| First Author | Pankonien I | Year | 2012 |
| Journal | Biochem Biophys Res Commun | Volume | 421 |
| Issue | 2 | Pages | 184-9 |
| PubMed ID | 22497893 | Mgi Jnum | J:183828 |
| Mgi Id | MGI:5319406 | Doi | 10.1016/j.bbrc.2012.03.132 |
| Citation | Pankonien I, et al. (2012) Ahnak1 interaction is affected by phosphorylation of Ser-296 on Cavbeta(2). Biochem Biophys Res Commun 421(2):184-9 |
| abstractText | Ahnak1 has been implicated in protein kinase A (PKA)-mediated control of cardiac L-type Ca(2+) channels (Cav1.2) through its interaction with the Cavbeta(2) regulatory channel subunit. Here we corroborate this functional linkage by immunocytochemistry on isolated cardiomyocytes showing co-localization of ahnak1 and Cavbeta(2) in the T-tubule system. In previous studies Cavbeta(2) attachment sites which impacted the channel's PKA regulation have been located to ahnak1's proximal C-terminus (ahnak1(4889-5535), ahnak1(5462-5535)). In this study, we mapped the ahnak1-interacting regions in Cavbeta(2) and investigated whether Cavbeta(2) phosphorylation affects its binding behavior. In vitro binding assays with Cavbeta(2) truncation mutants and ahnak1(4889-5535) revealed that the core region of Cavbeta(2) consisting of Src-homology 3 (SH3), HOOK, and guanylate kinase (GK) domains was important for ahnak1 interaction while the C- and N-terminal regions were dispensable. Furthermore, Ser-296 in the GK domain of Cavbeta(2) was identified as novel PKA phosphorylation site by mass spectrometry. Surface plasmon resonance (SPR) binding analysis showed that Ser-296 phosphorylation did not affect the high affinity interaction (K(D) approximately 35nM) between Cavbeta(2) and the alpha(1C) I-II linker, but affected ahnak1 interaction in a complex manner. SPR experiments with ahnak1(5462-5535) revealed that PKA phosphorylation of Cavbeta(2) significantly increased the binding affinity and, in parallel, it reduced the binding capacity. Intriguingly, the phosphorylation mimic substitution Glu-296 fully reproduced both effects, increased the affinity by approximately 2.4-fold and reduced the capacity by approximately 60%. Our results are indicative for the release of a population of low affinity interaction sites following Cavbeta(2) phosphorylation on Ser-296. We propose that this phosphorylation event is one mechanism underlying ahnak1 s modulator function on Cav1.2 channel activity. |
