Casein kinase 2 phosphorylation of protein kinase C and casein kinase 2 substrate in neurons (PACSIN) 1 protein regulates neuronal spine formation

Abstract

The PACSIN (protein kinase C and casein kinase 2 substrate in neurons) adapter proteins couple components of the clathrin-mediated endocytosis machinery with regulators of actin polymerization and thereby regulate the surface expression of specific receptors. The brain-specific PACSIN 1 is enriched at synapses and has been proposed to affect neuromorphogenesis and the formation and maturation of dendritic spines. In studies of how phosphorylation of PACSIN 1 contributes to neuronal function, we identified serine 358 as a specific site used by casein kinase 2 (CK2) in vitro and in vivo. Phosphorylated PACSIN 1 was found in neuronal cytosol and membrane fractions. This localization could be modulated by trophic factors such as brain-derived neurotrophic factor (BDNF). We further show that expression of a phospho-negative PACSIN 1 mutant, S358A, or inhibition of CK2 drastically reduces spine formation in neurons. We identified a novel protein complex containing the spine regulator Rac1, its GTPase-activating protein neuron-associated developmentally regulated protein (NADRIN), and PACSIN 1. CK2 phosphorylation of PACSIN 1 leads to a dissociation of the complex upon BDNF treatment and induces Rac1-dependent spine formation in dendrites of hippocampal neurons. These findings suggest that upon BDNF signaling PACSIN 1 is phosphorylated by CK2 which is essential for spine formation.

FIGURE 1.

Phosphorylation of PACSIN 1 at serine 358 by CK2. A, scheme of PACSIN 1 represents its domain structure and location of Ser³⁵⁸. B, recombinant wild-type PACSIN 1 (P1 wt) and PACSIN 1 S358A (P1 S358A) were purified as GST fusion proteins, and GST was removed by proteolytic cleavage. The proteins were incubated with CK2 and [γ-³²P]ATP for 20 min and analyzed by SDS-PAGE. The SDS-polyacrylamide gel was stained with Coomassie Brilliant Blue, dried, and exposed to an x-ray film. C, recombinant proteins including GST and an unrelated mutant PACSIN 1 T25A (P1 T25A) as controls were incubated with (+) or without (−) CK2, resolved by native PAGE, and immunoblotted with antibodies specific for PACSIN 1 or PACSIN 1 pS358. D, for kinetic analysis wild-type PACSIN 1 (P1 wt) was incubated with CK2 for the indicated time periods, separated by native PAGE and immunoblotted (WB) for PACSIN 1.

FIGURE 2.

In vivo phosphorylation of PACSIN 1 at serine 358. A, subcellular fraction of mouse brain homogenates (B) was used to produce a synaptosomal pellet and a nonsynaptosomal supernatant. Both fractions were separately subjected to ultracentrifugation to give the two high speed pellets SV (synaptosomal vesicles), V (nonsynaptosomal vesicles), and the combined supernatant C (cytosol). The three samples were further enriched for phosphorylated proteins (pV, pSV, and pC) by affinity chromatography. 8 μg of protein before and after phosphoprotein enrichment was separated by SDS-PAGE and immunoblotted for dynamin, PACSIN 1, and SNAP-47 as a negative control. B, PACSIN 1 was immunoprecipitated from a murine brain lysate and resolved by two-dimensional PAGE. Spots were manually picked and analyzed by LC-MS/MS. Four spots (encircled) were identified as PACSIN 1, and all spots contained PACSIN 1 pS358.

FIGURE 3.

Phospho-PACSIN 1 levels and localization in neuronal cells. A, N2a cells were transfected with a CK2α-specific siRNA mixture for 72 h and then incubated with (+) or without (−) BDNF (100 ng/ml) for 5 h and lysed. Comparable amounts of the individual lysates (50 μg/lane) were resolved by SDS-PAGE and immunoblotted (IB) with antibodies specific for the indicated proteins. As a control, the Ponceau staining of the membrane is shown. M, marker proteins. B, protein extracts from cultured mouse hippocampal neurons were subjected to centrifugation through a continuous sucrose density gradient and fractions analyzed for the distribution of total and phosphorylated PACSIN 1. Immunoblot (WB) analysis shows the shift of phosphorylated PACSIN 1 to lighter fractions upon BDNF-treatment of the neurons. C, quantification of immunoblot signal intensities for total (left graph) and phosphorylated (right graph) PACSIN 1 with Image Quant 5.0 for the relevant fractions (2, 3, and 4) of three independent experiments is shown (*, p < 0.05, t test). Error bars, S.D.

