Limits...
Elucidation of the evolutionary expansion of phosphorylation signaling networks using comparative phosphomotif analysis.

Yoshizaki H, Okuda S - BMC Genomics (2014)

Bottom Line: We compared the conservation of serine/threonine/tyrosine residues observed in humans and seven other species.Subsequently, we found that this zinc finger motif contributed to subcellular protein localization.Our study suggests that the phosphorylation networks acquired during evolution have added signaling network modules to the core signaling networks.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology I, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 920-0293, Japan. yossy@kanazawa-med.ac.jp.

ABSTRACT

Background: Protein phosphorylation is catalyzed by kinases and is involved in the regulation of a wide range of processes. The phosphosites in protein sequence motifs determine the types of kinases involved. The development of phosphoproteomics has allowed the identification of huge numbers of phosphosites, some of which are not involved in physiological functions.

Results: We developed a method for extracting phosphosites with important roles in cellular functions and determined 178 phosphomotifs based on the analysis of 34,366 phosphosites. We compared the conservation of serine/threonine/tyrosine residues observed in humans and seven other species. Consequently, we identified 16 phosphomotifs, where the level of conservation increased among species. The highly conserved phosphomotifs in humans and the worm were kinase regulatory sites. The motifs present in the fly were novel phosphomotifs, including zinc finger motifs involved in the regulation of gene expression. Subsequently, we found that this zinc finger motif contributed to subcellular protein localization. The motifs identified in fish allowed us to detect the expansion of phosphorylation signals related to alternative splicing. We also showed that the motifs present in specific species functioned in an additional network that interacted directly with the core signaling network conserved from yeast to humans.

Conclusions: Our method may facilitate the efficient extraction of novel phosphomotifs with physiological functions, thereby contributing greatly to the analysis of complex phosphorylation signaling cascades. Our study suggests that the phosphorylation networks acquired during evolution have added signaling network modules to the core signaling networks.

Show MeSH
Highly conserved motifs present in C2H2-type zinc finger motifs that regulate C2H2 localization in humans and the fly. (A) Structure of the C2H2-type zinc finger motif and the positions of motifs 82, 93, 121, and 129. (B) Sequence logos of the C2H2 motifs observed in all human proteins and those conserved in humans and the fly. (C) Number of C2H2 motifs observed in the genomes of species ranging from yeasts to humans. The colors in the bar plot indicate the lengths of the C2H2 motifs. (D) C2H2 motif sequence synthesized on the basis of the C2H2 motif in human ZNF24 protein. The ZNF24 proteins are known to be phosphorylated at tyrosine and threonine residues. The asterisk indicates a phosphosite. (E) Localization of m1Venus-2xC2H2 and m1CFP in Cos7 cells. The image showing m1Venus-2xC2H2 relative to m1CFP was produced to demonstrate their localization (upper images). Localizations of m1Venus-2xC2H2 and the m1CFP-2xC2H2YF mutant in Cos7 cells. The image of m1Venus-2xC2H2 relative to m1CFP-2xC2H2YF was produced to demonstrate their localization (lower images). The 2xC2H2YF mutant with a YF mutation at the tyrosine residue, which is indicated by an asterisk in Figure 4E. The arrows indicate nuclear locations with higher relative differences in CFP and YFP images.
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Fig4: Highly conserved motifs present in C2H2-type zinc finger motifs that regulate C2H2 localization in humans and the fly. (A) Structure of the C2H2-type zinc finger motif and the positions of motifs 82, 93, 121, and 129. (B) Sequence logos of the C2H2 motifs observed in all human proteins and those conserved in humans and the fly. (C) Number of C2H2 motifs observed in the genomes of species ranging from yeasts to humans. The colors in the bar plot indicate the lengths of the C2H2 motifs. (D) C2H2 motif sequence synthesized on the basis of the C2H2 motif in human ZNF24 protein. The ZNF24 proteins are known to be phosphorylated at tyrosine and threonine residues. The asterisk indicates a phosphosite. (E) Localization of m1Venus-2xC2H2 and m1CFP in Cos7 cells. The image showing m1Venus-2xC2H2 relative to m1CFP was produced to demonstrate their localization (upper images). Localizations of m1Venus-2xC2H2 and the m1CFP-2xC2H2YF mutant in Cos7 cells. The image of m1Venus-2xC2H2 relative to m1CFP-2xC2H2YF was produced to demonstrate their localization (lower images). The 2xC2H2YF mutant with a YF mutation at the tyrosine residue, which is indicated by an asterisk in Figure 4E. The arrows indicate nuclear locations with higher relative differences in CFP and YFP images.

Mentions: Motifs 82, 93, 121, and 129 were observed to be highly conserved motifs in species ranging from the fly to humans. Motifs 121 and 129 shared most of the proteins related to each motif, although these motifs were different. Motifs 82 and 93 also shared several of these proteins. A network of 194 proteins obtained from these motifs was extended using interaction information obtained from BioGRID and STRING, similar to Figure 2B, but only nine proteins were connected by known interaction information (data not shown). We investigated the domain structures of these motifs using Pfam search and observed that all phosphomotifs were related to a zinc finger motif (Figure 4A). The C2H2 motif is a structure where two cysteines and two histidines chelate zinc and proteins and has multiple tandem zinc finger motifs that can bind to DNA [37]. The C2H2 motifs were observed primarily in transcription factors, and over 6000 C2H2 motifs have been identified in the human genome [38]. We detected 6743 C2H2 motifs in our dataset. It has been reported that zinc finger proteins with C2H2 motifs regulate development and cell differentiation [39]. This motif was conserved in species ranging from the fly to humans, where a serine between cysteines and histidines, a threonine immediately downstream of the second histidine, and a tyrosine two amino acids upstream of the first cysteine in the C2H2 motif were phosphorylated.


Elucidation of the evolutionary expansion of phosphorylation signaling networks using comparative phosphomotif analysis.

Yoshizaki H, Okuda S - BMC Genomics (2014)

Highly conserved motifs present in C2H2-type zinc finger motifs that regulate C2H2 localization in humans and the fly. (A) Structure of the C2H2-type zinc finger motif and the positions of motifs 82, 93, 121, and 129. (B) Sequence logos of the C2H2 motifs observed in all human proteins and those conserved in humans and the fly. (C) Number of C2H2 motifs observed in the genomes of species ranging from yeasts to humans. The colors in the bar plot indicate the lengths of the C2H2 motifs. (D) C2H2 motif sequence synthesized on the basis of the C2H2 motif in human ZNF24 protein. The ZNF24 proteins are known to be phosphorylated at tyrosine and threonine residues. The asterisk indicates a phosphosite. (E) Localization of m1Venus-2xC2H2 and m1CFP in Cos7 cells. The image showing m1Venus-2xC2H2 relative to m1CFP was produced to demonstrate their localization (upper images). Localizations of m1Venus-2xC2H2 and the m1CFP-2xC2H2YF mutant in Cos7 cells. The image of m1Venus-2xC2H2 relative to m1CFP-2xC2H2YF was produced to demonstrate their localization (lower images). The 2xC2H2YF mutant with a YF mutation at the tyrosine residue, which is indicated by an asterisk in Figure 4E. The arrows indicate nuclear locations with higher relative differences in CFP and YFP images.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4117960&req=5

Fig4: Highly conserved motifs present in C2H2-type zinc finger motifs that regulate C2H2 localization in humans and the fly. (A) Structure of the C2H2-type zinc finger motif and the positions of motifs 82, 93, 121, and 129. (B) Sequence logos of the C2H2 motifs observed in all human proteins and those conserved in humans and the fly. (C) Number of C2H2 motifs observed in the genomes of species ranging from yeasts to humans. The colors in the bar plot indicate the lengths of the C2H2 motifs. (D) C2H2 motif sequence synthesized on the basis of the C2H2 motif in human ZNF24 protein. The ZNF24 proteins are known to be phosphorylated at tyrosine and threonine residues. The asterisk indicates a phosphosite. (E) Localization of m1Venus-2xC2H2 and m1CFP in Cos7 cells. The image showing m1Venus-2xC2H2 relative to m1CFP was produced to demonstrate their localization (upper images). Localizations of m1Venus-2xC2H2 and the m1CFP-2xC2H2YF mutant in Cos7 cells. The image of m1Venus-2xC2H2 relative to m1CFP-2xC2H2YF was produced to demonstrate their localization (lower images). The 2xC2H2YF mutant with a YF mutation at the tyrosine residue, which is indicated by an asterisk in Figure 4E. The arrows indicate nuclear locations with higher relative differences in CFP and YFP images.
Mentions: Motifs 82, 93, 121, and 129 were observed to be highly conserved motifs in species ranging from the fly to humans. Motifs 121 and 129 shared most of the proteins related to each motif, although these motifs were different. Motifs 82 and 93 also shared several of these proteins. A network of 194 proteins obtained from these motifs was extended using interaction information obtained from BioGRID and STRING, similar to Figure 2B, but only nine proteins were connected by known interaction information (data not shown). We investigated the domain structures of these motifs using Pfam search and observed that all phosphomotifs were related to a zinc finger motif (Figure 4A). The C2H2 motif is a structure where two cysteines and two histidines chelate zinc and proteins and has multiple tandem zinc finger motifs that can bind to DNA [37]. The C2H2 motifs were observed primarily in transcription factors, and over 6000 C2H2 motifs have been identified in the human genome [38]. We detected 6743 C2H2 motifs in our dataset. It has been reported that zinc finger proteins with C2H2 motifs regulate development and cell differentiation [39]. This motif was conserved in species ranging from the fly to humans, where a serine between cysteines and histidines, a threonine immediately downstream of the second histidine, and a tyrosine two amino acids upstream of the first cysteine in the C2H2 motif were phosphorylated.

Bottom Line: We compared the conservation of serine/threonine/tyrosine residues observed in humans and seven other species.Subsequently, we found that this zinc finger motif contributed to subcellular protein localization.Our study suggests that the phosphorylation networks acquired during evolution have added signaling network modules to the core signaling networks.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology I, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 920-0293, Japan. yossy@kanazawa-med.ac.jp.

ABSTRACT

Background: Protein phosphorylation is catalyzed by kinases and is involved in the regulation of a wide range of processes. The phosphosites in protein sequence motifs determine the types of kinases involved. The development of phosphoproteomics has allowed the identification of huge numbers of phosphosites, some of which are not involved in physiological functions.

Results: We developed a method for extracting phosphosites with important roles in cellular functions and determined 178 phosphomotifs based on the analysis of 34,366 phosphosites. We compared the conservation of serine/threonine/tyrosine residues observed in humans and seven other species. Consequently, we identified 16 phosphomotifs, where the level of conservation increased among species. The highly conserved phosphomotifs in humans and the worm were kinase regulatory sites. The motifs present in the fly were novel phosphomotifs, including zinc finger motifs involved in the regulation of gene expression. Subsequently, we found that this zinc finger motif contributed to subcellular protein localization. The motifs identified in fish allowed us to detect the expansion of phosphorylation signals related to alternative splicing. We also showed that the motifs present in specific species functioned in an additional network that interacted directly with the core signaling network conserved from yeast to humans.

Conclusions: Our method may facilitate the efficient extraction of novel phosphomotifs with physiological functions, thereby contributing greatly to the analysis of complex phosphorylation signaling cascades. Our study suggests that the phosphorylation networks acquired during evolution have added signaling network modules to the core signaling networks.

Show MeSH