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• W2035203431 abstract "Ca2+/calmodulin-dependent protein kinase II (CaMKII) acts in diverse cell types by phosphorylating proteins with key calcium-dependent functions such as synaptic plasticity, electrical excitability, and neurotransmitter synthesis. CaMKII displays calcium-dependent binding to proteins in vitro and translocation to synaptic sites after glutamatergic activity in neurons. We therefore hypothesized that subcellular targeting of CaMKII can direct its substrate specificity in an activity-dependent fashion. Here, we examined whether activity-dependent colocalization of CaMKII and its substrates could result in regulation of substrate phosphorylation in cells. We find that substrates localized at cellular membranes required CaMKII translocation to these compartments to achieve effective phosphorylation. Spatial barriers to phosphorylation could be overcome by translocation and anchoring to the substrate itself or to nearby target proteins within the membrane compartment. In contrast, phosphorylation of a cytoplasmic counterpart of the substrate does not require CaMKII translocation or stable protein-protein binding. Cytosolic phosphorylation is more permissive, exhibiting partial calcium-independence. Localization-dependent substrate specificity can also show more graded levels of regulation within signaling microdomains. We find that colocalization of translocated CaMKII and its substrate to lipid rafts in the plasma membrane can modulate the magnitude of phosphorylation. Thus, dynamic regulation of both substrate and kinase localization provides a powerful and nuanced way to regulate CaMKII signal specificity. Ca2+/calmodulin-dependent protein kinase II (CaMKII) acts in diverse cell types by phosphorylating proteins with key calcium-dependent functions such as synaptic plasticity, electrical excitability, and neurotransmitter synthesis. CaMKII displays calcium-dependent binding to proteins in vitro and translocation to synaptic sites after glutamatergic activity in neurons. We therefore hypothesized that subcellular targeting of CaMKII can direct its substrate specificity in an activity-dependent fashion. Here, we examined whether activity-dependent colocalization of CaMKII and its substrates could result in regulation of substrate phosphorylation in cells. We find that substrates localized at cellular membranes required CaMKII translocation to these compartments to achieve effective phosphorylation. Spatial barriers to phosphorylation could be overcome by translocation and anchoring to the substrate itself or to nearby target proteins within the membrane compartment. In contrast, phosphorylation of a cytoplasmic counterpart of the substrate does not require CaMKII translocation or stable protein-protein binding. Cytosolic phosphorylation is more permissive, exhibiting partial calcium-independence. Localization-dependent substrate specificity can also show more graded levels of regulation within signaling microdomains. We find that colocalization of translocated CaMKII and its substrate to lipid rafts in the plasma membrane can modulate the magnitude of phosphorylation. Thus, dynamic regulation of both substrate and kinase localization provides a powerful and nuanced way to regulate CaMKII signal specificity. Ca2+/calmodulin-dependent protein kinase II (CaMKII) 1The abbreviations used are: CaMKII, calmodulin-dependent protein kinase II; Vim, vimentin; CFP, cyan fluorescent protein; GFP, green fluorescent protein; EGFP, enhanced GFP; YFP, yellow fluorescent protein; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; AMPAR, AMPA receptor; NMDA, N-methyl-d-aspartate; NR2B, NMDA receptor 2B subunit; PSD, post-synaptic density; MAPK, mitogen-activated protein