PubMed · 9484606
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S K Maciver. 1998. How ADF/cofilin depolymerizes actin filaments.. https://doi.org/10.1016/s0955-0674(98)80097-5
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The molecular mechanisms leading to high-altitude pulmonary hypertension (HAPH) remains poorly understood. We previously analyzed the whole genome sequence of Kyrgyz highland population and identified eight genomic intervals having a potential role in HAPH. Tropomodulin 3 gene (TMOD3), which encodes a protein that binds and caps the pointed ends of actin filaments and inhibits cell migration, was one of the top candidates. Here we systematically sought additional evidence to validate the functional role of TMOD3. In-silico analysis reveals that some of the SNPs in HAPH associated genomic intervals were positioned in a regulatory region that could result in alternative splicing of TMOD3. In order to functionally validate the role of TMOD3 in HAPH, we exposed Tmod3-/+ mice to 4 weeks of constant hypoxia, i.e. 10% O2 and analyzed both functional (hemodynamic measurements) and structural (angiography) parameters related to HAPH. The hemodynamic measurements, such as right ventricular systolic pressure, a surrogate measure for pulmonary arterial systolic pressure, and right ventricular contractility (RV- ± dP/dt), increases with hypoxia did not separate between Tmod3-/+ and control mice. Remarkably, there was a significant increase in the number of lung vascular branches and total length of pulmonary vascular branches (P < 0.001) in Tmod3-/+ after 4 weeks of constant hypoxia as compared with controls. Notably, the Tmod3-/+ endothelial cells migration was also significantly higher than that from the wild-type littermates. Our results indicate that, under chronic hypoxia, lower levels of Tmod3 play an important role in the maintenance or neo-vascularization of pulmonary arteries.
In addition to its kinase activity, myosin light chain kinase has an actin-binding activity, which results in bundling of actin filaments [Hayakawa et al., Biochem. Biophys. Res. Commun. 199, 786-791, 1994]. There are two actin-binding sites on the kinase: calcium- and calmodulin-sensitive and insensitive sites [Ye et al., J. Biol. Chem. 272, 32182-32189, 1997]. The calcium/calmodulin-sensitive, actin-binding site is located at Asp2-Pro41 and the insensitive site is at Ser138-Met213. The cyanogen bromide fragment, consisting of Asp2-Met213, is furnished with both sites and is the actin-binding core of myosin light chain kinase. Cross-linking between the two sites assembles actin filaments into bundles. Breaking of actin-binding at the calcium/calmodulin-sensitive site by calcium/calmodulin disassembles the bundles.
1. Smooth muscle contraction is activated primarily by the Ca2+-calmodulin (CaM)-dependent phosphorylation of the 20 kDa light chains (LC20) of myosin. Activation can also occur in some instances without a change in intracellular free [Ca2+] or indeed in a Ca2+-independent manner. These signalling pathways often involve inhibition of myosin light chain phosphatase and unmasking of basal kinase activity leading to LC20 phosphorylation and contraction. 2. We have used demembranated rat caudal arterial smooth muscle strips and isolated chicken gizzard myofilaments in conjunction with the phosphatase inhibitor microcystin-LR to investigate the mechanism of Ca2+-independent phosphorylation of LC20 and contraction. 3. Treatment of Triton X-100-demembranated rat caudal arterial smooth muscle strips with microcystin at pCa 9 triggered a concentration-dependent contraction that was slower than that induced by pCa 4.5 or 6 but reached comparable steady-state levels of tension. 4. This Ca2+-independent, microcystin-induced contraction correlated with phosphorylation of LC20 at serine-19 and threonine-18. 5. Whereas Ca2+-dependent LC20 phosphorylation and contraction were inhibited by a synthetic peptide (AV25) based on the autoinhibitory domain of myosin light chain kinase (MLCK), Ca2+-independent, microcystin-induced LC20 phosphorylation and contraction were resistant to AV25. 6. Ca2+-independent LC20 kinase activity was also detected in chicken gizzard smooth muscle myofilaments and catalysed phosphorylation of endogenous myosin LC20 at serine-19 and/or threonine-18. This is in contrast to MLCK which phosphorylates threonine-18 only after prior phosphorylation of serine-19. 7. Gizzard Ca2+-independent LC20 kinase could be separated from MLCK by differential extraction from myofilaments and by CaM affinity chromatography. Its activity was resistant to AV25. 8. We conclude that inhibition of smooth muscle myosin light chain phosphatase (MLCP) unmasks the activity of a Ca2+-independent LC20 kinase associated with the myofilaments and distinct from MLCK. This kinase, therefore, probably plays a role in Ca2+ sensitization and Ca2+-independent contraction of smooth muscle in response to stimuli that act via Ca2+-independent pathways, leading to inhibition of MLCP.