Cardiac troponin T, a sarcomeric AKAP, tethers protein kinase A at the myofilaments.
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.
Department of Physiology, Center for Muscle Biology, University of Kentucky, Lexington, Kentucky 40536, USA.
C Amelia
CA
Sumandea
Mary L
ML
Garcia-Cazarin
Catherine H
CH
Bozio
Gail A
GA
Sievert
C William
CW
Balke
Marius P
MP
Sumandea
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D054746:Cyclic AMP-Dependent Protein Kinase Type II / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D009206:Myocardium / Q000166:cytology* / Q000378:metabolism*; D011487:Protein Conformation; D055550:Protein Stability; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D012518:Sarcomeres / Q000378:metabolism; D013379:Substrate Specificity; D020107:Troponin T / Q000737:chemistry / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D021122:Protein Subunits; D020107:Troponin T; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases