Interaction between cAMP-dependent protein kinase catalytic subunit and peptide inhibitors analyzed with lambda repressor fusions.
The lambda phage repressor is currently used as a genetic tool to analyze homodimeric interactions in Escherichia coli. We have applied this system to detect the interaction that takes place within an enzyme-protein inhibitor complex. The sequences encoding the catalytic subunit of the cAMP-dependent protein kinase and the active portion of the natural thermostable protein kinase inhibitor have been fused to the carboxy terminus of the repressor DNA binding domain and introduced into compatible plasmids. Co-expression of the two gene fusions in E. coli lead to the formation of heterodimers that confer a high level of protection from lambda phage infection. The level of lambda immunity depends specifically upon the amino acid sequence of the interacting proteins, as a single amino acid substitution in the inhibitor peptide (Phe10-Ala) restores the sensitivity phenotype.
Jappelli
R
R
Molecular Sciences Institute, La Jolla, CA 92037, USA.
Brenner
S
S
D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000235:genetics / Q000502:physiology; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000502:physiology / Q000821:virology; D005822:Genetic Vectors; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010641:Phenotype; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D014169:Transformation, Bacterial; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; C015482:protein kinase modulator; D017868:Cyclic AMP-Dependent Protein Kinases