title: The extent of cardiac myosin binding protein-C phosphorylation modulates actomyosin function in a graded manner.
abstract: Cardiac myosin binding protein-C (cMyBP-C), a sarcomeric protein with 11 domains, C0-C10, binds to the myosin rod via its C-terminus, while its N-terminus binds regions of the myosin head and actin. These N-terminal interactions can be attenuated by phosphorylation of serines in the C1-C2 motif linker. Within the sarcomere, cMyBP-C exists in a range of phosphorylation states, which may affect its ability to regulate actomyosin motion generation. To examine the functional importance of partial phosphorylation, we bacterially expressed N-terminal fragments of cMyBP-C (domains C0-C3) with three of its phosphorylatable serines (S273, S282, and S302) mutated in combinations to either aspartic acids or alanines, mimicking phosphorylation and dephosphorylation respectively. The effect of these C0-C3 constructs on actomyosin motility was characterized in both the unloaded in vitro motility assay and in the load-clamped laser trap assay where force:velocity (F:V) relations were obtained. In the motility assay, phosphomimetic replacement (i.e. aspartic acid) reduced the slowing of actin velocity observed in the presence of C0-C3 in proportion to the total number phosphomimetic replacements. Under load, C0-C3 depressed the F:V relationship without any effect on maximal force. Phosphomimetic replacement reversed the depression of F:V by C0-C3 in a graded manner with respect to the total number of replacements. Interestingly, the effect of C0-C3 on F:V was well fitted by a model that assumed C0-C3 acts as an effective viscous load against which myosin must operate. This study suggests that increasing phosphorylation of cMyBP-C incrementally reduces its modulation of actomyosin motion generation providing a tunable mechanism to regulate cardiac function.
authors: Department of Molecular Physiology & Biophysics, University of Vermont, HSRF, Room 116, 149 Beaumont Ave., Burlington, VT 05405, USA.
authors: Abbey E
authors: AE
authors: Weith
authors: Michael J
authors: MJ
authors: Previs
authors: Gregory J
authors: GJ
authors: Hoeprich
authors: Samantha Beck
authors: SB
authors: Previs
authors: James
authors: J
authors: Gulick
authors: Jeffrey
authors: J
authors: Robbins
authors: David M
authors: DM
authors: Warshaw
mesh_terms: D000205:Actomyosin / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002645:Chickens; D051379:Mice; D010766:Phosphorylation; D000099163:Myosin Binding Protein C
chemical_list: D000205:Actomyosin; D002352:Carrier Proteins; D000099163:Myosin Binding Protein C