kinase; ER, endoplasmic reticulum; HBSS, Hanks' balanced salt solution; BAPTA, bis-(o-aminophenoxy)-N,N,N′,N′-tetraacetic acid-acetoxymethyl; BAPTA-AM, BAPTA-acetoxymethyl; GluR1, glutamate receptor 1; MβCD, methyl-β-cyclodextrin; c, cytosolic; m, membrane.1The abbreviations used are: CaMKII, calmodulin-dependent protein kinase II; Vim, vimentin; CFP, cyan fluorescent protein; GFP, green fluorescent protein; EGFP, enhanced GFP; YFP, yellow fluorescent protein; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; AMPAR, AMPA receptor; NMDA, N-methyl-d-aspartate; NR2B, NMDA receptor 2B subunit; PSD, post-synaptic density; MAPK, mitogen-activated protein kinase; ER, endoplasmic reticulum; HBSS, Hanks' balanced salt solution; BAPTA, bis-(o-aminophenoxy)-N,N,N′,N′-tetraacetic acid-acetoxymethyl; BAPTA-AM, BAPTA-acetoxymethyl; GluR1, glutamate receptor 1; MβCD, methyl-β-cyclodextrin; c, cytosolic; m, membrane. is a multifunctional serine/threonine kinase important for a variety of cellular functions including cell division, differentiation, cardiac contraction, and synaptic plasticity (1Hudmon A. Schulman H. Annu. Rev. Biochem. 2002; 71: 473-510Crossref PubMed Scopus (513) Google Scholar). CaMKII is required for long term potentiation at CA1 synapses as well as in behavioral learning and memory tasks (2Malinow R. Schulman H. Tsien R.W. Science. 1989; 245: 862-866Crossref PubMed Scopus (1033) Google Scholar, 3Lledo P.M. Hjelmstad G.O. Mukherji S. Soderling T.R. Malenka R.C. Nicoll R.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11175-11179Crossref PubMed Scopus (353) Google Scholar, 4Lisman J. Schulman H. Cline H. Nat. Rev. Neurosci. 2002; 3: 175-190Crossref PubMed Scopus (1443) Google Scholar). A number of the prominent CaMKII substrates are membrane proteins. CaMKII phosphorylation of the AMPA receptor (AMPAR), ryanodine receptor, and several ion channels regulate their conductance properties (5Derkach V. Barria A. Soderling T.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3269-3274Crossref PubMed Scopus (677) Google Scholar, 6Lokuta A.J. Rogers T.B. Lederer W.J. Valdivia H.H. J. Physiol. (Lond.). 1995; 487: 609-622Crossref Scopus (149) Google Scholar, 7Park D. Coleman M.J. Hodge J.J. Budnik V. Griffith L.C. J. Neurobiol. 2002; 52: 24-42Crossref PubMed Scopus (36) Google Scholar). Thus, CaMKII has a wide repertoire of substrates and associated cellular functions, which raises the question of how CaMKII activity can discriminate among its many substrates. A possible point of regulation could be through the complex subcellular targeting of CaMKII. Several mechanisms of CaMKII subcellular targeting have been characterized as CaMKII splice variants and isoforms can contain targeting sequences such as a nuclear localization sequence and actin binding domains (8Srinivasan M. Edman C. Schulman H. J. Cell Biol. 1994; 126: 839-852Crossref PubMed Scopus (232) Google Scholar, 9Shen K. Teruel M.N. Subramanian K. Meyer T. Neuron. 1998; 21: 593-606Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). Membrane targeting can be mediated by assembly with proteins such as αCaMKII association protein (10Bayer K.U. Lohler J. Harbers K. Mol. Cell. Biol. 1996; 16: 29-36Crossref PubMed Google Scholar). Recently, CaMKII has shown calcium/calmodulin-dependent interactions with membrane proteins such as the NMDA receptor 2B subunit (NR2B) that can affect its distribution in heterologous cells (11Strack S. McNeill R.B. Colbran R.J. J. Biol. Chem. 2000; 275: 23798-23806Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 12Bayer K.U. De Koninck P. Leonard A.S. Hell J.W. Schulman H. Nature. 