Large-scale detection of ubiquitination substrates using cell extracts and protein microarrays Yifat Merbl and Marc W. Kirschner1 Department of Systems Biology, Harvard Medical School, Boston, MA 02115 Contributed by Marc W. Kirschner, December 19, 2008 (sent for review August 12, 2008) Identification of protein targets of post-translational modification is even though there is usually no attempt to reproduce exact an important analytical problem in biology. Protein microarrays cellular conditions. However, it is also likely that the use of exposed to cellular extracts could offer a rapid and convenient means purified enzymes is too permissive as it is well-known, for of identifying modified proteins, but this kind of biochemical assay, example, that purified kinases will label many proteins that are unlike DNA microarrays, depends on a faithful reconstruction of in not important in vivo targets. Furthermore, these in vitro vivo conditions. Over several years, concentrated cellular extracts reactions lack many cellular components, such as adapter and have been developed, principally for cell cycle studies that reproduce scaffold proteins, competing substrates, phosphatases, and in- very complex cellular states. We have used extracts that replicate the hibitors, that help specify targets. To ascertain whether concen- mitotic checkpoint and anaphase release to identify differentially trated and functional in vitro extracts can generate physiological regulated poyubiquitination. Protein microarrays were exposed to responses on solid-state arrays of full-length proteins, we studied these complex extracts, and the polyubiquitinated products were the well characterized ubiquitination of proteins at the met- detected by specific antibodies. We expected that among the sub- aphase-anaphase transition. Using what we would like to call an strates revealed by the microarray should be substrates of the extract-based functional assay (EFA), we have asked whether we anaphase promoting complex (APC). Among 8,000 proteins on the can identify new targets of ubiquitination at the metaphase- chip, 10% were polyubiquitinated. Among those, we found 11 known anaphase transition in mammalian cells. APC substrates (out of 16 present on the chip) to be polyubiquiti- nated. Interestingly, only 1.5% of the proteins were differentially Results ubiquitinated on exit from the checkpoint. When we arbitrarily chose It was shown previously that the mitotic checkpoint (CP) system 6 proteins thought to be involved in mitosis from the group of can be reproduced in extracts from nocodazole-arrested mam- differentially modified proteins, all registered as putative substrates malian cells (1, 6, 7). Nocodazole prevents the formation of the of the APC, and among 4 arbitrarily chosen non-mitotic proteins mitotic spindle and thus produces a strong checkpoint signal that picked from the same list, 2 were ubiquitinated in an APC-dependent inhibits the anaphase promoting complex (APC) which is the manner. The striking yield of potential APC substrates from a simple major ubiquitin ligase in mitosis and G1. APC inhibition can be assay with concentrated cell extracts suggests that combining mi- overcome by the addition of UbcH10 (7), an E2 that regulates croarray analysis of the products of post-translational modifications mitotic cyclin degradation (8). Thus, there is a well-characterized with extracts that preserve the physiological state of the cell can yield system for detecting APC-dependent polyubiquitination events information on protein modification under various in vivo conditions. that occur when the checkpoint is released and the cells enter anaphase. Checkpoint extracts from HeLa S3 cells arrested with anaphase promoting complex ͉ EFA ͉ post-translational modification ͉ nocodazole were divided into 3 aliquots, one was retained and proteomics denoted as the checkpoint extract (CP-extract), one was supple- mented with UbcH10 to relieve the checkpoint arrest (CP- rotein post-translational modifications have been implicated released), and the third also received UbcH0 along with a Pin virtually every aspect of cell regulation. Yet, work in this specific inhibitor of APC, emi1(APC-inhibited). To ascertain area, particularly on a genome-wide scale, has been hampered by the functionality of the extracts, an aliquot of each sample was a lack of suitable analytical methods. This is especially true for removed and S35 labeled-securin, a well-characterized mitotic polyubiquitination where the products are heterogeneous and substrate, was added (Fig. 1A). Securin remained stable in intermediates are found at low abundance in the cell. Hence, CP-extracts, consistent with the inhibition of APC by the spindle identifying polyubquitinated proteins in