Global analysis of phosphorylation networks in humans

Abstract

Phosphorylation-mediated signaling plays a crucial role in nearly every aspect of cellular physiology. A recent study based on protein microarray experiments identified a large number of kinase-substrate relationships (KSRs), and built a comprehensive and reliable phosphorylation network in humans. Analysis of this network, in conjunction with additional resources, revealed several key features. First, comparison of the human and yeast phosphorylation networks uncovered an evolutionarily conserved signaling backbone dominated by kinase-to-kinase relationships. Second, although most of the KSRs themselves are not conserved, the functions enriched in the substrates for a given kinase are often conserved. Third, the prevalence of kinase-transcription factor regulatory modules suggests that phosphorylation and transcriptional regulatory networks are inherently wired together to form integrated regulatory circuits. Overall, the phosphorylation networks described in this work promise to offer new insights into the properties of kinase signaling pathways, at both the global and the protein levels. This article is part of a Special Issue entitled: Computational Proteomics, Systems Biology & Clinical Implications. Guest Editor: Yudong Cai.

Keywords: Conservation; Kinase–substrate relationships (KSRs); Network module; Phosphorylation network.

Fig. 1. Statistics of human phosphorylation network

(A) The number of kinases per substrate and (B) the number of substrates per kinase are plotted. Mean and median values are shown. (C) The distribution of substrate overlap rate among all possible kinase pairs. Supposing A and B are the substrate sets of a pair of kinases, the overlap rate is defined as Jaccard Index (A∩B)/(A∪B). (D) The distribution of substrate numbers per kinase among different kinase groups. The bottom and top of the boxes represent the 25^th and 75^th percentile of the distribution, respectively. The line near the middle of the box represents the 50^th percentile. The empty squares in each box indicate the mean. The whiskers represent the 9^th percentile and the 91^st percentile. The dashed line across the figure indicates the overall average number of substrates per kinase identified in this study. (E) The distribution of phosphoproteins across various protein families. The Y-axis represents the fraction of proteins identified as kinase substrates in each of the six major protein families indicated. (F) The phosphoprotein percentage for different functional groups. Each point corresponds to one functional group (i.e., a GO term, such as “RNA-binding proteins”). The X-axis is the percentage calculated based on MS/MS data while the Y-axis is based on our protein microarray data. A good correlation between these two independent studies is observed. PCC, Pearson correlation coefficient.

Fig. 2. Evolutionarily conserved core

(A) Conserved core of phosphorylation networks identified by comparing the human and yeast KSR networks. Kinases and non-kinase proteins are represented in orange and green nodes, respectively. The size of the nodes is proportional to the number of associated interactions. (B) The overall conservation of K–K and K–S relationships. All human KSRs were compared with yeast and the percentage of conserved human KSRs was calculated for each cutoff. The conservation was defined as that the two proteins (kinase and substrate) have homologs in yeast and the homologous pairs also have the same KSR relationship in both species. (C) The conservation of human K–K and K–S in which the two proteins (kinase and substrate) both have homologs in yeast. Using this approach, we essentially removed the contribution from the protein sequence conservation and only considered the contribution from the kinase-substrate interaction conservation.

Fig. 3. Functional conservation between human PKA and yeast Tpk1

The substrates involved in cell differentiation are shown for these two kinases. Short lines indicate for homologous relationships between proteins from human and yeast.

Fig. 4. Representative conserved modulogs

A modulog is defined as evolutionarily conserved regulatory modules (see text for more details). The two numbers under each module represent the number of this module found in the human comKSRs (orange box) and that found in the conserved core KSR networks (grey boxes), respectively.

Fig. 5. List of 71 modules that include at least one transcription factor (TF) and one kinase

Arrows starting from kinases (orange) represent phosphorylation events, while arrows starting from TFs (blue) represent transcriptional activity. The three numbers below each module are the Z-score, the observed and expected numbers of the module in the network, respectively. The over- and under-represented modules are highlighted in red and blue, respectively.

Fig. 5. List of 71 modules that include at least one transcription factor (TF) and one kinase

Arrows starting from kinases (orange) represent phosphorylation events, while arrows starting from TFs (blue) represent transcriptional activity. The three numbers below each module are the Z-score, the observed and expected numbers of the module in the network, respectively. The over- and under-represented modules are highlighted in red and blue, respectively.