Polo-like kinase 1 (Plk1) is a serine/threonine-protein kinase that has been implicated in mitosis, cytokinesis, and smooth muscle cell proliferation. The role of Plk1 in smooth muscle contraction has not been investigated. Here, stimulation with acetylcholine induced Plk1 phosphorylation at Thr-210 (an indication of Plk1 activation) in smooth muscle. Contractile stimulation also activated Plk1 in live smooth muscle cells as evidenced by changes in fluorescence resonance energy transfer signal of a Plk1 sensor. Moreover, knockdown of Plk1 in smooth muscle attenuated force development. Smooth muscle conditional knock-out of Plk1 also diminished contraction of mouse tracheal rings. Plk1 knockdown inhibited acetylcholine-induced vimentin phosphorylation at Ser-56 without affecting myosin light chain phosphorylation. Expression of T210A Plk1 inhibited the agonist-induced vimentin phosphorylation at Ser-56 and contraction in smooth muscle. However, myosin light chain phosphorylation was not affected by T210A Plk1. Ste20-like kinase (SLK) is a serine/threonine-protein kinase that has been implicated in spindle orientation and microtubule organization during mitosis. In this study knockdown of SLK inhibited Plk1 phosphorylation at Thr-210 and activation. Finally, asthma is characterized by airway hyperresponsiveness, which largely stems from airway smooth muscle hyperreactivity. Here, smooth muscle conditional knock-out of Plk1 attenuated airway resistance and airway smooth muscle hyperreactivity in a murine model of asthma. Taken together, these findings suggest that Plk1 regulates smooth muscle contraction by modulating vimentin phosphorylation at Ser-56. Plk1 activation is regulated by SLK during contractile activation. Plk1 contributes to the pathogenesis of asthma.

Polo-like kinase 1 (Plk1) has been implicated in mitosis, cytokinesis, and proliferation. The mechanisms that regulate Plk1 expression remain to be elucidated. It is reported that miR-100 targets Plk1 in certain cancer cells. Here, treatment with miR-100 did not affect Plk1 protein expression in human airway smooth muscle cells. In contrast, treatment with miR-509 inhibited the expression of Plk1 in airway smooth muscle cells. Exposure to miR-509 inhibitor enhanced Plk1 expression in cells. Introduction of miR-509 reduced luciferase activity of a Plk1 3′UTR reporter. Mutation of miR-509 targeting sequence in Plk1 3′UTR resisted the reduction of the luciferase activity. Furthermore, miR-509 inhibited the PDGF-induced phosphorylation of MEK1/2 and ERK1/2, and cell proliferation without affecting the expression of c-Abl, a tyrosine kinase implicated in cell proliferation. Moreover, we unexpectedly found that vimentin filaments contacted paxillin-positive focal adhesions. miR-509 exposure inhibited vimentin phosphorylation at Ser-56, vimentin network reorganization, focal adhesion formation, and cell migration. The effects of miR-509 on ERK1/2 and vimentin were diminished in RNAi-resistant Plk1 expressing cells treated with miR-509. Taken together, these findings unveil previously unknown mechanisms that miR-509 regulates ERK1/2 and proliferation by targeting Plk1. miR-509 controls vimentin cytoskeleton reorganization, focal adhesion assembly, and cell migration through Plk1.

BackgroundAirway smooth muscle contraction is critical for maintenance of appropriate airway tone, and has been implicated in asthma pathogenesis. Smooth muscle contraction requires an “engine” (myosin activation) and a “transmission system” (actin cytoskeletal remodeling). However, the mechanisms that control actin remodeling in smooth muscle are not fully elucidated. The adapter protein Crk-associated substrate (CAS) regulates actin dynamics and the contraction in smooth muscle. In addition, profilin-1 (Pfn-1) and Abelson tyrosine kinase (c-Abl) are also involved in smooth muscle contraction. The interplays among CAS, Pfn-1 and c-Abl in smooth muscle have not been previously investigated.MethodsThe association of CAS with Pfn-1 in mouse tracheal rings was evaluated by co-immunoprecipitation. Tracheal rings from c-Abl conditional knockout mice were used to assess the roles of c-Abl in the protein-protein interaction and smooth muscle contraction. Decoy peptides were utilized to evaluate the importance of CAS/Pfn-1 coupling in smooth muscle contraction.ResultsStimulation with acetylcholine (ACh) increased the interaction of CAS with Pfn-1 in smooth muscle, which was regulated by CAS tyrosine phosphorylation and c-Abl. The CAS/Pfn-1 coupling was also modified by the phosphorylation of cortactin (a protein implicated in Pfn-1 activation). In addition, ACh activation promoted the spatial redistribution of CAS and Pfn-1 in smooth muscle cells, which was reduced by c-Abl knockdown. Inhibition of CAS/Pfn-1 interaction by a decoy peptide attenuated the ACh-induced actin polymerization and contraction without affecting myosin light chain phosphorylation. Furthermore, treatment with the Src inhibitor PP2 and the actin polymerization inhibitor latrunculin A attenuated the ACh-induced c-Abl tyrosine phosphorylation (an indication of c-Abl activation).ConclusionsOur results suggest a novel activation loop in airway smooth muscle: c-Abl promotes the CAS/Pfn-1 coupling and actin polymerization, which conversely facilitates c-Abl activation. The positive feedback may render c-Abl in active state after contractile stimulation.

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Allergic asthma is characterized by airway smooth muscle layer thickening, which is largely attributed to cell division that requires the formation of centrosomes. Centrosomes play a pivotal role in regulating bipolar spindle formation and cell division. Before mitosis, centrosomes undergo maturation characterized by expansion of pericentriolar material proteins, which facilitates spindle formation and mitotic efficiency of many cell types. Although polo-like kinase 1 (Plk1) has been implicated in centrosome maturation, the mechanisms by which Plk1 regulates the cellular process are incompletely elucidated. Here, we identified paxillin as a new Plk1-interacting protein in human airway smooth muscle cells. We unexpectedly found that phosphorylated paxillin (Ser-272) was localized in centrosomes of human smooth muscle cells, which regulated centrosome maturation and spindle assembly. Plk1 knockdown inhibited paxillin Ser-272 phosphorylation, centrosome maturation, and cell division. Furthermore, exposure to allergens enhanced airway smooth muscle layer and paxillin phosphorylation at this residue in mice, which was reduced by smooth muscle conditional knockout of Plk1. These findings suggest that Plk1 regulates centrosome maturation and cell division in part by modulating paxillin phosphorylation on Ser-272. Furthermore, Plk1 contributes to the pathogenesis of allergen-induced thickening of the airway smooth muscle layer by affecting paxillin phosphorylation at this position.