Abstract
The cyclic nucleotide cyclic guanosine monophosphate (cGMP) plays an important role in learning and memory, but its signaling mechanisms in the mammalian brain are not fully understood. Using mass-spectrometry-based proteomics, we evaluated how the cerebellum adapts its (phospho)proteome in a knockout mouse model of cGMP-dependent protein kinase type I (cGKI). Our data reveal that a small subset of proteins in the cerebellum (∼3% of the quantified proteins) became substantially differentially expressed in the absence of cGKI. More changes were observed at the phosphoproteome level, with hundreds of sites being differentially phosphorylated between wild-type and knockout cerebellum. Most of these phosphorylated sites do not represent known cGKI substrates. An integrative computational network analysis of the data indicated that the differentially expressed proteins and proteins harboring differentially phosphorylated sites largely belong to a tight network in the Purkinje cells of the cerebellum involving important cGMP/cAMP signaling nodes (e.g. PDE5 and PKARIIβ) and Ca2+ signaling (e.g. SERCA3). In this way, removal of cGKI could be linked to impaired cerebellar long-term depression at Purkinje cell synapses. In addition, we were able to identify a set of novel putative (phospho)proteins to be considered in this network. Overall, our data improve our understanding of cerebellar cGKI signaling and suggest novel players in cGKI-regulated synaptic plasticity.
Highlights
From ‡Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands; §Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, The Netherlands; ¶Interfakultares Institut fur Biochemie, Universitat Tubingen, D-72074 Tubingen, Germany; ʈGraduate School for Cellular and Molecular Neuroscience, Universitat Tubingen, D-72074 Tubingen, Germany; **German Center for Neurodegenerative diseases (DZNE), D-72076 Tubingen, Germany
We explored how mature state-of-the-artproteomics technology could be used to monitor the adaptation at the proteome level of the mouse cerebellum in a mouse line deficient for cyclic guanosine monophosphate (cGMP)-dependent protein kinase type I, a kinase that plays an important role in synaptic plasticity, motor learning, and other brain functions [14]
In this study we started to add much more molecular detail to this interesting field by investigating the changes that occur at the proteome and phosphoproteome levels of the cerebellum in a cGMPdependent protein kinase type I (cGKI)-deficient mouse model
Summary
Mouse Model—cGKI-KO mice carrying a null mutation of the prkg gene, termed cGKIL-/L- mice, were generated as described previously [24]. The three different labeled samples were reconstituted in 10% formic acid (FA) and mixed in a 1:1:1 ratio prior to fractionation using strong cation exchange (SCX) as described previously [26]. The enriched phosphopeptides were chromatographically separated with 150-min gradient as described previously and analyzed on an LTQ-Orbitrap Elite. To further filter for high-quality data, we used the following parameters: high-confidence peptide spectrum matches, minimal Mascot score of 20, minimal peptide length of six, only unique rank 1 peptide, and search rank 1 peptide. Antigen retrieval using the heating method was performed, and the sections were incubated overnight at 4 °C with primary antibodies: rabbit anti-cGKI (1:500) [36] or rabbit anti-SERCA3 (Thermo Fisher, 1:100). Sections were incubated with secondary antibody (Alexa 488, goat anti-rabbit, 1:200) for 1 h at room temperature and mounted with Shandon Immu-Mount (Thermo Fisher Scientific). Images were obtained under ϫ16 magnification using an Axiovert 200 (Zeiss)
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