FIGURE 4.

Reduced spine formation in the presence of the PACSIN 1 S358A mutant or after CK2 inhibition. A, rat hippocampal neurons were transfected with either PACSIN 1 wt (left). the mutant protein S358A (right) or the mutant protein S358D (data not shown) in combination with EGFP to visualize spines (upper panel). Enlargements of three dendrite segments of neurons transfected with either PACSIN 1 wt (left) or S358A mutant (right) are shown. Fewer spines can be detected in neurons expressing PACSIN 1 S358A (lower panel). B, quantification of two independent experiments. For each group five dendrites were counted on each of 12 cells, n = 60 dendrites; **, p < 0.01; ***, p < 0.001, t test. C, untransfected hippocampal neurons were treated either with dimethyl sulfoxide (DMSO) or the CK2 inhibitor TBCA in dimethyl sulfoxide and stained with the DiI plasma membrane dye to visualize the spines (upper panel). Enlargements of the indicated dendrite segments of neurons are shown (lower panel). D, quantification of three independent experiments is shown. For each group three dendrites were counted on each of five cells, n = 45 dendrites; ***, p < 0.001, t test. Error bars, S.D.

FIGURE 5.

PACSIN 1 pS358 is present in a complex with the spine regulator Rac1 and the GAP NADRIN. A, mouse brain homogenate was used for immunoprecipitation (IP) with an antibody specific for Rac1 (lower panel) and co-precipitated proteins analyzed by immunoblotting (WB). The bands marked IgG represent immunoglobulin light chains. Total PACSIN 1 (upper panel left) as well as PACSIN 1 pS358 (upper panel right) are detected in the Rac1-IP lane. B, the levels of GTP-bound Rac1 were determined in PACSIN 1 wt or PACSIN 1 S358A-transfected N2a cells which were lysed 24 h after transfection. Pulldown experiments were performed, and equal amounts of bead fractions and lysate fractions were resolved on SDS-polyacrylamide gels (15%). The GTP-bound forms of Rac1 in bead fractions and total Rac1 in lysate fractions were stained with mouse anti-Rac1 antibody (top and middle panels). Transfection efficiencies of both constructs were comparable (bottom panel). C, quantification of three independent experiments was performed using ImageJ. *, p < 0.05, t test. Error bar, S.D. D, N2a cells were co-transfected with hemagglutinin (HA)-tagged NADRIN and Myc-tagged PACSIN 1 wt, treated with (+) or without (−) BDNF (100 ng/ml) for 5 h, and lysed. NADRIN was precipitated with an antibody specific for HA, and comparable amounts of the individual precipitates were resolved by SDS-PAGE. After immunoblotting (IB), the precipitated proteins were visualized with antibodies specific for the indicated tags. E, N2a cells were treated with (+) or without (−) BDNF (100 ng/ml) for 5 h and lysed. Endogenous Rac1 was precipitated with a specific antibody, and comparable amounts of the individual precipitates were resolved by SDS-PAGE. After immunoblotting co-precipitated endogenous NADRIN was visualized with a specific antibody.

FIGURE 6.

Model of the mechanism by which PACSIN 1 affects spine formation. A, in the absence of BDNF, overexpression of PACSIN 1 S358A or inhibition of CK2 supports a stable Rac1-NADRIN-PACSIN 1 complex which leads to Rac1 GTP hydrolysis. B, in the presence of BDNF higher levels of activated CK2 phosphorylate PACSIN 1 at Ser³⁵⁸ which causes a destabilization of the protein complex. This leads to higher concentrations of Rac1 GTP which induces spine formation. TrkB, tropomyosin-related kinase B.