KANPHOS: A Database of Kinase-Associated Neural Protein Phosphorylation in the Brain

Abstract

Protein phosphorylation plays critical roles in a variety of intracellular signaling pathways and physiological functions that are controlled by neurotransmitters and neuromodulators in the brain. Dysregulation of these signaling pathways has been implicated in neurodevelopmental disorders, including autism spectrum disorder, attention deficit hyperactivity disorder and schizophrenia. While recent advances in mass spectrometry-based proteomics have allowed us to identify approximately 280,000 phosphorylation sites, it remains largely unknown which sites are phosphorylated by which kinases. To overcome this issue, previously, we developed methods for comprehensive screening of the target substrates of given kinases, such as PKA and Rho-kinase, upon stimulation by extracellular signals and identified many candidate substrates for specific kinases and their phosphorylation sites. Here, we developed a novel online database to provide information about the phosphorylation signals identified by our methods, as well as those previously reported in the literature. The "KANPHOS" (Kinase-Associated Neural Phospho-Signaling) database and its web portal were built based on a next-generation XooNIps neuroinformatics tool. To explore the functionality of the KANPHOS database, we obtained phosphoproteomics data for adenosine-A2A-receptor signaling and its downstream MAPK-mediated signaling in the striatum/nucleus accumbens, registered them in KANPHOS, and analyzed the related pathways.

Keywords: KANPHOS; adenosine signaling; intellectual disability; molecular mechanism; neurodevelopmental disorders; phosphoproteomics; phosphorylation; signal transduction.

Figure 1

The schema for the construction of KANPHOS. (A) Schematic representation of the Kinase-Interacting Substrate Screening (KISS). In KISS method, rodent brain lysates were extracted and incubated with specific kinase-tag beads and ATP. The bound proteins were subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify the phosphorylated proteins and their phosphorylation sites. (B) Schematic representation of the Kinase-Oriented Substrate Screening (KIOSS) method. In KIOSS method, mouse coronal brain slices were treated with specific kinase activator/receptor agonist or kinase inhibitor/receptor antagonist to stimulate a specific signaling pathway and then incubated with GST tag pS/T binding domain (such as 14-3-3ζ) to enrich phosphoproteins. The bound proteins were subjected to LC-MS/MS to identify the phosphorylated proteins and their phosphorylation sites. (C) In addition to our own developed phosphoproteomic method, data were also manually curated from the literature and protoarray.

Figure 2

Data statistics and content of KANPHOS database. (A) Number of phosphorylation sites that were enlisted in KANPHOS using different methods; (B) Percentage of phosphoserine (pS), phosphothreonine (pT), and phosphotyrosine (pY) distribution; (C) Number of Phosphoprotein and phosphorylation sites identified in different species; (D) Distribution of phosphorylation sites among kinase families; (E) Percentage of phosphorylation sites identified in vitro, in vivo, and both in vitro and in vivo.

Figure 3

Overview of the KANPHOS workflow. (A) Homepage and browse function; (B) Simple search options (1) Search by kinases,(2) Search by substrates, and (3) Search by pathways; (C) Result window containing information about the phosphorylation sites, possible upstream signal and kinase.

Figure 4

Identification of signaling molecules downstream of Adenosine-A2AR pathway in striatum/NAc. (A) Model of A2AR signaling pathway in D2R-MSN; (B) Scheme for the phosphoproteomic analysis used to identify candidate substrates downstream of the A2AR pathway. Striatal/accumbal slices were stimulated with CGS21680 (5 μM) for 5 min after pretreatment with quinpirole (1 μM) for 10 min to induce A2AR-mediated phosphorylation of the phosphoprotein and then subjected to 14-3-3ζ affinity chromatography followed by LC-MS/MS; (C) Stimulation of adenosine A2AR promotes PKA-mediated phosphorylation of Rap1gap, and Rasgrp2 in striatal/accumbal slices; (D) Motif analysis of the identified phosphopeptides downstream of A2AR signaling is shown.

Figure 5

Identification of MAPK candidate substrates downstream of A2AR signaling in striatum/NAc. (A) Screening of phosphoproteins downstream of MAPK-mediated signaling. Striatal/accumbal slices were treated with okadaic acid (1 μM) for 60 min after pretreatment of MEK inhibitor U0126 (10 µM) for 30 min. OA induced the phosphorylation ERK at T202/Y204 and PKA substrate GluR1 at S845, whereas the pretreatment of MEK inhibitor reduced the phosphorylation of ERK but had no effect on PKA substrate GluR1 phosphorylation; (B) In silico analysis of the identified MAPK candidate substrates using the Reactome database (available online: http://www.reactome.org/ (accessed on 7 November 2021)) is shown; (C) Overlapping of MAPK and A2AR candidate substrates; (D) List of MAPK candidate substrates downstream of A2AR signaling.

Figure 6

Workflow of KANPHOS for the identification of adenosine-A2A receptor-mediated signaling molecules. (A) Identification of Arhgap21 as a PKA candidate substrate downstream of adenosine signaling using “Kinases” search. Red circle represents the selected option and arrow indicates result outcome after each selection; (B) Identification of kinases which may phosphorylate Arhgap21 downstream of adenosine signaling using “Substrates” search. Red rectangle represents the selected search option and arrow indicates result outcome after each selection.

Figure 7

A case study of the KANPHOS revealed that phosphorylation of HCN and calcium channel were involved in the MEK-MAPK pathway downstream of A2AR. (A) Pathway viewer window showed dopamine signaling pathway in D1R-MSN and D2R-MSN; (B) List of MAPK candidate substrates downstream of A2AR in KANPHOS; (C) Results of pathway analysis using KANPHOS; (D) Advanced search option for the identification of channel protein in the MEK-MAPK pathway downstream of A2AR; (E) List of channel protein involved in the MEK-MAPK pathway downstream of A2AR. Here, number (1–7) indicate the workflow step by step, red circle and red rectangle represent the selected search options, and arrow indicates result output after each selection.