complex mixtures is very checkpoint. Extracts supplemented with UbcH10 (CP-released) difficult. One simple way of identifying proteins is to array them degraded securin rapidly while the addition of the APC inhibitor on a solid support where the position on the array serves as a emi1 (APC-inhibited) stabilized securin for at least 60 minutes. means of identification, thus obviating problems of analyzing The same 3 types of extracts were added to the protein microar- heterogeneous mixtures of products. However, to use a protein rays for 60 minutes at room temperature. The microarray, array to generate post-translational modifications on specific produced by Invitrogen contained approximately 8,000 proteins proteins relies on in vitro system for carrying out the chemical spotted in duplicate at a reported level of around 10 pg per spot reactions, in contrast to relying on a more physiological in vivo (median diameter ϭϷ150 ␮m). We used an anti-polyubiquitin analysis. Concentrated functional mammalian cell extracts have (FK1) antibody (see supporting information (SI) Fig. S1)to been shown to recapitulate complex events, such as the ordered detect ubiquitinated proteins on the microarray. Microarrays degradation of mitotic substrates, and, therefore, for many BIOCHEMISTRY purposes, bridge the gap between in vivo and in vitro conditions (1). Thus, we wished to see whether we could use these special Author contributions: Y.M. and M.W.K. designed research; Y.M. performed research; Y.M. cell-free systems and combine them with protein microarrays to analyzed data; and Y.M. and M.W.K. wrote the paper. identify targets of post-translational modification in a physio- The authors declare no conflict of interest. logical context. A few groups have pioneered protein microar- Freely available online through the PNAS open access option. rays in which full-length proteins are arrayed to identify the 1To whom correspondence should be addressed. E-mail: [email protected]. substrates of purified kinases (2, 3) or have used microarrays to This article contains supporting information online at www.pnas.org/cgi/content/full/ study binding interactions with small molecules or other proteins 0812892106/DCSupplemental. (4, 5). These assays can produce useful biochemical information © 2009 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812892106 PNAS ͉ February 24, 2009 ͉ vol. 106 ͉ no. 8 ͉ 2543–2548 Downloaded by guest on September 29, 2021 extract preparations (biological replicates) and with extracts under A different conditions (CP released vs. APC-inhibited). Fig. 2C shows scatter plots of the positive spot reactivities in each comparison (log scale). Visually, the different conditions (red dots) produced a signal that was more spread and variable compared with the biological replicates that are closer to the diagonal (black dots). As B expected, when we averaged the reactivity of spots per each protein, the variability between two biological replicate chips decreases in each of the conditions (Fig. S2). To determine which proteins are modified at release from the mitotic checkpoint, we compared the signals from the CP-released and the APC-inhibited extracts. Two microarrays from each con- dition were examined and a two-tailed t test was used to identify differentially modified proteins. To determine significance we used a permutation-based P value calculation (9–11) and corrected for false discovery rate (FDR) using Storey’s (12, 13) method (see Materials and Methods for details of the data analysis and functions that were used). Over 132 proteins were differentially modified (q-value Ͻ0.096) and these proteins are listed in Table S1.Alistof the top 20 proteins in our list (sorted by q-value) is presented in Table 1. Using an algorithm for clustering by functional annotation [DAVID (14)], we identified which biological terms/functions are specifically enriched in our gene list compared to the background list of proteins that were spotted on the microarray. Fig. 3 presents a summary of the enriched functional terms (P Ͻ 0.05) and the Fig. 1. (A) Assaying the biological state and competence of the extracts. To proteins that were classified to each GO annotation (black squares). evaluate the activity of the extracts, S35-labeled securin was added to extract The percentage (%) of proteins that correspond to each GO samples to follow its degradation. The reactions were stopped at the indicted category is presented. Interestingly, among the most enriched times by the addition of sample buffer and were then analyzed by on SDS/ biological terms in our predicted list were ‘post-translational pro- PAGE and autoradiography. The star (*) labeled lanes reflect the state of the tein modification’ (GO:0043687, P value ϭ 4.02EϪ10)