Catalytic Subunit of Protein Kinase A Caged at the Activating Phosphothreonine Keyong Zou, ² Stephen Cheley, ² Richard S. Givens, ‡ and Hagan Bayley* ,²,§ Contribution from the Department of Medical Biochemistry and Genetics, The Texas A&M UniVersity System Health Science Center, College Station, Texas 77843-1114, Department of Chemistry, UniVersity of Kansas, Lawrence, Kansas 66045-7582, and Department of Chemistry, Texas A&M UniVersity, College Station, Texas 77843-3255 Received March 19, 2002 Abstract: Caged reagents are photoactivatable molecules with applications in biological research. While a great deal of work has been carried out on small caged molecules, less has been done on caged macromolecules, such as proteins. Caged proteins would be especially useful in signal transduction research. Since most proteins involved in cell signaling are regulated by phosphorylation, a means to cage phosphorylated proteins would be generally applicable. Here we show that the catalytic subunit of protein kinase A can be activated by thiophosphorylation at Thr-197. The modified protein can then be caged with 4-hydroxyphenacyl bromide to yield a derivative with a specific catalytic activity that is reduced by ∼17- fold. Upon photolysis at near UV wavelengths, an ∼15-fold increase in activity is observed, representing an ∼85-90% yield of uncaged product with a quantum yield φ P ) 0.21. Because protein kinases belong to a superfamily with structurally related catalytic domains, the protein chemistry demonstrated here should be applicable to a wide range of signaling proteins. Introduction A caged reagent 1 is a molecule in which the activity has been blocked by chemical modification with a photolabile reagent. Photolysis removes the protecting group and restores activity. Because the photorelease of an effector can be temporally and spatially controlled, caged molecules, such as caged ATP, are widely applied in biological research. 2,3 While a great deal of work has been carried out on small caged molecules, less has been done on caged macromolecules. 4,5 In the experiments described here, a catalytic subunit (C) of cAMP-dependent protein kinase (PKA) caged on an activating phosphate was prepared with the recently developed 4-hydroxyphenacyl re- agent. 6 The idea of gaining photochemical control of protein activity is not new 7-9 and approaches other than direct caging have been taken including the use of proteins derivatized with photo- isomerizable reagents 10-18 and the application of caged 19 or photoisomerizable 20,21 inhibitors and activators. In general, it has been difficult to achieve all-or-none effects with photo- isomerizable reagents, although the prospect of reversible activation is intriguing. 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CHEM. SOC. 2002, 124, 8220-8229 10.1021/ja020405e CCC: $22.00 © 2002 American Chemical Society