Exploiting the Unique ATP-Binding Pocket of Toxoplasma Calcium-Dependent Protein Kinase 1 To Identify Its Substrates

详细信息    查看全文

• 作者：Sebastian Lourido ; Grace R. Jeschke ; Benjamin E. Turk ; L. David Sibley
• 刊名：ACS Chemical Biology
• 出版年：2013
• 出版时间：June 21, 2013
• 年：2013
• 卷：8
• 期：6
• 页码：1155-1162
• 全文大小：368K
• 年卷期：v.8,no.6(June 21, 2013)
• ISSN：1554-8937

文摘

Apicomplexan parasites rely on calcium as a second messenger to regulate a variety of essential cellular processes. Calcium-dependent protein kinases (CDPK), which transduce these signals, are conserved among apicomplexans but absent from mammalian hosts, making them attractive targets for therapeutic intervention. Despite their importance, the signaling pathways CDPK regulate remain poorly characterized, and their protein substrates are completely unknown. In Toxoplasma gondii, CDPK1 is required for calcium-regulated secretion from micronemes, thereby controlling motility, invasion, and egress from host cells. CDPK1 is unique among parasite and mammalian kinases in containing glycine at the key 鈥済atekeeper鈥?residue, which results in an expanded ATP-binding pocket. In the present study, we use a synthetic ATP纬S analogue that displays steric complementarity to the ATP-binding pocket and hence allows identification of protein substrates based on selective thiophosphorylation. The specificity of this approach was validated by the concordance between the identified phosphorylation sites and the in vitro substrate preference of CDPK1. We further demonstrate that the phosphorylation of predicted substrates is dependent on CDPK1 both in vivo and in vitro. This combined strategy for identifying the targets of specific protein kinases provides a platform for defining the roles of CDPKs in apicomplexans.