期刊
JOURNAL OF MUSCLE RESEARCH AND CELL MOTILITY
卷 33, 期 6, 页码 449-459出版社
SPRINGER
DOI: 10.1007/s10974-012-9312-y
关键词
Force-velocity; Viscosity; Motility assay; Laser trap; Protein kinase A; Contractility
类别
资金
- National Institutes of Health [HL007944, HL07647, HL059408]
- Fondation Leducq
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.
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