isoforms and most abundant in the developing nervous system. Brain Res Mol Brain Res 70: 147–154.
[4]  Lisman J, Schulman H, Cline H (2002) The molecular basis of CaMKII function in synaptic and behavioural memory. Nat Rev Neurosci 3: 175–190.
[5]  Hudmon A, Schulman H (2002) Neuronal CA2+/calmodulin-dependent protein kinase II: the role of structure and autoregulation in cellular function. Annu Rev Biochem 71: 473–510.
[6]  Lee YS, Silva AJ (2009) The molecular and cellular biology of enhanced cognition. Nat Rev Neurosci 10: 126–140.
[7]  Malinow R, Schulman H, Tsien RW (1989) Inhibition of postsynaptic PKC or CaMKII blocks induction but not expression of LTP. Science 245: 862–866.
[8]  Silva AJ, Stevens CF, Tonegawa S, Wang Y (1992) Deficient hippocampal long-term potentiation in alpha-calcium-calmodulin kinase II mutant mice. Science 257: 201–206.
[9]  Derkach V, Barria A, Soderling TR (1999) Ca2+/calmodulin-kinase II enhances channel conductance of alpha-amino-3-hydroxy-5-methyl-4-isoxazo?lepropionatetype glutamate receptors. Proc Natl Acad Sci U S A 96: 3269–3274.
[10]  Hayashi Y, Shi SH, Esteban JA, Piccini A, Poncer JC, et al. (2000) Driving AMPA receptors into synapses by LTP and CaMKII: requirement for GluR1 and PDZ domain interaction. Science 287: 2262–2267.
[11]  Opazo P, Labrecque S, Tigaret CM, Frouin A, Wiseman PW, et al. (2010) CaMKII Triggers the Diffusional Trapping of Surface AMPARs through Phosphorylation of Stargazin. Neuron 67: 239–252.
[12]  Kristensen AS, Jenkins MA, Banke TG, Schousboe A, Makino Y, et al. (2011) Mechanism of Ca2+/calmodulin-dependent kinase II regulation of AMPA receptor gating. Nat Neurosci 14: 727–735.
[13]  Giese KP, Fedorov NB, Filipkowski RK, Silva AJ (1998) Autophosphorylation at Thr286 of the alpha calcium-calmodulin kinase II in LTP and learning. Science 279: 870–873.
[14]  Buard I, Coultrap SJ, Freund RK, Lee YS, Dell'Acqua ML, et al. (2010) CaMKII “autonomy” is required for initiating but not for maintaining neuronal long-term information storage. J Neurosci 30: 8214–8220.
[15]  Miller SG, Kennedy MB (1986) Regulation of brain type II Ca2+/calmodulin-dependent protein kinase by autophosphorylation: a Ca2+-triggered molecular switch. Cell 44: 861–870.
[16]  Lou LL, Lloyd SJ, Schulman H (1986) Activation of the multifunctional Ca2+/calmodulin-dependent protein kinase by autophosphorylation: ATP modulates production of an autonomous enzyme. Proc Natl Acad Sci U S A 83: 9497–9501.
[17]  Schworer CM, Colbran RJ, Soderling TR (1986) Reversible generation of a Ca2+-independent form of Ca2+(calmodulin)-dependent protein kinase II by an autophosphorylation mechanism. J Biol Chem 261: 8581–8584.
[18]  Coultrap SJ, Buard I, Kulbe JR, Dell'Acqua ML, Bayer KU (2010) CaMKII autonomy is substrate-dependent and further stimulated by Ca2+/calmodulin. J Biol Chem 285: 17930–17937.
[19]  De Koninck P, Schulman H (1998) Sensitivity of CaM kinase II to the frequency of Ca2+ oscillations. Science 279: 227–230.
[20]  Bayer KU, De Koninck P, Schulman H (2002) Alternative splicing modulates the frequency-dependent response of CaMKII to Ca(2+) oscillations. EMBO J 21: 3590–3597.
[21]  Chao LH, Stratton MM, Lee IH, Rosenberg OS, Levitz J, et al. (2011) A mechanism for tunable autoinhibition in the structure of a human Ca2+/calmodulin- dependent kinase II holoenzyme. Cell 146: 732–745.