2001; 411: 801-805Crossref PubMed Scopus (552) Google Scholar). Activity-dependent CaMKII translocation in neurons has also been observed. Synaptic calcium influx through NMDA receptors in cultured, dissociated neurons and in vivo can cause rapid accumulation of CaMKII in post-synaptic sites (13Shen K. Teruel M.N. Connor J.H. Shenolikar S. Meyer T. Nat. Neurosci. 2000; 3: 881-886Crossref PubMed Scopus (176) Google Scholar, 14Gleason M.R. Higashijima S. Dallman J. Liu K. Mandel G. Fetcho J.R. Nat. Neurosci. 2003; 6: 217-218Crossref PubMed Scopus (73) Google Scholar). Furthermore, induction of long term potentiation results in increased post-synaptic density (PSD) association of autophosphorylated CaMKII that displays calcium-independent activity (15Strack S. Choi S. Lovinger D.M. Colbran R.J. J. Biol. Chem. 1997; 272: 13467-13470Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). Disruptions in the synaptic localization of CaMKII have been correlated with behavioral defects in transgenic mice carrying mutated CaMKII or lacking local CaMKII synthesis in dendrites (16Elgersma Y. Fedorov N.B. Ikonen S. Choi E.S. Elgersma M. Carvalho O.M. Giese K.P. Silva A.J. Neuron. 2002; 36: 493-505Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 17Miller S. Yasuda M. Coats J.K. Jones Y. Martone M.E. Mayford M. Neuron. 2002; 36: 507-519Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar). A synaptic localization and function is supported by the finding of 28 CaMKII substrates in the PSD, including receptors, cytoskeletal proteins, enzymes, and scaffolds (18Yoshimura Y. Shinkawa T. Taoka M. Kobayashi K. Isobe T. Yamauchi T. Biochem. Biophys. Res. Commun. 2002; 290: 948-954Crossref PubMed Scopus (83) Google Scholar) and is consistent with CaMKII regulation of cellular processes such as dendritic spine remodeling and AMPAR exocytosis that span several subcellular compartments (19Fink C.C. Bayer K.U. Myers J.W. Ferrell Jr., J.E. Schulman H. Meyer T. Neuron. 2003; 39: 283-297Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 20Hayashi Y. Shi S.H. Esteban J.A. Piccini A. Poncer J.C. Malinow R. Science. 2000; 287: 2262-2267Crossref PubMed Scopus (1229) Google Scholar). The significance of CaMKII targeting for substrate phosphorylation has not been fully examined. Targeting to selected subcellular domains is crucial for effective cAMP-dependent protein kinase, protein kinase C, and MAPK signaling (21Gao T. Yatani A. Dell'Acqua M.L. Sako H. Green S.A. Dascal N. Scott J.D. Hosey M.M. Neuron. 1997; 19: 185-196Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar, 22Yedovitzky M. Mochly-Rosen D. Johnson J.A. Gray M.O. Ron D. Abramovitch E. Cerasi E. Nesher R. J. Biol. Chem. 1997; 272: 1417-1420Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 23Mahanty S.K. Wang Y. Farley F.W. Elion E.A. Cell. 1999; 98: 501-512Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). For example, phosphorylation and subsequent regulation of L-type calcium channels by cAMP-dependent protein kinase requires expression of the appropriate cAMP-dependent protein kinase anchoring protein, AKAP79, and targeting of the kinase to the channel (21Gao T. Yatani A. Dell'Acqua M.L. Sako H. Green S.A. Dascal N. Scott J.D. Hosey M.M. Neuron. 1997; 19: 185-196Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). Although it is widely speculated that CaMKII translocation might result in similar signal specificity, this has not been directly tested. Furthermore, CaMKII is expressed at extremely high levels in the hippocampal neurons, estimated to be from 1 to 2% of total protein and is enriched in synaptic sites. Since the kinase is at such high concentration for a signaling protein, it is unclear whether dynamic localization would add much specificity since the kinase may be at saturating levels throughout such neurons. In this work, we tested the hypothesis that access to certain substrates requires a directed translocation of CaMKII. We first determined whether phosphorylation of a substrate requires subcellular targeting of both the kinase and its substrate in a heterologous cell system in which both substrate and kinase concentrations are high. We targeted CaMKII to selected subcellular compartments using the calcium-dependent interaction between CaMKII and the C-terminal domain of NR2B (12Bayer K.U. De Koninck P. Leonard A.S. Hell J.W. Schulman H. Nature. 