Identification of the interface between cGMP-dependent protein kinase Ibeta and its interaction partners TFII-I and IRAG reveals a common interaction motif.
Protein-protein interactions have emerged as an important mechanism providing for specificity in cellular signal transduction. Two splice variants of type I cGMP-dependent protein kinase (PKG Ialpha and Ibeta) differ only in their N-terminal approximately 100 amino acids, which mediate binding to different target proteins. PKG Ibeta, but not Ialpha, binds to the general transcriptional regulator TFII-I and the inositol 1,4,5-trisphosphate receptor-associated PKG substrate IRAG. Using a combination of site-directed mutagenesis and in vitro binding assays, we identified a group of acidic amino acids in the N-terminal leucine zipper dimerization domain of PKG Ibeta required for its binding to both TFII-I and IRAG. Small clusters of basic amino acids in possible alpha-helical regions in TFII-I and IRAG were found to mediate their interaction with PKG Ibeta. Mutation of two negatively charged residues in the PKG Ibeta leucine zipper (D26K/E31R) to positively charged residues, found in corresponding positions in PKG Ialpha, completely abrogated binding to TFII-I and IRAG without disrupting PKG dimerization. Mutation of specific basic residues in TFII-I or IRAG abolished binding of the full-length proteins to PKG Ibeta in intact cells. Based on these results, we propose a model for specific PKG Ibeta interaction with target proteins.
Casteel
Darren E
DE
Department of Medicine and Cancer Center, University of California at San Diego, La Jolla, California 92093-0652, USA.
Boss
Gerry R
GR
Pilz
Renate B
RB
D017398:Alternative Splicing; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D015220:Calcium Channels / Q000737:chemistry; D002417:Cattle; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007930:Leucine / Q000737:chemistry; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry; D015398:Signal Transduction; D055672:Static Electricity; D019473:Transcription Factors, TFII / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D015220:Calcium Channels; C104259:GTF2I protein, human; C485784:IRAG1 protein, human; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008565:Membrane Proteins; D010750:Phosphoproteins; D018160:Receptors, Cytoplasmic and Nuclear; D019473:Transcription Factors, TFII; D004247:DNA; D005982:Glutathione Transferase; D017869:Cyclic GMP-Dependent Protein Kinases; D007930:Leucine