Biochemical and Biophysical Research Communications
CaMKIIα phosphorylation of Shank3 modulates ABI1-Shank3 interaction
Introduction
The scaffolding protein Shank3 interacts directly or indirectly with receptors, ion channels, cytoskeletal proteins, and signaling molecules to regulate their targeting and anchoring to postsynaptic densities and dendritic spines in excitatory neurons [1,2]. These interactions are mediated by well-defined binding domains in Shank3, including multiple ankyrin repeats, SH3 and PDZ domains, a novel CaMKII binding motif, distinct proline-rich regions for binding to ABI1, homer, and cortactin, and a C-terminal SAM domain [[3], [4], [5], [6], [7]]. Mutations in the SHANK3 gene are often linked to neurodevelopmental and neuropsychiatric disorders, such as autism spectrum disorder (ASD) [8]. Moreover, disruptions of Shank3 expression in multiple mouse models result in deficits in synaptic transmission, abnormal neuronal morphology, and diverse behavioral phenotypes [9,10]. Since phenotypes of these mouse models vary with the specific Shank3 mutation, which often target different exons, understanding physiological signaling mechanisms coupled to different regions of Shank3 will provide better targeted therapy for Shank3-related neuropsychiatric disorders.
Phosphorylation of scaffolding proteins can regulate their binding interactions, subcellular distribution, and ultimately their physiological function [11]. Several phospho-proteomics studies have reported that Shank3 is phosphorylated at multiple sites when isolated from brain tissues, and in some instances the kinase and residue have been identified. For example, ribosomal S6 kinase 2 (RSK2) phosphorylates Shank3 at Ser1648 in vitro and in primary neurons [12]. Recently, Shank3 was shown to be phosphorylated in vivo at Ser685, immediately adjacent to the ABI1 binding motif [13]. A phospho-null S685A mutation or a missense S685I mutation, identified in a patient with ASD, reduced the ABI1-Shank3 interaction and impaired dendritic spine development and synaptic transmission. In vitro studies indicated that PKA, but not ERK2, GSK3β, or casein kinase 2, phosphorylated Ser685 [13]. However, a direct effect of Shank3 Ser685 phosphorylation, or of a phospho-mimetic mutation of Ser685 to an acidic residue, on ABI1 binding was not reported.
Synaptic plasticity involves dynamic changes in the actin cytoskeleton and spine morphology that are modulated by both Ca2+ and cAMP signaling [[14], [15], [16], [17], [18]]. While CaMKII and PKA can phosphorylate the same residues in some shared substrates, such as connexin43 [19,20] or the Rpt6 subunit of the 26 S proteasome [21,22], they also target distinct sites in other shared substrates, such as the GluA1 subunit of the AMPA-type glutamate receptor [23,24]. Notably, phospho-proteomics analyses revealed that phosphorylation of several sites in Shank3 is increased following CaMKII activation in isolated synaptic fractions [25,26]. Thus, it seems likely that both PKA and CaMKII can target Shank3, but the potential role of CaMKII in Shank3 Ser685 phosphorylation has not been investigated.
In this study, we tested the hypothesis that Shank3 Ser685 is also targeted by CaMKII. We found that both PKA and CaMKII can phosphorylate a GST-Shank3 fusion protein containing residues 572–691, and that this phosphorylation is prevented by mutation of Ser685 to a phospho-null alanine residue. Consistent with prior studies, any mutation of Ser685 disrupts Shank3 interactions with ABI1. However, we show for the first time that Ser685 phosphorylation by CaMKII significantly enhances ABI1 interaction with Shank3.
Section snippets
Antibodies
The following antibodies and dilutions were used for immunoblotting: mouse anti-GFP (Vanderbilt Antibody and Protein Resource catalog 1C9A5, 1:3000), mouse anti-HA (Biolegend catalog 901503, 1:3000), mouse anti-CaMKIIα 6G9 (Thermo Fisher Scientific catalog MA1-048, 1:3000), goat anti-GST (GE Healthcare Life Sciences catalog 27-4577-01, 1:5000), HRP-conjugated anti-mouse (Promega catalog W4021, 1:3000), and HRP-conjugated anti-goat (Abcam catalog ab6741, 1:5000).