2001; 411: 801-805Crossref PubMed Scopus (552) Google Scholar). We showed that CaMKII targeting is required for effective phosphorylation of plasma membrane and ER substrates. By contrast, a cytoplasmic substrate does not require anchoring of the kinase for phosphorylation to occur. Furthermore, the cytoplasmic compartment seemed more permissive for phosphorylation at basal calcium levels. We also found that lipid raft microdomains can influence the degree of phosphorylation when both kinase and substrate are targeted to the plasma membrane. These results characterize for the first time how colocalization of CaMKII and its substrates to various cellular compartments regulates substrate phosphorylation. Gene Construction—All Vim constructs (Fig. 1) were derived from a PRK-5 vector containing a Myc-tag-labeled vimentin head. To generate the construct Vim-CFP-F, the coding sequence for EGFP was replaced by CFP through NheI and BamHI sites in the construct EGFP-F containing the farnesylation sequence from c-Ha-Ras (Clontech) CFP-F was then subcloned into NheI and BamHI of the cVim vector. CFP contains the A206K mutation to prevent any dimerization between fluorophores (24Zacharias D.A. Violin J.D. Newton A.C. Tsien R.Y. Science. 2002; 296: 913-916Crossref PubMed Scopus (1750) Google Scholar). mNR2B-Vim was generated by PCR amplifying the Myc-tagged vimentin head sequence with ApaI sites at the 5′ ends of both sense and antisense primers. PCR products were subcloned into the ApaI site at 4613 of pRK5-NR2B. cNR2B-Vim was generated by PCR from mNR2B-Vim and subcloned into pRK5. cNR2B was similarly generated by PCR from pRK5-NR2B. lyn-cNR2B was generated by PCR from pRK5-NR2B with the palmitoylation signal sequence from Lyn kinase. Yellow fluorescent protein tagged (YFP-CaMKII) was generated by replacing EGFP with YFP containing the A206K mutation (24Zacharias D.A. Violin J.D. Newton A.C. Tsien R.Y. Science. 2002; 296: 913-916Crossref PubMed Scopus (1750) Google Scholar) by PCR. Cell Culture, Transfection, and Stimulation Protocols—HEK293 cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum and were transfected with plasmids using Lipofectamine Plus™ (Invitrogen) according to the manufacturer's instructions. 24 h after the transfection, the cells were preincubated for 10 min with HBSS, 25 mm Hepes, pH 7.4, alone or 10 μm methyl-β-cyclodextrin (MβCD, Sigma) and stimulated with 10 μm ionomycin (Calbiochem) and 2 mm calcium in the absence or presence of MβCD. Some were loaded with 5 μm BAPTA-AM (Molecular Probes) in HBSS-Hepes for 20 min prior to fixation and immunostain. For GluR1 phosphorylation experiments, cells were preincubated for 30 min in 5 μm bisindolyl-maleimide I (Calbiochem). Immunocytochemistry and Immunoblotting—Cells were fixed with 4% paraformaldehyde in 100 mm phosphate buffer for 10 min followed by treatment with –20 °C methanol for 10 min. They were then incubated with the phosphorylation-specific vimentin antibody MO82 (0.2 μg/ml) diluted in phosphate-buffered saline with 2% normal goat serum. The cells were also counterstained with a polyclonal anti-Myc antibody (Santa Cruz Biotechnology) diluted 1:300 to assess expression level of vimentin constructs. The immunoreactivities were visualized by incubation with Alexa Fluor 594-conjugated anti-mouse antibodies and Alexa Fluor 