Rational Redesign of a Functional Protein Kinase-Substrate Interaction.
Eukaryotic protein kinases typically phosphorylate substrates in the context of specific sequence motifs, contributing to specificity essential for accurate signal transmission. Protein kinases recognize their target sequences through complementary interactions within the active site cleft. As a step toward the construction of orthogonal kinase signaling systems, we have re-engineered the protein kinase Pim1 to alter its phosphorylation consensus sequence. Residues in the Pim1 catalytic domain interacting directly with a critical arginine residue in the substrate were substituted to produce a kinase mutant that instead accommodates a hydrophobic residue. We then introduced a compensating mutation into a Pim1 substrate, the pro-apoptotic protein BAD, to reconstitute phosphorylation both in vitro and in living cells. Coexpression of the redesigned kinase with its substrate in cells protected them from apoptosis. Such orthogonal kinase-substrate pairs provide tools to probe the functional consequences of specific phosphorylation events in living cells and to design synthetic signaling pathways.
Department of Pharmacology, Yale University School of Medicine , New Haven, Connecticut 06520, United States.
Catherine
C
Chen
Department of Pharmacology, Yale University School of Medicine , New Haven, Connecticut 06520, United States.
Wutigri
W
Nimlamool
Department of Pharmacology, Yale University School of Medicine , New Haven, Connecticut 06520, United States.
Chad J
CJ
Miller
Department of Pharmacology, Yale University School of Medicine , New Haven, Connecticut 06520, United States.
Hua Jane
HJ
Lou
Department of Pharmacology, Yale University School of Medicine , New Haven, Connecticut 06520, United States.
Benjamin E
0000-0001-9275-4069
BE
Turk
D017209:Apoptosis / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D020125:Mutation, Missense; D015202:Protein Engineering / Q000379:methods*; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D051573:Proto-Oncogene Proteins c-pim-1 / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics; D051025:bcl-Associated Death Protein / Q000235:genetics / Q000378:metabolism
D051025:bcl-Associated Death Protein; D011494:Protein Kinases; D051573:Proto-Oncogene Proteins c-pim-1