Identification of the sites phosphorylated by cyclic AMP-dependent protein kinase on the β2 subunit of L-type voltage-dependent calcium channels

Voltage-dependent L-type calcium (Ca) channels are heteromultimeric proteins that are regulated through phosphorylation by cAMP-dependent protein kinase (PKA). We demonstrated that the beta(2) subunit was a substrate for PKA in intact cardiac myocytes through back-phosphorylation experiments. In addition, a heterologously expressed rat beta(2a) subunit was phosphorylated at two sites in vitro by purified PKA. This beta(2a) subunit contains two potential consensus sites for PKA-mediated phosphorylation at Thr(164) and Ser(591). However, upon mutation of both of these residues to alanines, the beta(2a) subunit remained a good substrate for PKA. The actual sites of phosphorylation on the beta(2a) subunit were identified by phosphopeptide mapping and microsequencing. Phosphopeptide maps of a bacterially expressed beta(2a) subunit demonstrated that this subunit was phosphorylated similarly to the beta(2) subunit isolated from heart tissue and that the phosphorylation sites were contained in the unique C-terminal region. Microsequencing identified three serine residues, each of which conformed to loose consensus sites for PKA-mediated phosphorylation. Mutation of these residues to alanines resulted in the loss of the PKA-mediated phosphorylation of the beta(2a) subunit. The results suggest that phosphorylation of the beta(2a) subunit by PKA occurs at three loose consensus sites for PKA in the C-terminus and not at either of the two strong consensus sites for PKA. The results also highlight the danger of assuming that consensus sites represent actual sites of phosphorylation. The actual sites of PKA-mediated phosphorylation are conserved in most beta(2) subunit isoforms and thus represent potential sites for regulation of channel activity. The sites phosphorylated by PKA are not substrates for protein kinase C (PKC), as the mutated beta(2) subunits lacking PKA sites remained good substrates for PKC.