647 anti-rabbit antibodies (Molecular Probes), and the samples were examined under a confocal microscope (Zeiss LSM-510). Images were quantitated on Metamorph by first producing a ratio image by dividing the phospho-vimentin image by the Myc image. All treatment ratio values were then normalized to control ratio values. Thresholding the Myc image to select for cells resulted in a mask that was then applied to the ratio image. For Vim-CFP-F experiments, intensity quantitation was complicated by the variable appearance of intracellular clusters of the Vim-CFP-F construct. Instead, the thresholded ratio image was blindly evaluated in a binary manner for the appearance of a visible plasma membrane ring of phospho-staining at least three times background levels. Immunoblotting was performed as described previously (12Bayer K.U. De Koninck P. Leonard A.S. Hell J.W. Schulman H. Nature. 2001; 411: 801-805Crossref PubMed Scopus (552) Google Scholar), using horseradish peroxidase-conjugated secondary antibodies and the ECL Western blotting detection system (Amersham Biosciences). Anti-Myc antibody was used at 1:750, MO82 was used at 0.2 μg/ml, anti-αCaMKII was used at 1:10,000, anti-phospho-Ser-831 (Upstate Biotechnology) was used at 1:500, anti-GluR1 (Chemicon) was used at 1:1000, and anti-GFP (Molecular Probes) was used at 1:1000. Membranes were stripped between blots with Restore Western blot stripping buffer (Pierce) as per the manufacturer's instructions. We developed an assay to monitor CaMKII activity in different subcellular compartments. We utilized a protein sequence derived from vimentin to selectively measure CaMKII activity in cells (25Inagaki N. Nishizawa M. Arimura N. Yamamoto H. Takeuchi Y. Miyamoto E. Kaibuchi K. Inagaki M. J. Biol. Chem. 2000; 275: 27165-27171Abstract Full Text Full Text PDF PubMed Google Scholar). Vimentin is an intermediate filament protein, the assembly of which is regulated by phosphorylation of several protein kinases at multiple sites. One of these sites, Ser-82, has been characterized as a substrate for CaMKII, but not other kinases, including CaMKI, CaMKIV, protein kinase C, and cAMP-dependent protein kinase (25Inagaki N. Nishizawa M. Arimura N. Yamamoto H. Takeuchi Y. Miyamoto E. Kaibuchi K. Inagaki M. J. Biol. Chem. 2000; 275: 27165-27171Abstract Full Text Full Text PDF PubMed Google Scholar, 26Ando S. Tokui T. Yamauchi T. Sugiura H. Tanabe K. Inagaki M. Biochem. Biophys. Res. Commun. 1991; 175: 955-962Crossref PubMed Scopus (60) Google Scholar, 27Inagaki M. Matsuoka Y. Tsujimura K. Ando S. Tokui T. Takahashi T. Inagaki N. BioEssays. 1996; 18: 481-487Crossref Scopus (160) Google Scholar). Using an antibody against the phosphorylated form of Ser-82, ectopically expressed vimentin has proved to be a sensitive probe for CaMKII activity both in heterologous cells and in neurons (25Inagaki N. Nishizawa M. Arimura N. Yamamoto H. Takeuchi Y. Miyamoto E. Kaibuchi K. Inagaki M. J. Biol. Chem. 2000; 275: 27165-27171Abstract Full Text Full Text PDF PubMed Google Scholar). By attaching an 88-amino-acid segment containing Ser-82 derived from the N terminus of the vimentin head to proteins with known subcellular distribution, we measured CaMKII activity in the vicinity of the fusion protein by immunocytochemistry against phospho-vimentin. CaMKII has been shown to bind to the NMDA receptor subunit NR2B in a calcium-dependent manner (11Strack S. McNeill R.B. Colbran R.J. J. Biol. Chem. 2000; 275: 23798-23806Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 12Bayer K.U. De Koninck P. Leonard A.S. Hell J.W. Schulman H. Nature. 2001; 411: 801-805Crossref PubMed Scopus (552) Google Scholar, 28Gardoni F. Caputi A. Cimino M. Pastorino L. Cattabeni F. Di Luca M. J. Neurochem. 