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C. elegans phosphoproteome sticks out Protein phosphorylation patterns in C. elegans diverge significantly from those seen in other higher eukaryotes, such as Drosophila and humans, as Matthias Mann and colleagues at the Max Planck Institute of Biochemistry (Germany) report in JPR (2009, DOI http:// dx.doi.org/10.1021/pr900384k). Phosphorylation, which is modulated by kinases and phosphatases, plays many essential roles in cell growth and signaling during development. Phosphoproteomessinventories of phosphorylation sites on an organism’s proteinss have recently been reported for yeast, fly, mouse, and human, but not C. elegans. C. elegans has proven to be an ideal model organism for studying topics such as programmed cell death and the relationship between metabolism and longevity. But even among worms, C. elegans sticks out. Based on its genome, the nematode appears to have split from its closest known relatives around 30 million years ago, and its self-fertilizing reproductive strategy has shaped its genome in peculiar ways. So when Florian Gnad, a bioinformatics specialist in Mann’s group, sat down to analyze phosphorylation in C. elegans’ proteins, he was gratified to see how different the organism was compared to other eukaryotes. “Our initial plan was to look at phosphorylation sites in worm, to see if they were conserved in the same way as in other organisms,” says Gnad, who is now at Harvard University. “It turns out they are not.” Evidence suggests that the worm’s phosphorylation patterns have changed since the evolutionary time when C. elegans separated from other multicellular organisms, he says. To reach this conclusion, Gnad teamed up with Dorota Zielinska and Jacek Wis´niewski, who had developed a versatile sample preparation method that allows full solubilization of membrane proteins by using strong detergents. They combined Wis´niewski’s filter-aided sample preparation method of removing detergents with titanium oxide chromatography, which selects for phosphorylated peptides.
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They identified almost 6800 phosphorylation sites on 2400 proteins in adult worms and analyzed the sites based on the protein sequences around the phosphorylation sites. Many kinases prefer a characteristic sequence motif around their substrate sites. Protein kinase A, which is responsible for responding to the messenger cyclic AMP, prefers the
The phosphorylation sites in C. elegans are cataloged in www.phosida.com, an online repository for phosphorylation information.
motif R-R/K-X-S/T (where X stands for any amino acid), for example. C. elegans’ phosphoproteome contains numerous examples of kinase motifs common to other eukaryotes, but it also contains motifs not seen elsewhere. “While C. elegans has many kinases not found in Drosophila or human, almost all of the extras in worm come from expansions of families that are shared with fly and human,” says Gerard Manning, who specializes in kinase bioinformatics at the Salk Institute. “So these new motifs and the generally different pattern of worm phosphosites are interesting and mysterious.” The authors also catalogued phosphorylated proteins on the basis of their presumed function and saw a strong representation of sequences predicted to have activities such as ATPase and nucleic acid binding. What was distinct about C. elegans,
Journal of Proteome Research • Vol. 8, No. 8, 2009
they write, was an overrepresentation of proteins involved in sex determination and differentiation. “In the mouse and human phosphoproteomes, this is not the case,” Gnad says. In these worms, a unique dosage compensation complex makes sure cells do not get too much expression from the genes on the X chromosome. The complex represses gene transcription from the X chromosome in hermaphrodites, which have two X chromosomes, but is inactive in males. The dosage compensation complex resembles the condensin complex, which is involved in chromosome assembly and segregation and is found in all eukaryotes. The authors propose that the dosage complex is regulated by phosphorylation, because its subunits are phosphorylated on sites that are not conserved in the condensin complex in other eukaryotes. Eric Haag, who studies C. elegans sex differentiation at the University of Maryland College Park, says phosphorylation could play a regulatory role in dosage compensation, but little direct evidence exists for it. He adds that analysis of samples of all-male worms or worms in specific developmental states would be necessary to extend the initial findings. Haag says he was intrigued by the results on sex determination pathways, because a key phosphatase required for male development, fem-2, has substrates that have not yet been identified. “Our field was founded and has been dominated by some really smart geneticists. But it appears that genetics is reaching the point of diminishing returns,” he says. “This will inspire other C. elegans researchers who are willing to grind up some worms and do some biochemistry.” Manning says that phosphoproteomic analysis comparing C. elegans with other eukaryotes is just beginning. “This is a very fast-moving field,” he says. “Overall, I see this as a resource that will pay off for many years to come, and probably the first of many such studies, with later studies focusing on particular developmental states, mutations, or stresses.” —Quinn Eastman
10.1021/pr900559f
© 2009 American Chemical Society
