Characterization of a central Ca2+/calmodulin-dependent protein kinase IIalpha/beta binding domain in densin that selectively modulates glutamate receptor subunit phosphorylation

Abstract

The densin C-terminal domain can target Ca(2+)/calmodulin-dependent protein kinase IIα (CaMKIIα) in cells. Although the C-terminal domain selectively binds CaMKIIα in vitro, full-length densin associates with CaMKIIα or CaMKIIβ in brain extracts and in transfected HEK293 cells. This interaction requires a second central CaMKII binding site, the densin-IN domain, and an "open" activated CaMKII conformation caused by Ca(2+)/calmodulin binding, autophosphorylation at Thr-286/287, or mutation of Thr-286/287 to Asp. Mutations in the densin-IN domain (L815E) or in the CaMKIIα/β catalytic domain (I205/206K) disrupt the interaction. The amino acid sequence of the densin-IN domain is similar to the CaMKII inhibitor protein, CaMKIIN, and a CaMKIIN peptide competitively blocks CaMKII binding to densin. CaMKII is inhibited by both CaMKIIN and the densin-IN domain, but the inhibition by densin is substrate-selective. Phosphorylation of a model peptide substrate, syntide-2, or of Ser-831 in AMPA receptor GluA1 subunits is fully inhibited by densin. However, CaMKII phosphorylation of Ser-1303 in NMDA receptor GluN2B subunits is not effectively inhibited by densin in vitro or in intact cells. Thus, densin can target multiple CaMKII isoforms to differentially modulate phosphorylation of physiologically relevant downstream targets.

FIGURE 1.

The densin C-terminal domain binds selectively to CaMKIIα. A, domain structure of densin and the GST-D-CTA fusion protein. TM, originally proposed putative transmembrane domain; CaMKIIBD, known C-terminal CaMKIIα binding domain. B, CaMKII isoforms were pre-autophosphorylated at Thr-286/7 and then incubated with GST alone, GST-D-CTA, or GST-N2B. Left, the protein-stained membrane shows the GST protein preparations used (top: an asterisk on the left marks the full-length GST-D-CTA protein) and a representative immunoblot for CaMKII bound to the GST proteins (bottom). Right, quantification of CaMKII isoform binding to GST-D-CTA, expressed as a percentage of the binding of CaMKIIα (mean ± S.E., n = 3. *, p < 0.001). Binding of CaMKIIδ₂ could not be accurately quantified because it co-migrates with GST-D-CTA. C, WT and monomeric CaMKIIα mutants were Thr-286-autophosphorylated, incubated with the indicated GST proteins, and then analyzed as in panel B. Binding of truncation mutants was expressed as a percentage of WT CaMKIIα binding (mean ± S.E., n = 3. *, p < 0.001).

FIGURE 2.

Full-length densin associates with both CaMKIIα and CaMKIIβ. A, comparison of the major adult densin splice variant (densin-FLA) with a natural splice variant lacking the C-terminal CaMKII binding domain (densin-FLC). B, lysates from HEK293 cells expressing CaMKIIα and either GFP-tagged densin-FLA or densin-FLC were immunoprecipitated using densin Ab450 or control goat IgG. Extracts (Input) and immune complexes were immunoblotted for densin and CaMKIIα. C, lysates from HEK293 cells expressing CaMKIIβ and either GFP-tagged densin-FLA or densin-FLC were analyzed as in panel B. D, forebrain extracts from WT or CaMKIIα-KO mice were immunoprecipitated using mouse antibodies to CaMKIIα or CaMKIIβ or control IgG. Extracts and immune complexes were immunoblotted for densin and both CaMKII isoforms.

FIGURE 3.

Identification of a novel CaMKII binding domain in densin. A, summary of truncation mutants created to identify the densin-IN domain. Letters B–D in parentheses indicate GST-densin protein families used in panels B–D; major CaMKII binding fragments are highlighted with thicker lines. Amino acid residues included in each fusion protein are below. B–D, GST or GST fusion proteins were incubated with Thr-286-autophosphorylated CaMKIIα, and proteins isolated using glutathione-agarose were analyzed by protein stain and/or CaMKII immunoblot as indicated. Full-length GST fusion proteins are indicated by asterisks located immediately to their left in each panel. B, first round analysis is shown. CaMKIIα binds to residues 708–962 >GST-D-CTA > residues 963–1290 but not to residues 482–707 or the N-terminal T482 (LRR) domain. C, second round analysis of densin-708–962 region is shown. CaMKIIα binds to residues 708–824 but not to residues 825–915 or 916–962. D, third round analysis of the densin-708–824 region is shown. CaMKIIα binds comparably to residues 761–824 and 793–824, with no detectable interaction with other fragments. Quantification (n ≥ 3) shows that CaMKII binding to densin-708–824 is fully recapitulated in the densin-793–824 fragment, hereafter referred to as the densin-IN domain.

FIGURE 4.

CaMKII activation is required for binding to the densin-IN domain. A, GST-D-IN or GST-N2B were incubated with non-phosphorylated or Thr-286-autophosphorylated CaMKIIα and the indicated additional reagents: 2 mm EDTA, 2 μm calmodulin (CaM), 2 mm calcium chloride (Ca²⁺),5 mm magnesium acetate (Mg²⁺), 0.1 mm AMP-PNP. Complexes were isolated using glutathione-agarose and then washed using buffers containing the respective concentrations of EDTA or divalent cations. Binding (mean ± S.E., n = 3) was expressed as a percentage of Thr-286-autophosphorylated CaMKIIα binding to GST-N2B in the presence of excess EDTA (condition g) (*, p < 0.05; **, p < 0.01; ***, p < 0.001; as indicated). B, CaMKIIα or CaMKIIβ were incubated with GST-D-IN or GST-N2B after autophosphorylation at Thr-286/287 and in the presence of excess EDTA (left panel) or without autophosphorylation and in the presence of calcium chloride (2 mm) and calmodulin (0.1 or 5 μm: L or H, respectively) (right panel). Complexes were isolated using glutathione-agarose. Aliquots of inputs, supernatants, and pellets were analyzed on SDS-polyacrylamide gels and stained for protein in parallel. Data are representative of two similar experiments.

FIGURE 5.

Densin-IN binding to the CaMKII catalytic domain is blocked by CaMKIIN-tide. A, extracts of HEK293 cells expressing GFP-CaMKIIα-(1–290) were incubated with GST, GST-D-IN, or GST-D-CTA. Aliquots of the extract (Input) and complexes isolated using glutathione-agarose were analyzed by GFP immunoblot and by protein staining to detect GST fusion proteins. B, Thr-286-autophosphorylated CaMKIIα was incubated with GST or GST-D-IN (equimolar) in the absence or presence of the indicated peptides (50 μm). Complexes isolated using glutathione-agarose were analyzed by protein staining of nitrocellulose membranes. CaMKII binding (mean ± S.E., n ≥ 3) was expressed as a percentage of binding to GST-D-IN in absence of peptides (*, p < 0.05 versus control). C, extracts of HEK293 cells expressing mCherry-CaMKIIβ(T287D) or mCherry-CaMKIIβ(T287D) with additional I206K or D239R mutations where indicated were incubated with GST-D-IN or GST-N2B. Aliquots of inputs and of complexes isolated using glutathione-agarose were immunoblotted for mCherry using RFP antibodies. Binding of catalytic domain mutants was expressed as a percentage of binding of control mCherry-CaMKIIβ(T287D) (mean ± S.E., n = 3). *, p < 0.05; ***, p < 0.001 compared with control; †, p < 0.001 compared with GST-D-IN.

FIGURE 6.

L815E mutation of the densin IN domain disrupts CaMKII binding. A, amino acid sequences of the densin-IN domain, the CaMKIIN inhibitory domain, and the CaMKII binding domain of GluN2B are aligned with identical or similar residues in gray boxes. The underlined region of CaMKIIN-tide was resolved in an x-ray crystal structure of the CaMKII-CaMKIIN-tide complex (44). Densin-IN domain mutants characterized in panel B are indicated above. A color version of this panel is available online as supplemental Fig. 1. B, Thr-286/287-autophosphorylated CaMKIIα or CaMKIIβ were incubated with GST-D-IN (WT or mutated) or GST. Complexes isolated on glutathione-agarose were analyzed by protein staining of nitrocellulose membranes. Binding of CaMKII isoforms to mutated GST-D-INs was expressed as a percentage of the binding to WT (mean ± S.E., n = 3). †, p < 0.05 compared with binding of CaMKIIβ (2-way analysis of variance with Bonferroni post-test). *, p < 0.001 compared with WT (1-way analysis of variance with Dunnett's multiple comparison test). ¶, p < 0.05 compared with WT (1-way analysis of variance with Dunnett's multiple comparison test). C, extracts of HEK293 cells expressing mCherry-CaMKIIα-T286D and GFP-tagged densin-FLA (WT or L815E) were incubated with densin Ab450 or control goat IgG. Inputs and immune complexes were immunoblotted for densin and CaMKII. IP, immunoprecipitated. D, lysates from HEK293 cells co-expressing mCherry-CaMKIIβ-T287D with GFP-tagged densin-FLA (WT or L815E) were analyzed as in panel C. Samples were analyzed in parallel with those in panel C but were loaded in a different order to avoid confusion during analysis. The Input, IgG, and Densin lane pairs were each flipped horizontally during preparation of the figure so that all lanes are in the same order as in panel C; lines on the blots indicate these discontinuities.

FIGURE 7.

Densin IN potently inhibits CaMKII phosphorylation of GluA1 but not GluN2B. A, GST-GluA1 was incubated with Thr-286-autophosphorylated CaMKIIα, [γ-³²P]ATP, and the indicated concentrations of GST-D-IN (WT or L815E), N-tide or GST alone. Top, reaction aliquots were analyzed by SDS-PAGE followed by Coomassie Blue staining and autoradiography. Bottom, quantitative analysis using phosphocellulose papers (see “Experimental Procedures”); the mean of duplicates is plotted. Data are representative of four independent experiments. B, GST-N2Bs was incubated with Thr-286-autophosphorylated CaMKIIα and the indicated concentrations of GST-D-IN (WT or L815E), N-tide, or GST alone and analyzed as described in panel A.

FIGURE 8.

Densin inhibits phosphorylation of GluA1, but not GluN2B, in intact cells. Triton soluble fractions (Input) of HEK293 cells expressing GluA1 or FLAG-GluN2B with or without CaMKIIα and/or densin were immunoprecipitated (IP) (see “Experimental Procedures”). A, inputs and GluA1 immune complexes were immunoblotted for CaMKII, densin, total GluA1, or phospho-Ser-831 GluA1 (P-Ser-831), as indicated. The graph summarizes the quantification of Ser-831 phosphorylation normalized to total GluA1 from 5 similar experiments. *, p < 0.05 compared with GluA1 alone. **, p < 0.01 compared with GluA1+CAMKII and to GluA1+CAMKII+L815E-densin. B, inputs or FLAG-GluN2B complexes were immunoblotted for CaMKII, densin, total GluN2B, or phospho-Ser-1303 GluN2B (P-Ser1303) as indicated. The graph summarizes the quantification of Ser-1303 phosphorylation normalized to total GluN2B from 4 similar experiments. ***, p < 0.0001 compared with GluN2B alone.