Large-scale detection of ubiquitination substrates using cell extracts and protein microarrays.
Identification of protein targets of post-translational modification is an important analytical problem in biology. Protein microarrays exposed to cellular extracts could offer a rapid and convenient means of identifying modified proteins, but this kind of biochemical assay, unlike DNA microarrays, depends on a faithful reconstruction of in vivo conditions. Over several years, concentrated cellular extracts have been developed, principally for cell cycle studies that reproduce very complex cellular states. We have used extracts that replicate the mitotic checkpoint and anaphase release to identify differentially regulated poyubiquitination. Protein microarrays were exposed to these complex extracts, and the polyubiquitinated products were detected by specific antibodies. We expected that among the substrates revealed by the microarray should be substrates of the anaphase promoting complex (APC). Among 8,000 proteins on the chip, 10% were polyubiquitinated. Among those, we found 11 known APC substrates (out of 16 present on the chip) to be polyubiquitinated. Interestingly, only 1.5% of the proteins were differentially ubiquitinated on exit from the checkpoint. When we arbitrarily chose 6 proteins thought to be involved in mitosis from the group of differentially modified proteins, all registered as putative substrates of the APC, and among 4 arbitrarily chosen non-mitotic proteins picked from the same list, 2 were ubiquitinated in an APC-dependent manner. The striking yield of potential APC substrates from a simple assay with concentrated cell extracts suggests that combining microarray analysis of the products of post-translational modifications with extracts that preserve the physiological state of the cell can yield information on protein modification under various in vivo conditions.
Merbl
Yifat
Y
Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.
Kirschner
Marc W
MW
D064173:Anaphase-Promoting Complex-Cyclosome; D006367:HeLa Cells; D006801:Humans; D040081:Protein Array Analysis; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism*; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism; D054875:Ubiquitination
D011506:Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome