title: Ahnak1 interaction is affected by phosphorylation of Ser-296 on Cavβ₂.
abstract: 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)≈35 nM) 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 ahnak1's modulator function on Cav1.2 channel activity.
authors.affiliation: Max Delbrück Center for Molecular Medicine, Department of Molecular Muscle Physiology, Robert-Rössle-Strasse 10, 13125 Berlin, Germany. i_pankonien@hotmail.com
authors.forename: Ines
authors.initials: I
authors.lastname: Pankonien
authors.forename: Albrecht
authors.initials: A
authors.lastname: Otto
authors.forename: Nathan
authors.initials: N
authors.lastname: Dascal
authors.forename: Ingo
authors.initials: I
authors.lastname: Morano
authors.forename: Hannelore
authors.initials: H
authors.lastname: Haase
mesh_terms: D000818:Animals; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008815:Mice, Inbred Strains; D032383:Myocytes, Cardiac / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism*; D010766:Phosphorylation; D060066:Protein Interaction Maps; D017434:Protein Structure, Tertiary; D012694:Serine / Q000235:genetics / Q000378:metabolism*
chemical_list: C489572:Ahnak protein, mouse; C491528:Cacnb2 protein, mouse; D020746:Calcium Channels, L-Type; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases