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Identifying Substrates of the Pho85-Pcl1 Kinase

出版	University of California, San Francisco, 2006
ISBN	05428289609780542828966
URL	http://books.google.com.hk/books?id=KGslNFxV5YQC&hl=&source=gbs_api

註釋Phosphorylation is a ubiquitous protein modification important for regulating nearly every aspect of cellular biology. Identifying the substrates of a kinase is essential for understanding its cellular role, but doing so remains a difficult task. Pho85 is a non-essential yeast cyclin dependent kinase which when bound to the cyclin Pho80 plays a well characterized role in the cellular response to phosphate starvation. However, cells lacking PHO85 have additional phenotypes unrelated to the phosphate starvation pathway, including a role in the G'1 phase of the cell-division cycle. Ten different Pho85 cyclins, termed Pcl's, associate with Pho85 and are thought to direct it to phosphorylate distinct substrates involved in different cellular processes. To better understand the role of the Pho85-Pcl1 cyclin dependent kinase, I performed a high-throughput screen of the yeast proteome for kinases substrates. To do so, I helped plan, construct, and verify an epitope tagged protein library composed of 4250 yeast strains each harboring a distinct detectable fusion protein. Using this library and an AS version of Pho85, Pho85(F82G), which functionally substitutes for wild-type Pho85 in vivo, and which is able to utilize the ATP analog N6-benzyl ATP in vitro, I developed a high-throughput method to screen for protein kinase substrates and used it to identify 24 direct substrates of the yeast protein kinase Pho85-Pcl1, including the known substrate Rvs167. The power of this method to identify true kinase substrates is strongly supported by functional overlap and colocalization of candidate substrates and the kinase, as well as by the specificity of Pho85-Pcl1 for some of the substrates over another Pho85-cyclin kinase complex. This method is readily adaptable to other yeast kinases.