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Student Contribution  |   January 2005
Protein-Protein Interactions in Vasopressin Signal Transduction: New Evidence for Cytoskeletal Dynamics
Author Affiliations
  • Kenneth L. Byron, PhD
    Midwestern University's Chicago College of Osteopathic Medicine, Downers Grove, IL 60515; Loyola University Chicago, Department of Medicine, Cardiovascular Institute, Maywood, IL 60153
Article Information
Neuromusculoskeletal Disorders
Student Contribution   |   January 2005
Protein-Protein Interactions in Vasopressin Signal Transduction: New Evidence for Cytoskeletal Dynamics
The Journal of the American Osteopathic Association, January 2005, Vol. 105, 24-25. doi:10.7556/jaoa.2005.105.1.24
The Journal of the American Osteopathic Association, January 2005, Vol. 105, 24-25. doi:10.7556/jaoa.2005.105.1.24
Web of Science® Times Cited: 73
The [Arg8]vasopressin (AVP)–induced signal transduction pathway that stimulates oscillations (spikes) in cytosolic free Ca2+ concentrations ([Ca2+]i) was examined in confluent cultures of rat A7r5 vascular smooth muscle (VSM) cells. Recent studies have suggested that AVP signal transduction may involve dynamic protein-protein interactions, ultimately leading to the phosphorylation and inhibition of delayed rectifier potassium channels (Kv1.2). Several likely protein candidates involved in this signaling cascade have been identified, including proline-rich tyrosine kinase 2 (PYK2), which may phosphorylate the Kv1.2 channel protein and protein kinase C (PKC) family members, which may phosphorylate PYK2. In the present study, A7r5 VSM cells were treated for varying times (0-30 min) with and without physiological vasoconstrictor concentrations of AVP (100 pM) or the PKC activator 4β-phorbol 12-myristate 12-acetate (PMA). Following these treatments, protein-protein interactions were examined by immunoprecipitation of PYK2 and by Western blot analysis to identify proteins that become associated with PYK2 during the time course of the treatments. Western blot analysis of untreated A7r5 VSM cells confirmed previous studies, which found that PYK2 is constitutively bound to the Kv1.2 channel. Western blot analysis of the AVP time-course treatment of A7r5 VSM cells also revealed that PKC ϵ and PKC δ transiently associate with PYK2. PMA treatment induced a similar PKC translocation. These results extend previous findings that suggested that PKC plays an active role in the signal transduction pathway leading to Ca2+ spiking. Additional new evidence of protein-protein interactions was observed in AVP treated A7r5 cells where an unknown protein band was visible on a Ponceau red stain of a Western blot membrane. This unknown protein in the PYK2 immunoprecipitate was identified as actin based on the apparent molecular weight (43 kDa), and subsequently confirmed by Western blot analysis using an antibody specific for smooth muscle α-actin. The transient association of actin with PYK2 was maximal at 10 min of AVP treatment and significantly decreased by 30 min. This novel result suggests a dynamic role of actin in vasopressin signal transduction and emphasizes the importance of the cell cytoskeleton in signaling cascades.