TY - JOUR
T1 - Ahnak1 interaction is affected by phosphorylation of Ser-296 on Cavβ 2
AU - Pankonien, Ines
AU - Otto, Albrecht
AU - Dascal, Nathan
AU - Morano, Ingo
AU - Haase, Hannelore
N1 - Funding Information:
The study was supported by the German-Israeli Foundation for Scientific Research and Development (GIF). A special thanks goes to Dr. Patricia C. Hidalgo for providing the Cavβ 2 -SH3 construct, Dr. Anje Sporbert (Microscopy Core Facility of the Max Delbrück Center for Molecular Medicine, Berlin) and Petra Domaing for confocal imaging. We greatly appreciate excellent technical assistance by Steffen Lutter and Karin Karczewski.
PY - 2012/5/4
Y1 - 2012/5/4
N2 - 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 Cavβ 2 regulatory channel subunit. Here we corroborate this functional linkage by immunocytochemistry on isolated cardiomyocytes showing co-localization of ahnak1 and Cavβ 2 in the T-tubule system. In previous studies Cavβ 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 Cavβ 2 and investigated whether Cavβ 2 phosphorylation affects its binding behavior. In vitro binding assays with Cavβ 2 truncation mutants and ahnak1 4889-5535 revealed that the core region of Cavβ 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 Cavβ 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≈35nM) between Cavβ 2 and the α 1C I-II linker, but affected ahnak1 interaction in a complex manner. SPR experiments with ahnak1 5462-5535 revealed that PKA phosphorylation of Cavβ 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 ≈2.4-fold and reduced the capacity by ≈60%. Our results are indicative for the release of a population of low affinity interaction sites following Cavβ 2 phosphorylation on Ser-296. We propose that this phosphorylation event is one mechanism underlying ahnak1́s modulator function on Cav1.2 channel activity.
AB - 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 Cavβ 2 regulatory channel subunit. Here we corroborate this functional linkage by immunocytochemistry on isolated cardiomyocytes showing co-localization of ahnak1 and Cavβ 2 in the T-tubule system. In previous studies Cavβ 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 Cavβ 2 and investigated whether Cavβ 2 phosphorylation affects its binding behavior. In vitro binding assays with Cavβ 2 truncation mutants and ahnak1 4889-5535 revealed that the core region of Cavβ 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 Cavβ 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≈35nM) between Cavβ 2 and the α 1C I-II linker, but affected ahnak1 interaction in a complex manner. SPR experiments with ahnak1 5462-5535 revealed that PKA phosphorylation of Cavβ 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 ≈2.4-fold and reduced the capacity by ≈60%. Our results are indicative for the release of a population of low affinity interaction sites following Cavβ 2 phosphorylation on Ser-296. We propose that this phosphorylation event is one mechanism underlying ahnak1́s modulator function on Cav1.2 channel activity.
KW - Ahnak
KW - Cavβ
KW - L-type calcium channel
KW - PKA phosphorylation
KW - Protein-protein interaction
UR - http://www.scopus.com/inward/record.url?scp=84860468788&partnerID=8YFLogxK
U2 - 10.1016/j.bbrc.2012.03.132
DO - 10.1016/j.bbrc.2012.03.132
M3 - ???researchoutput.researchoutputtypes.contributiontojournal.article???
AN - SCOPUS:84860468788
SN - 0006-291X
VL - 421
SP - 184
EP - 189
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 2
ER -