Regulation of phosphorylation at the postsynaptic density during different activity states of Ca2+/calmodulin-dependent protein kinase II

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Abstract

Ca2+/calmodulin-dependent protein kinase II (CaMKII), the most abundant kinase at the postsynaptic density (PSD), is expected to be involved in activity-induced regulation of synaptic properties. CaMKII is activated when it binds calmodulin in the presence of Ca2+ and, once autophosphorylated on T-286/7, remains active in the absence of Ca2+ (autonomous form). In the present study we used a quantitative mass spectrometric strategy (iTRAQ) to identify sites on PSD components phosphorylated upon CaMKII activation. Phosphorylation in isolated PSDs was monitored under conditions where CaMKII is: (1) mostly inactive (basal state), (2) active in the presence of Ca2+, and (3) active in the absence of Ca2+. The quantification strategy was validated through confirmation of previously described autophosphorylation characteristics of CaMKII. The effectiveness of phosphorylation of major PSD components by the activated CaMKII in the presence and absence of Ca2+ varied. Most notably, autonomous activity in the absence of Ca2+ was more effective in the phosphorylation of three residues on SynGAP. Several PSD scaffold proteins were phosphorylated upon activation of CaMKII. The strategy adopted allowed the identification, for the first time, of CaMKII-regulated sites on SAPAPs and Shanks, including three conserved serine residues near the C-termini of SAPAP1, SAPAP2, and SAPAP3. Involvement of CaMKII in the phosphorylation of PSD scaffold proteins suggests a role in activity-induced structural re-organization of the PSD.

Introduction

The postsynaptic density (PSD) is a multiprotein complex lining the plasma membrane on the dendritic side of the synapse. The PSD contains numerous receptors and signal transduction elements organized in a tight array by specialized scaffold proteins (review [1]). Certain types of activity-dependent changes in synaptic strength, including long-term potentiation (LTP), require an increase in postsynaptic Ca2+-levels and activation of CaMKII, an abundant component of the PSD (review [2]). CaMKII-mediated phosphorylation of PSD components is likely to underlie changes in synaptic strength. In the present study we sought to identify those phosphorylation events at the PSD that are regulated through CaMKII.

PSDs are rigidly organized complexes with components attached to one-another, presumably at defined protein–protein interaction sites. This type of organization makes enzymatic reactions essentially different from those in soluble systems where all components can have access to each other via diffusion. Thus, phosphorylation of purified proteins by purified CaMKII in a soluble system does not necessarily imply that the reaction would occur at the PSD, even if both kinase and substrate were present. We therefore chose to observe endogenous phosphorylation in isolated, intact PSDs where native appositions of components of the PSD are maintained.

CaMKII exhibits unique activation characteristics in its response to Ca2+ (review [3]). The holoenzyme is composed of ∼12 identical/similar subunits, each with catalytic and regulatory domains that allow activation upon binding of Ca2+/calmodulin. Organization within a multimeric unit allows intra-holoenzyme phosphorylation (autophosphorylation) of one subunit by another on multiple residues. Autophosphorylation of a particular residue – T-286 on the alpha subunit, T-287 on the beta subunit – confers autonomous activity, that is, CaMKII maintains the capacity to phosphorylate in the absence of Ca2+. We took advantage of the unique activation properties of the kinase to identify CaMKII-mediated phosphorylation events in isolated PSDs.

In recent years, implementation of mass spectrometric techniques in conjunction with an enrichment strategy for phosphopeptides yielded an extensive catalogue of phosphorylation sites at the PSD [4], [5], [6], [7], [8], [9]. While these approaches identified sites phosphorylated in vivo, information on the regulation of phosphorylation, including identities of the kinases involved, is, in many instances, still lacking. A previous study [10], [11] reported CaMKII-mediated phosphorylation of several PSD proteins, but in this case, the residues phosphorylated could not be identified by the methods employed. In the present study we applied a mass spectrometric strategy combined with the iTRAQ method for relative quantification to monitor the relative levels of phosphorylation at specific residues in isolated PSDs during different activity states of CaMKII.

Section snippets

Material and methods

Preparation of PSD fraction from rat cerebral cortex, as well as procedures following in vitro phosphorylation, including protein digestion, iTRAQ labeling, phosphopeptide enrichment, LC/MS/MS, and data analysis are detailed in Supplementary material section.

In vitro phosphorylation experiments. Prior to phosphorylation, PSD fractions were treated with 0.1 M dithiothreitol and let to stand on ice for 2–4 h. Reactions were started by the addition of PSD fraction on phosphorylation media as

Results

Autophosphorylation characteristics of CaMKII in the PSD fraction were studied using two sets of protocols comparing a group of samples subjected to four different incubation conditions each (Fig. 1). Under each protocol, Samples 1–4 were differentially labelled with iTRAQ reagents for comparison of phosphopeptide levels. Protocol I was designed to produce different activity states of CaMKII. Incubation for 5 min on ice in the presence of Ca2+/calmodulin was intended to switch on autonomous

Discussion

In vitro protein phosphorylation in intact organelles offers two advantages: retention of the native appositions of kinases and phosphatases vis a vis their substrates and the ability for precise manipulation of phosphorylation conditions which allows the tracking of particular enzymatic pathways. The present study demonstrates that application of quantitative mass spectrometry by the iTRAQ technique for the analysis of differentially phosphorylated intact organelles can lead to the global

Acknowledgments

This work was supported by the intramural research program at NIH/NINDS. We would like to thank Dr. Thomas Reese for support and discussion.

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