Abl silencing inhibits CAS-mediated process and constriction in resistance arteries
Yana Anfinogenova1, Ruping Wang, Qing-fen Li
1Center for Cardiovascular Sciences, Albany Medical College, Albany, NY 12208, USA.
Abstract:
The tyrosine phosphorylated protein Crk-associated substrate (CAS) has previously been shown to participate in the cellular processes regulating dynamic changes in the actin architecture and arterial constriction. In the present study, treatment of rat mesenteric arteries with phenylephrine (PE) led to the increase in CAS tyrosine phosphorylation and the association of CAS with the adapter protein CrkII. CAS phosphorylation was catalyzed by Abl in an in vitro study. To determine the role of Abl tyrosine kinase in arterial vessels, plasmids encoding Abl short hairpin RNA (shRNA) were transduced into mesenteric arteries by chemical loading plus liposomes. Abl silencing diminished increases in CAS phosphorylation on PE stimulation. Previous studies have shown that assembly of the multiprotein compound containing CrkII, neuronal Wiskott-Aldrich Syndrome Protein (N-WASP) and the Arp2/3 (Actin Related Protein) complex triggers actin polymerization in smooth muscle as well as in nonmuscle cells. In this study, Abl silencing attenuated the assembly of the multiprotein compound in resistance arteries on contractile stimulation. Furthermore, the increase in F/G-actin ratios (an index of actin assembly) and constriction on contractile stimulation were reduced in Abl-deficient arterial segments compared with control arteries. However, myosin regulatory light chain phosphorylation (MRLCP) elicited by contractile activation was not inhibited in Abl-deficient arteries. These results suggest that Abl may play a pivotal role in mediating CAS phosphorylation, the assembly of the multiprotein complex, actin assembly, and constriction in resistance arteries. Abl does not participate in the regulation of myosin activation in arterial vessels during contractile stimulation.
Insights
Abl tyrosine kinase regulates arterial constriction by mediating Crk-associated substrate phosphorylation and actin assembly. Abl deficiency reduces CAS phosphorylation and actin polymerization, impacting arterial smooth muscle function without affecting myosin activation.
Area of Science:
- Vascular Biology
- Molecular Cell Biology
- Biochemistry
Background:
- Crk-associated substrate (CAS) is involved in actin dynamics and arterial constriction.
- Phenylephrine (PE) stimulation increases CAS tyrosine phosphorylation and its association with CrkII in rat mesenteric arteries.
- Abl tyrosine kinase catalyzes CAS phosphorylation in vitro.
Purpose of the Study:
- To investigate the role of Abl tyrosine kinase in arterial constriction and associated signaling pathways.
- To determine if Abl mediates CAS phosphorylation and actin assembly in response to contractile stimulation.
- To elucidate Abl's specific role in smooth muscle contraction regulation.
Main Methods:
- Silencing of Abl tyrosine kinase using short hairpin RNA (shRNA) delivered via chemical loading and liposomes in rat mesenteric arteries.
- Assessment of CAS tyrosine phosphorylation and its association with CrkII.
- Analysis of multiprotein complex assembly (CrkII, N-WASP, Arp2/3) and F/G-actin ratios as indicators of actin polymerization.
- Measurement of arterial constriction and myosin regulatory light chain phosphorylation (MRLCP).
Main Results:
- Abl silencing significantly diminished PE-induced increases in CAS tyrosine phosphorylation.
- Abl deficiency attenuated the assembly of the CrkII, N-WASP, and Arp2/3 complex upon contractile stimulation.
- Abl-deficient arteries showed reduced actin assembly (F/G-actin ratios) and constriction compared to controls.
- Myosin regulatory light chain phosphorylation (MRLCP) was not affected by Abl silencing during contractile activation.
Conclusions:
- Abl tyrosine kinase plays a critical role in mediating CAS phosphorylation, multiprotein complex assembly, and actin assembly in resistance arteries.
- Abl is essential for contractile stimulation-induced arterial constriction.
- Abl's function in arterial vessels is specific to regulating actin dynamics and does not involve myosin activation pathways.
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