[22]  Hanson PI, Meyer T, Stryer L, Schulman H (1994) Dual role of calmodulin in autophosphorylation of multifunctional CaM kinase may underlie decoding of calcium signals. Neuron 12: 943–956.
[23]  Rich RC, Schulman H (1998) Substrate-directed function of calmodulin in autophosphorylation of Ca2+/calmodulin-dependent protein kinase II. J Biol Chem 273: 28424–28429.
[24]  Bradshaw JM, Hudmon A, Schulman H (2002) Chemical quenched flow kinetic studies indicate an intraholoenzyme autophosphorylation mechanism for Ca2+/calmodulin-dependent protein kinase II. J Biol Chem 277: 20991–20998.
[25]  Kwiatkowski AP, Shell DJ, King MM (1988) The role of autophosphorylation in activation of the type II calmodulin-dependent protein kinase. J Biol Chem 263: 6484–6486.
[26]  Katoh T, Fujisawa H (1991) Autoactivation of calmodulin-dependent protein kinase II by autophosphorylation. J Biol Chem 266: 3039–3044.
[27]  Ishida A, Kitani T, Fujisawa H (1996) Evidence that autophosphorylation at Thr-286/Thr-287 is required for full activation of calmodulin-dependent protein kinase II. Biochim Biophys Acta 1311: 211–217.
[28]  Tzortzopoulos A, Torok K (2004) Mechanism of the T286A-mutant alphaCaMKII interactions with Ca2+/calmodulin and ATP. Biochemistry 43: 6404–6414.
[29]  Hanson PI, Kapiloff MS, Lou LL, Rosenfeld MG, Schulman H (1989) Expression of a multifunctional Ca2+/calmodulin-dependent protein kinase and mutational analysis of its autoregulation. Neuron 3: 59–70.
[30]  Ohsako S, Nakazawa H, Sekihara S, Ikai A, Yamauchi T (1991) Role of threonine-286 as autophosphorylation site for appearance of Ca2(+)-independent activity of calmodulin-dependent protein kinase II alpha subunit. J Biochem 109: 137–143.
[31]  Fong YL, Taylor WL, Means AR, Soderling TR (1989) Studies of the regulatory mechanism of Ca2+/calmodulin-dependent protein kinase II. Mutation of threonine 286 to alanine and aspartate. J Biol Chem 264: 16759–16763.
[32]  Bayer KU, De Koninck P, Leonard AS, Hell JW, Schulman H (2001) Interaction with the NMDA receptor locks CaMKII in an active conformation. Nature 411: 801–805.
[33]  Singla SI, Hudmon A, Goldberg JM, Smith JL, Schulman H (2001) Molecular characterization of calmodulin trapping by calcium/calmodulin-dependent protein kinase II. J Biol Chem 276: 29353–29360.
[34]  Coultrap SJ, Bayer KU (2012) Ca2+/Calmodulin-Dependent Protein Kinase II (CaMKII). In: Mukai H, editor. Neuromethods: Protein Kinase Technologies: Humana Press. pp. in press.
[35]  Vest RS, Davies KD, O'Leary H, Port JD, Bayer KU (2007) Dual Mechanism of a Natural CaMKII Inhibitor. Mol Biol Cell 18: 5024–5033.
[36]  Colbran RJ (1993) Inactivation of Ca2+/calmodulin-dependent protein kinase II by basal autophosphorylation. J Biol Chem 268: 7163–7170.
[37]  Hanson PI, Schulman H (1992) Inhibitory autophosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase analyzed by site-directed mutagenesis. J Biol Chem 267: 17216–17224.
[38]  Vest RS, O'Leary H, Coultrap SJ, Kindy MS, Bayer KU (2010) Effective post-insult neuroprotection by a novel Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor. J Biol Chem 285: 20675–20682.
[39]  Hashimoto Y, Schworer CM, Colbran RJ, Soderling TR (1987) Autophosphorylation of Ca2+/calmodulin-dependent protein kinase II. Effects on total and Ca2+-independent activities and kinetic parameters. J Biol Chem 262: 8051–8055.
[40]  Patton BL, Miller SG, Kennedy MB (1990) Activation of type II calcium/calmodulin-dependent protein kinase by Ca2+/calmodulin is inhibited by autophosphorylation of threonine within the calmodulin-binding domain. J Biol Chem 265: 11204–11212.
