cDNA cloning and complete primary structure of skeletal muscle phosphorylase kinase (alpha subunit).

Abstract

We have isolated and sequenced a cDNA encoding the a subunit of phosphorylase kinase from rabbit fast-twitch skeletal muscle. The cDNA molecule consists of 388 nucleotides of 5'-nontranslated sequence, the complete coding sequence of 3711 nucleotides, and 342 nucleotides of 3'-nontranslated sequence followed by a poly(dA) tract. It encodes a polypeptide of 1237 amino acids and a deduced molecular mass of 138,422 Da. Nearly half of the deduced amino acid sequence is confirmed by peptide sequencing. Seven positions of endogenously phosphorylated serine residues and autophosphorylation sites, identified by peptide sequencing, could be assigned. They cluster in a segment of only 60 amino acids. RNA blot hybridization analysis demonstrates a predominant RNA species of -4500 nucleotides and a less abundant RNA of 8700 nucleotides. (19,21,22), and rats (23). Defects of the muscular and of the hepatic form of the enzyme and X chromosomal as well as autosomal transmission are observed. The isolation of DNA sequences encoding the subunits of phosphorylase kinase would make it possible to characterize the molecular nature of these deficiencies and may improve clinical diagnosis and genetic counseling.To these ends, we have determined large parts of the peptide sequence of the a subunit of phosphorylase kinase from rabbit fast-twitch skeletal muscle. We have used these sequences to design oligonucleotide probes and to isolate and identify cDNA molecules encoding this subunit. Here, we present the nucleotide sequence of a nearly complete mRNA copy, and the complete primary structure of the a subunitA Phosphorylase kinase (ATP:phosphorylase-b phosphotransferase, EC 2.7.1.38) was the first regulatory protein kinase to be discovered (1) and the starting point for revealing, during the last three decades, reversible protein phosphorylation as an important control mechanism in eukaryotic cells. It is a large oligomeric enzyme (1.3 MDa) with the subunit structure (afOyS)4. In linking glycogen breakdown to both nervous and endocrine stimulation, the enzyme is regulated in a complex way by phosphorylation of various serine residues and by calcium (2-4). The subunit 8 is identical to calmodulin and confers Ca2+ sensitivity to the enzyme (5). The subunit y carries catalytic activity (6) and is similar to other known protein kinases (7). The primary structures of these subunits have been determined, and cDNAs encoding the subunit y have been cloned (8-10). The two large subunits, a and X3, comprise 80%o of the total protein. They carry all phosphorylation sites; furthermore, they have been implicated in catalytic function (4, 11) as well as binding of nucleotides (11, 12), calmodulin (13), and troponin (14). Only small partial amino acid sequences have been published from a and ,8. To provide a structural basis for the wealth of enzymological data that has been accumulated and to correlate the various functional properties to structural features, it is necessary to determine the primary structures of these subunits.There are c...

Self-association of the α Subunit of Phosphorylase Kinase as Determined by Two-hybrid Screening

“…Second, the stretch from 1012 to 1056 is a region of very low homology between the rabbit muscle and liver ␣ subunits (40). Third, at least seven phosphorylation sites occur between residues 972 and 1030 (20). The sum of this evidence strongly suggests that the overlapping region contains regulatory domains, as opposed to an association domain.…”

“…Of the 7.8 ϫ 10 6 independent clones obtained, 73% contained inserts averaging 1.6 kilobases in size. Rabbit PhK ␣ cDNA (␣D1) was kindly provided by Dr. Manfred W. Kilimann (Ruhr-Universitä t Bochum, Bochum, Germany) (20) and used as the template for the preparation of all ␣ truncations shown in Fig. 2.…”

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

“…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. Some minor differences in 2°structure were observed between ␣ and ␤, however, and these likely reflect the presence of several nonhomologous stretches, some of which include phosphorylatable regulatory domains specific to each subunit (20,21). The ␣ subunit has a large phosphorylatable C-terminal regulatory domain with multiple phosphorylation sites (1), whereas the ␤ subunit contains two known phosphorylatable regions, Ser-700 plus Ser-11/Ser-26 in the N-terminal phosphorylatable domain (NB1, residues 1-40) (7,28).…”

“…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).…”

cDNA encoding a 59 kDa homolog of ribosomal protein S6 kinase from rabbit liver

“…Size-fractionated cDNA (larger than 1800 nucleotides (nt)) was synthesized as described [5] and inserted into &tlO with EcoRl linkers. A primary library of 700 000 plaques from rabbit liver was screened under reduced stringency with the phosphorylase kinase y subunit cDNA (complete coding sequence plus 228 nt of 3 '-noncoding sequence) from mouse muscle (donated by J. Chamberlain [2]).…”