Journal of Biological Chemistry
Volume 285, Issue 8, 19 February 2010, Pages 5674-5682
Journal home page for Journal of Biological Chemistry

Signal Transduction
Distinct Sarcomeric Substrates Are Responsible for Protein Kinase D-mediated Regulation of Cardiac Myofilament Ca2+ Sensitivity and Cross-bridge Cycling*

https://doi.org/10.1074/jbc.M109.066456Get rights and content
Under a Creative Commons license
open access

Protein kinase D (PKD), a serine/threonine kinase with emerging cardiovascular functions, phosphorylates cardiac troponin I (cTnI) at Ser22/Ser23, reduces myofilament Ca2+ sensitivity, and accelerates cross-bridge cycle kinetics. Whether PKD regulates cardiac myofilament function entirely through cTnI phosphorylation at Ser22/Ser23 remains to be established. To determine the role of cTnI phosphorylation at Ser22/Ser23 in PKD-mediated regulation of cardiac myofilament function, we used transgenic mice that express cTnI in which Ser22/Ser23 are substituted by nonphosphorylatable Ala (cTnI-Ala2). In skinned myocardium from wild-type (WT) mice, PKD increased cTnI phosphorylation at Ser22/Ser23 and decreased the Ca2+ sensitivity of force. In contrast, PKD had no effect on the Ca2+ sensitivity of force in myocardium from cTnI-Ala2 mice, in which Ser22/Ser23 were unavailable for phosphorylation. Surprisingly, PKD accelerated cross-bridge cycle kinetics similarly in myocardium from WT and cTnI-Ala2 mice. Because cardiac myosin-binding protein C (cMyBP-C) phosphorylation underlies cAMP-dependent protein kinase (PKA)-mediated acceleration of cross-bridge cycle kinetics, we explored whether PKD phosphorylates cMyBP-C at its PKA sites, using recombinant C1C2 fragments with or without site-specific Ser/Ala substitutions. Kinase assays confirmed that PKA phosphorylates Ser273, Ser282, and Ser302, and revealed that PKD phosphorylates only Ser302. Furthermore, PKD phosphorylated Ser302 selectively and to a similar extent in native cMyBP-C of skinned myocardium from WT and cTnI-Ala2 mice, and this phosphorylation occurred throughout the C-zones of sarcomeric A-bands. In conclusion, PKD reduces myofilament Ca2+ sensitivity through cTnI phosphorylation at Ser22/Ser23 but accelerates cross-bridge cycle kinetics by a distinct mechanism. PKD phosphorylates cMyBP-C at Ser302, which may mediate the latter effect.

Contractile Protein
Heart
Protein Phosphorylation
Serine/Threonine Protein Kinase
Signal Transduction
Calcium Sensitivity
Cardiac Contraction
Sarcomeric Proteins

Cited by (0)

*

This work was supported by British Heart Foundation Project Grant PG/07/056/23150 and in part by the King's British Heart Foundation Centre of Research Excellence.

The on-line version of this article (available at http://www.jbc.org) contains supplemental text, Tables S1 and S2, and Fig. S1.