Functional effects of rho-kinase-dependent phosphorylation of specific sites on cardiac troponin.
We tested the hypothesis that activation of Rho-A-dependent kinase (ROCK-II) alters cardiac myofilament response to Ca2+ by mechanisms involving phosphorylation of thin filament proteins. We determined effects of a constitutively active form of ROCK-II on ATPase activity and tension development in detergent-extracted (skinned) fiber bundles isolated from mouse left ventricular papillary muscles. ROCK-II induced a depression in maximum ATPase rate and tension, which was associated with phosphorylation of troponin T (TnT), troponin I (TnI), and myosin-binding protein C (C-protein). This effect of ROCK-II was retained in fiber bundles isolated from transgenic (TG) mice in which phosphorylation sites (S14, S15, and S19) of myosin light chain 2 were mutated to alanine. Moreover, exchange of ROCK-II-phosphorylated Tn complex with the native Tn complex in the fiber bundles resulted in inhibition of maximal Ca2+ activation of tension and ATPase activity. Mass spectrometric analysis demonstrated that ROCK-II phosphorylated cardiac TnI (cTnI) at S23, S24, and T144 and cardiac TnT (cTnT) at S278 and T287. An important role for these cTnT sites is indicated by results demonstrating that ROCK-II induced a depression in tension and ATPase activity in skinned fiber bundles from a TG model in which cTnI is replaced by slow skeletal TnI, which lacks S23 and S24 and in which T144 is replaced by proline. Our data provide the first evidence that ROCK-II phosphorylation of the Tn complex, most likely at cTnT, has an important role in functional effects of signaling through the Rho-A pathway.
Vahebi
Susan
S
University of Illinois at Chicago Department of Physiology and Biophysics and Center for Cardiovascular Research College of Medicine, Chicago, Ill 60612-7342, USA.
Kobayashi
Tomoyoshi
T
Warren
Chad M
CM
de Tombe
Pieter P
PP
Solaro
R John
RJ
D008841:Actin Cytoskeleton / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D024722:Cardiac Myosins / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008822:Mice, Transgenic; D018994:Myosin Light Chains / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D019210:Troponin I / Q000378:metabolism; D020107:Troponin T / Q000378:metabolism*; D054460:rho-Associated Kinases
D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains; D019210:Troponin I; D020107:Troponin T; C107184:myosin light chain 2; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D024722:Cardiac Myosins; D002118:Calcium