The plant phosphoproteome: no longer a black box?
study the effects of stress on kinase activities. H2O2 and other oxidative stress compounds are produced in plants In the current rush to discover cliniby various stressors, such as extreme cally relevant biomarkers of diseases, temperatures or pathogen exposure. To proteomic research on nonmodel orobserve early signaling responses, the ganisms, such as plants, is often left on researchers focused on kinase activities the sidelines. But plants are more than in the cytoplasm. A. thaliana cells were just decoration for your office window treated for 5 minutes with H2O2 or were or backyard garden. They impact human health by producing oxygen for us left untreated, and cytoplasmic extracts to breathe and food for us to eat. Some from both types of cells were applied plants even have medicinal qualities. to separate peptide arrays. Of the 47 To help unlock the secrets of plant peptides on the chip, 3 were specifically proteins, Sergio de la Fuente van phosphorylated when exposed to the Bentem and colleagues at the Univertreated cytoplasmic extract. A peptide sity of Vienna, the Medical University from the mRNA splicing factor SCL30 of Vienna, the Research was the most strongly Institute of Molecular phosphorylated protein, so Pathology, the Gregor the group focused on this Identification of in vivo Putative Mendel Institute of Mosubstrate. phosphorylation phosphorylation sites by MS lecular Plant Biology To quickly home in on network (all in Austria), Pepscan the kinase responsible for Presto (The Netherlands), phosphorylating SCL30, and URGV Plant Genomthe researchers first ics (France) studied the reviewed the literature. global phosphoproteome SRPK4 phosphorylates a of Arabidopsis thaliana by protein in the same family combining immobilized as SCL30, but it is unknown PeptideIdentification of protein kinase metal-ion affinity chromawhether SRPK4 is activated chip analysis tography (IMAC) and MS by H2O2. On the other Induced analysis with data obtained hand, MAP kinases are acspot from peptide arrays. In JPR tivated by H2O2, but SCL30 (2008, 7, 2458–2470), they is not a known substrate for Matching up kinases and phosphosites. Motif analysis and peptide chips report the identification of these kinases. Therefore, allowed scientists to home in on kinases that probably phosphorylate vari303 phosphosites, >80% of SRPK4 and three MAP kious proteins in plants. which were novel. In addinases were tested for their tion, they discovered that ability to phosphorylate van Bentem points out. phosphosites on certain proteins may full-length or truncated forms of SCL30 Motif analyses and peptide array be involved in the stress response. in vitro. All of the kinases phosphorylatexperiments were performed to match When de la Fuente van Bentem and ed all of the SCL30 constructs, but they up kinases with their substrates. By colleagues began this project 3 years did not modify a control peptide. In adexamining the amino acid sequences ago, scientists weren’t paying much dition, SRPK4 and MPK3 (MAP kinase of phosphorylated substrates in the attention to plant phosphoproteomes 3) phosphorylated the SCL30 peptide on data set, the researchers found that or even plant proteomics in general. a chip. Additional studies are necessary, mitogen-actived protein (MAP) kinases “Although a lot of research was carried but the researchers hypothesize that and casein kinase II probably can phos out in this field on animals, phosphoMAP kinases phosphorylate SCL30 in phorylate many of the identified phosproteomics was a complete black box vivo under stressful conditions and that phosites. Next, nuclear and cytoplasmic in plants,” recollects de la Fuente van SRPK4 constitutively phosphorylates extracts were applied to separate pepBentem. Some groups had identified this substrate under normal conditions. tide chips that contained 47 of the idena few sites on a few proteins. However, Next up for de la Fuente van Bentem tified phosphopeptides. The cytosolic no large-scale, proteome-wide studies and colleagues is a quantitative phosextract phosphorylated 17 peptides, of phosphorylation had been reported, phoproteomics study. “Our analysis whereas the nuclear extract phosphoryand only ~50 phosphosites were known was not quantitative, so now we want lated 20 peptides. The activities overat that time. After this project was off to apply a stress and see what changes,” lapped, but some peptides were preferthe ground, two other independent he says. With these projects, perhaps entially modified by one of the extracts. groups identified a total of ~1000 phosthe black box of the plant phosphoproThis finding suggested that certain phosites in A. thaliana, but these were teome will be completely ripped open to phosphorylation events occur primarily restricted to sites on plasma membrane reveal the secrets of protein regulation in the nucleus or cytoplasm. proteins. hidden within. Peptide arrays also were used to To discover more phosphoproteins —Katie Cottingham throughout A. thaliana cells and pinpoint phosphosites, de la Fuente van Bentem and colleagues extracted proteins from root cell cultures. The proteins were digested, and phosphopeptides were enriched with IMAC. The isolated peptides were analyzed by MS/ MS, and 303 phosphosites were identified on 205 proteins. Some overlap with other studies was observed, but most of the sites had never been described before. Interestingly, two phosphotyrosine residues were discovered. “In plants, hardly any phosphotyrosine sites have been described, and very few specific sites have been identified,” de la Fuente
Journal of Proteome Research • Vol. 7, No. 6, 2008 2189