1998; 71: 1733-1741Crossref PubMed Scopus (152) Google Scholar). Such targeting of the kinase may facilitate modulation of AMPAR nearby at synaptic sites. We therefore designed a probe of CaMKII activity at membrane-targeted NMDA receptors. We inserted a Myc-tagged vimentin head fragment (1–97) at positions 1420–1421 of NR2B beyond a second CaMKII binding site, one that is not autophosphorylation-dependent (11Strack S. McNeill R.B. Colbran R.J. J. Biol. Chem. 2000; 275: 23798-23806Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 12Bayer K.U. De Koninck P. Leonard A.S. Hell J.W. Schulman H. Nature. 2001; 411: 801-805Crossref PubMed Scopus (552) Google Scholar). We expressed the mNR2B-Vim construct together with GFP-CaMKII in HEK293 cells and showed that upon a calcium stimulation induced by ionomycin treatment, GFP-CaMKII translocates to mNR2B-Vim (Fig. 2A, right panels). The pattern of mNR2B-Vim expression was consistent with previously reported ER membrane retention of wild-type NR2B in the absence of NR1 (Fig. 2A) (29Fukaya M. Kato A. Lovett C. Tonegawa S. Watanabe M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4855-4860Crossref PubMed Scopus (154) Google Scholar). We then examined Ser-82 phosphorylation of the vimentin tag on NR2B and found that it was phosphorylated in a robust and calcium-dependent manner (Fig. 2A, right panels, and 2B). Calcium-ionomycin stimulation causes both CaMKII activation and translocation to membrane-localized mNR2B-Vim. To determine whether CaMKII translocation is required for mNR2B-Vim phosphorylation, we utilized a CaMKII point mutant, CaMKII(I205K), that has been previously shown to be defective in binding NR2B in vitro and unable to translocate to NR2B in situ (Fig. 1A) (12Bayer K.U. De Koninck P. Leonard A.S. Hell J.W. Schulman H. Nature. 2001; 411: 801-805Crossref PubMed Scopus (552) Google Scholar) while retaining calcium-dependent activation and autophosphorylation (30Yang E. Schulman H. J. Biol. Chem. 1999; 274: 26199-26208Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). When GFP-CaMKII(I205K) is cotransfected with mNR2B-Vim, it has the same initial cytosolic distribution as with wild-type GFP-CaMKII (Fig. 3A, left panels). However, upon calcium-ionomycin stimulation, GFP-CaMKII(I205K) does not translocate to mNR2B-Vim, as reported previously (Fig. 3A, right panels) (12Bayer K.U. De Koninck P. Leonard A.S. Hell J.W. Schulman H. Nature. 2001; 411: 801-805Crossref PubMed Scopus (552) Google Scholar). Although the kinase should be activated under this stimulation protocol, immunostain with the Ser-82 phospho-antibody reveals no significant mNR2B-Vim phosphorylation over background levels (Fig. 3A, right panels, and 3B). Similar results were obtained from immunoblot of transfected cell lysates (Supplementary Fig. 1). Taken together, these data demonstrate that translocation is necessary for phosphorylation of mNR2B-Vim. Is translocation required for mNR2B-Vim phosphorylation because the substrate is anchored to a membrane? To investigate CaMKII phosphorylation of an equivalent cytosolic substrate, we created a cytosol-targeted mNR2B-Vim substrate (cNR2B-Vim) by generating a truncated construct (residues 1120–1482) that lacks the membrane domain of NR2B but contains the CaMKII binding site and the vimentin tag (Fig. 1B). We cotransfected cells with cNR2B-Vim and GFP-CaMKII or GFP-I205K. Both wild-type and CaMKII (I205K) remained cytoplasmic in basal conditions and upon calcium-ionomycin stimulation (Fig. 4A,B). We compared cNR2B-Vim phosphorylation between wild-type and CaMKII (I205K) both with and without calcium-ionomycin stimulation and found them to be comparable (Fig. 4C). Since CaMKII (I205K) was equally competent in phosphorylating cNR2B-Vim, although it was unable to stably bind the NR2B tail, we