Cloning and protein expression
Constructs to express GFP-Shank3
CaMKIIα and PKA phosphorylate Shank3 in vitro
As an initial test of our hypothesis that CaMKII can phosphorylate Shank3 at Ser685, a site previously shown to be phosphorylated by PKA, we generated a GST fusion protein containing Shank3 amino acids 572–691, which includes the PDZ domain and the adjacent ABI1-binding motif. GST was used as a negative control and a GST fusion protein containing the AMPA receptor subunit GluA1 C-terminal residues 827–901 was used as a positive control because CaMKII and PKA specifically phosphorylate Ser831 [23
Discussion
Dynamic changes in the size and morphology of dendritic spines and postsynaptic densities during synaptic development and synaptic plasticity are mediated in part by the re-organization of multiprotein complexes that are assembled by numerous scaffolding proteins [29]. Phosphorylation of some synaptic proteins has been shown to modulate their interaction with scaffolding proteins [30,31]. Here, we provide direct evidence that phosphorylation of a major synaptic scaffolding protein, Shank3,
Author contributions
TLP prepared the initial drafts of the manuscript. TLP and RJC edited and revised the manuscript with comments and contributions from all authors. TLP, PES, and KLS performed experiments. TLP and RJC conceived the study. All authors approved the final version of this manuscript.
Funding
This work was supported in part by the National Institutes of Health [T32-DK007563 to TLP, R01-MH063232 and R01-NS078291 to RJC] and the American Heart Association [18PRE33960034 to TLP].
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We thank Drs. Craig Garner, Winship Herr, and Jackie Corbin for generously providing various plasmids and reagents, as detailed above.
References (37)
- et al.
Regulation of dendritic spine morphology and synaptic function by Shank and Homer
Neuron
(2001) - et al.
Shank, a novel family of postsynaptic density proteins that binds to the NMDA receptor/PSD-95/GKAP complex and cortactin
Neuron
(1999) - et al.
Coupling of mGluR/Homer and PSD-95 complexes by the Shank family of postsynaptic density proteins
Neuron
(1999) - et al.
Transcriptional and functional complexity of Shank3 provides a molecular framework to understand the phenotypic heterogeneity of SHANK3 causing autism and Shank3 mutant mice
Mol. Autism.
(2014) Translocation of autophosphorylated calcium/calmodulin-dependent protein kinase II to the postsynaptic density
J. Biol. Chem.
(1997)- et al.
Regulation of actin dynamics during structural plasticity of dendritic spines: signaling messengers and actin-binding proteins
Mol. Cell. Neurosci.
(2018) - et al.
Regulation of neuronal PKA signaling through AKAP targeting dynamics
Eur. J. Cell Biol.
(2006) - et al.
Subcellular dynamics of type II PKA in neurons
Neuron
(2009) - et al.
The effects of connexin phosphorylation on gap junctional communication
Int. J. Biochem. Cell Biol.
(2004) - et al.
Proteasome function is regulated by cyclic AMP-dependent protein kinase through phosphorylation of Rpt6
J. Biol. Chem.
(2007)
Regulation of the proteasome by neuronal activity and calcium/calmodulin-dependent protein kinase II
J. Biol. Chem.
Characterization of multiple phosphorylation sites on the AMPA receptor GluR1 subunit
Neuron
Identification of novel phosphorylation sites on postsynaptic density proteins
Biochem. Biophys. Res. Commun.
Regulation of phosphorylation at the postsynaptic density during different activity states of Ca2+/calmodulin-dependent protein kinase II
Biochem. Biophys. Res. Commun.
Differential modulation of Ca2+/calmodulin-dependent protein kinase II activity by regulated interactions with N-methyl-D-aspartate receptor NR2B subunits and alpha-actinin
J. Biol. Chem.
Characterization of the Shank family of synaptic proteins. Multiple genes, alternative splicing, and differential expression in brain and development
J. Biol. Chem.
SHANK proteins: roles at the synapse and in autism spectrum disorder
Nat. Rev. Neurosci.
Abelson interacting protein 1 (Abi-1) is essential for dendrite morphogenesis and synapse formation
EMBO J.
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