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Dissertation
Mitogen-activated protein kinase and protein kinase C-catalyzed caldesmon phosphorylation in vascular smooth muscle: is it more important in the regulation of resting tone or stimulation-induced contraction?
Doctor of Philosophy (Ph.D.), Drexel University
Apr 2012
DOI:
https://doi.org/10.17918/00009894
Abstract
Caldesmon is hypothesized to modulate contractile activity of smooth muscle by inhibiting actin-activated myosin ATPase activity, and inhibition is reversed by phosphorylation. Mitogen-activated protein (MAP) kinase is proposed to be the endogenous caldesmon kinase; however, MAP kinase-mediated phosphorylation can only account for Ser789 phosphorylation and not total caldesmon phosphorylation. We tested the hypothesis that MAP kinase and protein kinase C (PKC) are involved in stimulation-induced caldesmon phosphorylation and force development in swine carotid artery. We examined changes in resting and stimulated MAP kinase catalyzed Ser 789-caldesmon and total caldesmon phosphorylation levels in the presence of MAP kinase and/or PKC inhibitors. Our results demonstrated that caldesmon is regulated in the resting state and during agonist stimulation, albeit by different mechanisms. At rest, there is a basal level of activated p44 MAP kinase that phosphorylates caldesmon at Ser789. p42 MAP kinase phosphorylates caldesmon at a site(s) other than Ser789 and is regulated by a constitutively active PKC, potentially via modulation of MAP kinase phosphatase activity. In stimulated tissues, MAP kinase phosphorylates caldesmon at Ser789 and potentially other sites in response to histamine stimulation, whereas PKC-catalyzed caldesmon phosphorylation in response to phorbol 12,13-dibutyrate (PDBu) stimulation is at a site(s) other than Ser789. These data provide evidence that MAP kinase and PKC are endogenous caldesmon kinases but PKC may be more important in regulating MAP kinase-catalyzed caldesmon phosphorylation in resting as compared to stimulated tissues. Additionally, we examined the effect of MAP kinase and PKC-catalyzed caldesmon regulation on active force development using inhibitors of MAP kinase and/or PKC in siRNA caldesmon knockdown muscle strips. Our results show that caldesmon does not directly inhibit actin-activated myosin ATPase activity. Instead, caldesmon may regulate myosin ATPase activity and force development through a novel thin filament-associated inhibitory protein and inhibition is reversed by MAP kinase and PKC-mediated caldesmon phosphorylation. We have provided evidence that MAP kinase and PKC-mediated caldesmon phosphorylation has a critical role in regulating agonist-stimulated smooth muscle contraction and crossbridge cycling but may have a more significant role in regulating resting vessel tone and therefore resting blood pressure.
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Details
- Title
- Mitogen-activated protein kinase and protein kinase C-catalyzed caldesmon phosphorylation in vascular smooth muscle
- Creators
- Danielle M. Trappanese
- Contributors
- Robert S. Moreland (Advisor) - Drexel University, Drexel University (1970-)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xi, 200 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- College of Medicine; Pharmacology and Physiology; Drexel University
- Other Identifier
- 991021889106304721