Designing isoform-specific peptide disruptors of protein kinase A localization.
A kinase-anchoring proteins (AKAPs) coordinate cAMP-mediated signaling by binding and localizing cAMP-dependent protein kinase (PKA), using an amphipathic helical docking motif. Peptide disruptors of PKA localization that mimic this helix have been used successfully to assess the involvement of PKA in specific signaling pathways. However, these peptides were developed as disruptors for the type II regulatory subunit (RII) even though both RI and RII isoforms can bind to AKAPs and have discrete functions. To evaluate the effects of each localized isoform, we designed peptides that specifically bind to either RI or RII. Using a peptide array, we have defined the minimal binding sequence of dual specific-AKAP 2 (d-AKAP2), which binds tightly to both RI and RII. Side-chain requirements for affinity and isoform specificity were evaluated by using a peptide substitution array where each position along the A kinase binding domain of d-AKAP2 was substituted by the other 19 l-amino acids. This array comprises 513 single-site substitution analogs of the d-AKAP2 sequence. Peptides containing single and multiple mutations were evaluated in a quantitative fluorescence binding assay and a cell-based colocalization assay. This strategy has allowed us to design peptides with high affinity (K(D) = 1-2 nM) and high specificity for RIalpha versus RIIalpha. These isoform-specific peptides will be invaluable tools to evaluate functional differences between localized RI and RII PKA and are RIalpha-specific disruptors. This array-based analysis also provides a foundation for biophysical analysis of this docking motif.
Burns-Hamuro
Lora L
LL
Howard Hughes Medical Institute and Department of Chemistry and Biochemistry, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0654, USA.
Ma
Yuliang
Y
Kammerer
Stefan
S
Reineke
Ulrich
U
Self
Chris
C
Cook
Charles
C
Olson
Gary L
GL
Cantor
Charles R
CR
Braun
Andreas
A
Taylor
Susan S
SS
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics*; D002417:Cattle; D003001:Cloning, Molecular; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics; D019281:Dimerization; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D010455:Peptides; D021122:Protein Subunits; C470173:ROPN1L protein, human; D011994:Recombinant Proteins; C487792:Ropn1l protein, mouse; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases