JAOA • Vol 105 • No 1 • January 2005 • 24-25
Protein-Protein Interactions in Vasopressin Signal Transduction: New Evidence for Cytoskeletal Dynamics
Marc A. Bjurlin, BA*;
Kenneth L. Byron, PhD
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 4ß-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
and PKC 
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 
-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.
* Midwestern University's Chicago College of
Osteopathic Medicine, Downers Grove, IL 60515;
Loyola University Chicago, Department of
Medicine, Cardiovascular Institute, Maywood, IL 60153
