Binding of Protein Phosphatase 2A to the L-Type Calcium Channel Ca_v1.2 next to Ser1928, Its Main PKA Site, Is Critical for Ser1928 Dephosphorylation

Abstract

The cAMP-dependent protein kinase (PKA) controls a large number of cellular functions. One critical PKA substrate in the brain and heart is the L-type Ca(2+) channel Ca(v)1.2, the activity of which is upregulated by PKA. The main PKA phosphorylation site is serine 1928 in the central pore forming alpha(1)1.2 subunit of Ca(v)1.2. PKA is bound to Ca(v)1.2 within a macromolecular signaling complex consisting of the beta(2) adrenergic receptor, trimeric G(s) protein, and adenylyl cyclase for fast, localized, and hence specific signaling [Davare, M. A., Avdonin, V., Hall, D. D., Peden, E. M., Buret, A., Weinberg, R. J., Horne, M. C., Hoshi, T., and Hell, J. W. (2001) Science 293, 98-101]. Protein phosphatase 2A (PP2A) serves to effectively balance serine 1928 phosphorylation by PKA through its association with the Ca(v)1.2 complex [Davare, M. A., Horne, M. C., and Hell, J. W. (2000) J. Biol. Chem. 275, 39710-39717]. We now show that native PP2A holoenzymes, as well as the catalytic subunit itself, bind to alpha(1)1.2 immediately downstream of serine 1928. Of those holoenzymes, only heterotrimeric PP2A containing B' and B' ' subunits copurify with alpha(1)1.2. Preventing the binding of PP2A by truncating alpha(1)1.2 28 residues downstream of serine 1928 hampers its dephosphorylation in intact cells. Our results demonstrate for the first time that a stable interaction of PP2A with Ca(v)1.2 is required for effective reversal of PKA-mediated channel phosphorylation. Accordingly, PKA as well as PP2A are constitutively associated with Ca(v)1.2 for its proper regulation by phosphorylation and dephosphorylation of serine 1928.

Molecular basis for PP2A regulatory subunit B56α targeting in cardiomyocytes

“…There is little information on the particular domains of the PP2A holoenzyme trimeric complex that define such subcellular targeting. Although yeast two-hybrid studies have shown that PP2A/c, and not B, subunits interact directly with the ␣ 1 -subunit of the L-type Ca 2ϩ channel (11,22), a more common targeting motif is seen in ryanodine receptor/PP2A interactions where PP2A is tethered through a B-targeting subunit, PR130 (43,65). Yeast two-hybrid studies have defined distinct binding activities for B56 subunits although the sites have not been defined with precision.…”

“…In heart, PP2A function is necessary for normal excitation-contraction coupling, and PP2A levels are altered in heart failure (1,57,58). Numerous studies have suggested that PP2A activity is associated with critical cardiac receptors, ion channels, and transporters including ␤-adrenergic receptor (10,12,22), L-type Ca 2ϩ -channels (11,22), ryanodine receptor (43), Na/Ca exchanger (70), inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3 ] receptor (13), and Na/K ATPase (35).…”

Cyclin G2 is a centrosome-associated nucleocytoplasmic shuttling protein that influences microtubule stability and induces a p53-dependent cell cycle arrest

“…Polyclonal antibodies were affinity purified as described [1]. The affinity and specificity of the antibodies were tested by immunoprecipitation and immunoblotting of HA-tagged PP2A B' subunits expressed in HEK cells [1] and brain lysate [52]. This pan-specific antibody specifically detects the HA-tagged forms expressed in HEK cells as well as several endogenous B' subunit isotypes ranging from 52-72 kDa present in brain lysate.…”

Displacement of α-Actinin from the NMDA Receptor NR1 C0 Domain By Ca2+/Calmodulin Promotes CaMKII Binding

“…Basically, doubling of the exposure time has to lead to a doubling of the signal as we published earlier (41,42). Signals were scanned using an Epson Perfection 4180 Photo flatbed scanner and EPSONscan software (42).…”

“…Basically, doubling of the exposure time has to lead to a doubling of the signal as we published earlier (41,42). Signals were scanned using an Epson Perfection 4180 Photo flatbed scanner and EPSONscan software (42). To ensure that signals were comparable between different films and not distorted by using non-linear contrast functions, neither autocontrast nor any other manipulations of contrast such as non-linear transformation were applied throughout the whole procedure.…”