Catalytic subunit of protein kinase A caged at the activating phosphothreonine.
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 approximately 17-fold. Upon photolysis at near UV wavelengths, an approximately 15-fold increase in activity is observed, representing an approximately 85-90% yield of uncaged product with a quantum yield phi(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.
Zou
Keyong
K
Department of Medical Biochemistry and Genetics, The Texas A&M University System Health Science Center, College Station, Texas 77843-1114, USA.
Cheley
Stephen
S
Givens
Richard S
RS
Bayley
Hagan
H
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000098:Acetophenones / Q000737:chemistry; D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D017868:Cyclic AMP-Dependent Protein Kinases / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010769:Phosphothreonine / Q000737:chemistry / Q000378:metabolism*; D010782:Photolysis; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction
C465291:4-hydroxyphenacyl bromide; D000098:Acetophenones; D010769:Phosphothreonine; D064413:3-Phosphoinositide-Dependent Protein Kinases; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases