Abstract
Efficient and specific phosphorylation of PKA substrates, elicited in response to β-adrenergic stimulation, require spatially confined pools of PKA anchored in proximity of its substrates. PKA-dependent phosphorylation of cardiac sarcomeric proteins has been the subject of intense investigations. Yet, the identity, composition, and function of PKA complexes at the sarcomeres have remained elusive. Here we report the identification and characterization of a novel sarcomeric AKAP (A-kinase anchoring protein), cardiac troponin T (cTnT). Using yeast two-hybrid technology in screening two adult human heart cDNA libraries, we identified the regulatory subunit of PKA as interacting with human cTnT bait. Immunoprecipitation studies show that cTnT is a dual specificity AKAP, interacting with both PKA-regulatory subunits type I and II. The disruptor peptide Ht31, but not Ht31P (control), abolished cTnT/PKA-R association. Truncations and point mutations identified an amphipathic helix domain in cTnT as the PKA binding site. This was confirmed by a peptide SPOT assay in the presence of Ht31 or Ht31P (control). Gelsolin-dependent removal of thin filament proteins also reduced myofilament-bound PKA-type II. Using a cTn exchange procedure that substitutes the endogenous cTn complex with a recombinant cTn complex we show that PKA-type II is troponin-bound in the myofilament lattice. Displacement of PKA-cTnT complexes correlates with a significant decrease in myofibrillar PKA activity. Taken together, our data propose a novel role for cTnT as a dual-specificity sarcomeric AKAP.
Highlights
Summary of human heart cDNA prey clones interacting with human cTnT bait Y2H screens of more than 4 ϫ 106 cDNA clones identified over 1000 colonies that grew on high-stringency plates, of which 375 turned blue in the presence of ␣galactosidase, indicating positive interactions with hcTnT
DNA sequencing led to the identification of 19 gene products, including multiple hits for cTnI and tropomyosin, known binding partners of cTnT
Using gelsolin and cTn exchange assays we show that PKA-RII associates with thin filaments and endogenous cTnT, in isolated rat myofibrils
Summary
Primer sequence 5Ј-3Ј hcTnT hcTnT hcTnT-(1–117) hcTnT-(1–180) hcTnT-(118–228) hcTnT-(181–228) hcTnT-(225–228) hcTnT-K210-del hcTnT-V218P hcTnT-R216I hcTnT-A220N hPKA-RI hPKA-RI hPKA-RI hPKA-RII hPKA-RII hPKA-RII pGBKT7 pCMV6-AN-Myc pGBKT7 pGBKT7 pGBKT7 pGBKT7 pGBKT7 pGBKT7 pGBKT7 pGBKT7 pGBKT7 pGADT7 pCMV6-AN-HA pCMV6-AC-HA pGADT7 pCMV6-AN-HA pCMV6-AC-HA
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