conclude that unlike mNR2B-Vim, phosphorylation of cNR2B-Vim does not require CaMKII anchoring. Thus, when CaMKII and cNR2B-Vim are freely diffusible, phosphorylation does not require anchoring of the kinase to the substrate. Furthermore, these results indicate that the dependence of substrate phosphorylation on translocation demonstrated in Fig. 2 using the CaMKII (I205K) mutant is not due to an intrinsic inability of this mutant to recognize the vimentin substrate. These results suggest that CaMKII translocation can exert extremely tight regulation over substrate phosphorylation, provided that substrates are spatially restricted into subcellular compartments or less accessible conformations. Phosphorylation of CaMKII substrates in cytosolic and membrane compartments under unstimulated (basal) conditions appears to be differentially regulated. HEK293 cells have low endogenous expression of CaMKII, which results in a barely detectable amount of basal phosphorylation for cNR2B-Vim and no phosphorylation above background for mNR2B-Vim (Fig. 5A). Basal phosphorylation is dependent on CaMKII concentration because both were elevated upon coexpression of GFP-CaMKII (Fig. 5B). However, although both mNR2B-Vim and cNR2B-Vim basal phosphorylation is increased by coexpression of the kinase, the effect is far more dramatic for cNR2B-Vim (Fig. 5, B and D, middle). Basal phosphorylation of cNR2B-Vim in the cytosol is much higher than basal phosphorylation of the mNR2B-Vim at the membrane. To elucidate whether basal phosphorylation was entirely due to continued calcium activation of CaMKII or to calcium-independent CaMKII activity, we determined whether phosphorylation is sensitive to BAPTA-AM treatment, which should eliminate most of the free intracellular calcium. A significant portion of basal cNR2B-Vim phosphorylation was reduced when cells were pretreated for 20 min with 5 μm BAPTA-AM, but a significant fraction of the phosphorylation was BAPTA-resistant, perhaps due to a population of autophosphorylated CaMKII with calcium-independent activity (31Miller S.G. Patton B.L. Kennedy M.B. Neuron. 1988; 1: 593-604Abstract Full Text PDF PubMed Scopus (225) Google Scholar). Interestingly, mNR2B-Vim phosphorylation did not display any sensitivity to BAPTA (Fig. 5, C and D, right), although it is possible that changes may have been obscured by the smaller magnitude of starting basal phosphorylation for this construct as compared with cNR2B-Vim. Similar results were obtained by the coexpression of vimentin constructs with GFP-CaMKII(I205K), suggesting that basal phosphorylation was not mediated by persistent activity due to CaMKII/NR2B anchoring (data not shown.) Taken together, these data indicated that the cytosolic compartment can be much more permissive than membrane compartments for submaximal calcium signaling. To further characterize the nature of localization-restricted phosphorylation, we investigated whether a docking interaction between kinase and substrate was required for membrane-delimited phosphorylation or whether mere compartmental co-localization is sufficient. To this end, we constructed a cyan fluorescent protein-tagged farnesylated, plasma membrane-targeted vimentin substrate (Vim-CFP-F) that is not expected to interact with CaMKII or NR2B (Fig. 1B). Indeed, coexpression of Vim-CFP-F and YFP-CaMKII did not result in plasma membrane CaMKII translocation (data not shown). Similarly, coexpression of Vim-CFP-F, YFP-CaMKII, and cNR2B did not allow for plasma membrane CaMKII translocation (Fig. 6A, bottom row). Thus, we could observe whether CaMKII translocated to the plasma membrane via binding interactions with a plasma membrane-targeted NR2B C-tail enhances phosphorylation of an independent plasma membrane substrate that is capable of lateral mobility in the membrane. We tagged a portion of the NR2B C-tail (residues 1120–1482) that had previously been shown to be sufficient to direct CaMKII subcellular localization (11Strack S. McNeill R.B. Colbran R.J. J. Biol. Chem. 2000; 275: 23798-23806Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar) with an N-terminal palmitoylation signal from lyn kinase (lyn-cNR2B) to direct this construct to the plasma membrane (Fig. 1B). YFP-CaMKII was used instead of the GFP-CaMKII constructs in previous experiments to allow spectral distinction between YFP-labeled kinase and CFP-labeled vimentin substrate. In the unstimulated state, YFP-CaMKII is cytosolic, and there is little basal phosphorylation of Vim-CFP-F, as expected based on previous findings with mNR2B-Vim and a cytosolic CaMKII. With lyn-cNR2B coexpressed to provide a membrane-anchoring site for CaMKII and upon calcium stimulation, YFP-CaMKII translocates to the plasma membrane and phosphorylates Vim-CFP-F (Fig. 6A, top row). Phosphorylation of Vim-CFP-F required coexpression of a CaMKII plasma membrane-targeting construct as both CaMKII translocation and Vim-CFP-F phosphorylation were absent if cytosolic NR2B tail (cNR2B) were substituted for lyn-cNR2B (Fig. 6A, bottom row). Cells assessed as positive for the appearance of plasma membrane phosphorylation demonstrated a clear requirement for lyn-cNR2B (Fig. 6B, see “Experimental Procedures”). These data demonstrate that, as seen for the ER, the plasma membrane also displays a localization requirement for CaMKII substrate phosphorylation. Furthermore, simple colocalization of kinase and substrate to the membrane compartment is sufficient to fulfill localization restrictions placed by membrane localization; direct anchoring interactions of kinase on substrate are not required. These findings have implications for known plasma membrane substrates of CaMKII that do not exhibit direct anchoring to the kinase. In the post-synaptic membrane, one such example is the AMPA receptor subunit GluR1, which can be phosphorylated by CaMKII on Ser-831 (32Barria A. Derkach V. Soderling T. J. Biol. Chem. 1997; 272: 32727-32730Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). To investigate whether a similar translocation-dependent phosphorylation by CaMKII can occur on this natural substrate, we immunoblotted lysates of cells cotransfected with hemagglutinin (HA)-GluR1, GFP-CaMKII, and cNR2B, lyn-cNR2B, or full-length NR2B and probed for phosphorylation of Ser-831 after calcium-ionomycin stimulation. As Ser-831 is also phosphorylated by protein kinase C, cells were pretreated for 30 min with 5 μm protein kinase C inhibitor bisindolylmaleimide I. Ser-831 phosphorylation was greatly enhanced if either lyn-cNR2B or NR2B was present but not with cNR2B (Supplemental Fig. 2). This suggests that translocation of CaMKII to a membrane anchor facilitates phosphorylation of membrane proteins, consistent with our vimentin reporter construct results. Within a subcellular compartment, signaling microdomains can further influence access of enzymes to their substrates. Lipid rafts have been found to play important roles in organizing signaling at the plasma membrane, through clustering or segregating signaling components (33Pi" @default.
• W2035203431 created "2016-06-24" @default.
• W2035203431 creator A5006973998 @default.
• W2035203431 creator A5038123003 @default.
• W2035203431 creator A5058557647 @default.
• W2035203431 date "2005-03-01" @default.
• W2035203431 modified "2023-10-16" @default.
• W2035203431 title "Calcium/Calmodulin-dependent Protein Kinase II (CaMKII) Localization Acts in Concert with Substrate Targeting to Create Spatial Restriction for Phosphorylation" @default.
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