The alpha and beta subunits of phosphorylase kinase are homologous: cDNA cloning and primary structure of the beta subunit.

Abstract

We have cloned cDNA molecules encoding the P subunit of phosphorylase kinase (ATP:phosphorylase-b phosphotransferase; EC 2.7.1.38) from rabbit fast-twitch skeletal muscle and have determined the complete primary structure of the polypeptide by a combination of peptide and DNA sequencing. In the mature 13 subunit, the initial methionine is replaced by an acetyl group. The subunit is composed of 1092 amino acids and has a calculated molecular mass of 125,205 Da. Alignment of its sequence with the a subunit of phosphorylase kinase reveals extensive regions of homology, but each molecule also possesses unique sequences. Two of the three phosphorylation sites known for the P subunit and all seven phosphorylation sites known for the a subunit are located in these unique domains.

Structure and Location of the Regulatory β Subunits in the (αβγδ)4 Phosphorylase Kinase Complex

“…Previous analyses of the primary structures of ␣ and ␤ noted that the two subunits were homologous and putatively products of gene duplication, but each was unique as a complete protein (20). Predictions of 2°structure for both subunits suggest them to be predominantly ␣-helical, and our CD measurements of the ␣␥␦ and (␣␤␥␦) 4 complexes are in agreement, showing ␣ and ␤ to have similar 2°structure content.…”

“…The sum of these data indicates that residues in the ␤ C terminus, the NB1 domain, and ␥CRD are surface-accessible and proximal to one another within the (␣␤␥␦) 4 complex. Another likely surface-accessible region of ␤ is Ser-700, which is reportedly autophosphorylated by PhK (20). We also report herein an epitope in the PhK complex for an anti-␤-specific mAb that has been localized through ␤-truncation to within a region of this subunit from residues 704 to 815.…”

“…Although the primary structures are known for both ␣ and ␤ (20,21), essentially all higher order secondary (2°) and tertiary (3°) structures reported for these subunits are based on predicted rather than experimental data (22)(23)(24). In contrast, high resolution structural information is available for the catalytic core of the ␥ subunit and for the ␦ subunit (3,25); on the other hand, the small size and location of these subunits in the complex make them unlikely candidates for bridging the two (␣␤␥␦) 2 lobes (14,16).…”

“…It is also the subunit that is predominantly responsible for the regulation of PhK activity by phosphorylation (7). The major regulatory phosphorylatable serine, Ser-26 (28), which is targeted in vivo by cAMP-dependent protein kinase (PKA) and is part of the ␤ subunit's unique N-terminal phosphorylatable domain (NB1) (20), is also reportedly autophosphorylated, along with Ser-11, by the ␥ subunit within the complex (7,20), triggering a conformational change in ␤ that is detected by its cross-linking with 1,5-difluoro-2,4-dinitrobenzene (DFDNB) to form homodimers (29). This possible association of ␤ subunits is consistent with phospho-mimetic S11E/S26E joint mutations that promote self-association of ␤ chimeras in two-hybrid assays (7).…”

“…A comparison of the sequences of the ␤ subunit and its ␣ subunit homolog indicated that both are multidomain structures, containing subunit-specific regulatory regions and large distinct sequence-similar domains, the latter of which suggested that the subunits are products of early gene duplication events (20). Since these earlier reports, both N-terminal glycosyl hydrolase (GH) clan 15 member-like (GHL) domains and C-terminal calcineurin-B like (CBL) domains have been predicted for ␣ and ␤ (22-24); however, attempts to model the entire structures were unsuccessful (22,23).…”