Submitted on July 19, 2007
Accepted on October 13, 2007
C-terminal association of human smooth muscle calcium channel Cav1.2b with c-src kinase protein binding domains: Effect of nitrotyrosylation
Minho Kang1, Gracious R Ross1, and Hamid I. Akbarali1*
1 Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia, United States
* To whom correspondence should be addressed. E-mail: hiakbarali{at}vcu.edu.
The carboxyl terminus of the calcium channel plays an important role in the regulation of calcium entry, signal transduction and gene expression. Potential protein-protein interaction sites within the c-terminus of the L-type calcium channel include those for the SH3 and SH2 binding domains of c-src kinase that can regulate calcium currents in smooth muscle. In this study we examined the binding sites involved in src kinase mediated phosphorylation of the human Cav1.2b and the effect of nitrotyrosylation. Co-transfection of HEK293 cells with hCav1.2b and c-src resulted in tyrosine phosphorylation of the calcium channel which was prevented by nitration of tyrosine residues by peroxynitrite. Whole-cell calcium currents were reduced by 58 + 5% by the src kinase inhibitor, PP2 and 64 + 6% by peroxynitrite. Nitrotyrosylation prevented src-mediated regulation of the currents. GST-fusion protein of the distal c-terminus of hCav1.2b (1809-2138) bound to SH2 domain of src following tyrosine phosphorylation while SH3 binding required the presence of the proline-rich motif. Site-directed mutation of Y2134 prevented SH2 binding and resulted in reduced phosphorylation of hCav1.2b. Within the distal c-terminus, single, double or triple mutations of Y1837, Y1861 and Y2134 were constructed and expressed in HEK293 cells. The inhibitory effects of PP2 and peroxynitrite on calcium currents were significantly reduced in the double mutant Y1837-2134F. These data demonstrate that the c-terminus of hCav1.2b contains sites for the SH2 and SH3 binding of src kinase. Nitrotyrosylation of these sites prevents src kinase regulation and may be importantly involved in calcium influx regulation during inflammation