[41]  Elgersma Y, Fedorov NB, Ikonen S, Choi ES, Elgersma M, et al. (2002) Inhibitory autophosphorylation of CaMKII controls PSD association, plasticity, and learning. Neuron 36: 493–505.
[42]  Meyer T, Hanson PI, Stryer L, Schulman H (1992) Calmodulin trapping by calcium-calmodulin-dependent protein kinase. Science 256: 1199–1202.
[43]  Glazewski S, Giese KP, Silva A, Fox K (2000) The role of alpha-CaMKII autophosphorylation in neocortical experience-dependent plasticity. Nat Neurosci 3: 911–918.
[44]  Hardingham N, Glazewski S, Pakhotin P, Mizuno K, Chapman PF, et al. (2003) Neocortical long-term potentiation and experience-dependent synaptic plasticity require alpha-calcium/calmodulin-dependent protein kinase II autophosphorylation. J Neurosci 23: 4428–4436.
[45]  Wilbrecht L, Holtmaat A, Wright N, Fox K, Svoboda K (2010) Structural plasticity underlies experience-dependent functional plasticity of cortical circuits. J Neurosci 30: 4927–4932.
[46]  Gustin RM, Shonesy BC, Robinson SL, Rentz TJ, Baucum AJ, 2nd , et al. (2011) Loss of Thr286 phosphorylation disrupts synaptic CaMKIIalpha targeting, NMDAR activity and behavior in pre-adolescent mice. Mol Cell Neurosci 47: 286–292.
[47]  Radwanska K, Medvedev NI, Pereira GS, Engmann O, Thiede N, et al. (2011) Mechanism for long-term memory formation when synaptic strengthening is impaired. Proc Natl Acad Sci U S A 108: 18471–18475.
[48]  Yamagata Y, Kobayashi S, Umeda T, Inoue A, Sakagami H, et al. (2009) Kinase-dead knock-in mouse reveals an essential role of kinase activity of Ca2+/calmodulin-dependent protein kinase IIalpha in dendritic spine enlargement, long-term potentiation, and learning. J Neurosci 29: 7607–7618.
[49]  Otmakhov N, Griffith LC, Lisman JE (1997) Postsynaptic inhibitors of calcium/calmodulin-dependent protein kinase type II block induction but not maintenance of pairing-induced long-term potentiation. J Neurosci 17: 5357–5365.
[50]  Chen HX, Otmakhov N, Strack S, Colbran RJ, Lisman JE (2001) Is persistent activity of calcium/calmodulin-dependent kinase required for the maintenance of LTP? J Neurophysiol 85: 1368–1376.
[51]  Sanhueza M, Fernandez-Villalobos G, Stein IS, Kasumova G, Zhang P, et al. (2011) Role of the CaMKII/NMDA receptor complex in the maintenance of synaptic strength. J Neurosci 31: 9170–9178.
[52]  Ikeda A, Okuno S, Fujisawa H (1991) Studies on the generation of Ca2+/calmodulin-independent activity of calmodulin-dependent protein kinase II by autophosphorylation. Autothiophosphorylation of the enzyme. J Biol Chem 266: 11582–11588.
[53]  Colbran RJ (2004) Targeting of calcium/calmodulin-dependent protein kinase II. Biochem J 378: 1–16.
[54]  Merrill MA, Chen Y, Strack S, Hell JW (2005) Activity-driven postsynaptic translocation of CaMKII. Trends Pharmacol Sci 26: 645–653.
[55]  Rosenberg OS, Deindl S, Sung RJ, Nairn AC, Kuriyan J (2005) Structure of the autoinhibited kinase domain of CaMKII and SAXS analysis of the holoenzyme. Cell 123: 849–860.
[56]  Chao LH, Pellicena P, Deindl S, Barclay LA, Schulman H, et al. (2010) Intersubunit capture of regulatory segments is a component of cooperative CaMKII activation. Nat Struct Mol Biol 17: 264–272.
[57]  Rellos P, Pike AC, Niesen FH, Salah E, Lee WH, et al. (2010) Structure of the CaMKIIdelta/calmodulin complex reveals the molecular mechanism of CaMKII kinase activation. PLoS Biol 8: e1000426.


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