Sequencing of phosphoserine‐containing peptides yields normally no identifiable PTH‐derivatives at those positions where phosphoserine is located. Here a new method is described which allows identification of the position of phosphoserine by chemical modification just before sequence analysis. In a one‐step microbatch reaction, phosphoserine present in the intact peptide can be transformed quantitatively into stable derivatives such as β‐methylaminoalanine (MAA), S‐ethanolcysteine or S‐ethylcysteine. These derivatives are detectable during microsequencing with less than 100 pmol peptide using an Applied Biosystems gas‐phase sequencer equipped with an on‐line PTH amino acid analyzer.

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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...

The primary structure of the a and (3 subunits ofphosphorylase kinase reveals that both proteins contain a carboxyl-terminal CA1A2X motif (where C is cysteine, Al and A2 are aliphatic amino acids, and X is an uncharged amino acid), the recognition signal for a protein polyisoprenyltransferase. The product, a polyisoprenylated cysteine, can be detected by phenylthiocarbamoylamino acid analysis and by microsequencing following conversion to S-ethylcysteine. Mass spectrometry confirms a covalently linked farnesyl residue in both subunits. Tandem mass spectrometry localizes these modifications at the cysteine residues present in the carboxylterminal CAMQ and CLVS sequences of the a and 3 subunits, respectively. Membrane association of phosphorylase kinase, probably mediated by these farnesyl residues, is discussed. be dictated by the amino acid X. If X represents methionine or serine, the motifis recognized by farnesyltransferases (24), whereas a carboxyl-terminal leucine or phenylalanine specifies geranylgeranylation (25,26 Phosphorylase kinase is found in two distinct compartments in muscle cells. The bulk of the enzyme is present in the cytosol, associated preferentially with glycogen particles. There, it can trigger a rapid conversion of glycogen phosphorylase b to the active a form, switching on glycogenolysis to meet energy demands of working muscle (1). A small fraction of phosphorylase kinase, however, is membraneassociated (2-4). Monoclonal or polyclonal monospecific antibodies localize all four subunits (a, A, y, and 8) of phosphorylase kinase at the sarcoplasmic reticulum, especially at the surface of terminal cisternae facing the T-tubule (5, 6).Phosphorylase kinase can enhance the activity of the sarcoplasmic reticulum Ca2+-transport ATPase (7) due to generation of phosphatidylinositol 4-phosphate, a lipophilic effector of this ATPase (8). Indeed, purified phosphorylase kinase catalyzes phosphorylation of phosphatidylinositol; attempts to separate this phosphatidylinositol 4-kinase from phosphorylase kinase were not successful (9).The carboxyl terminus of both the a and (3 subunits of phosphorylase kinase contains a CA1A2X motif (10,11), where C is cysteine, Al and A2 are aliphatic residues, and X is an uncharged amino acid. In this motif cysteine can be polyisoprenylated (12,13). Upon thioether formation, further posttranslational modifications may occur, such as removal ofthe three terminal amino acids and subsequent methylation of the newly formed terminal carboxyl group (14-17). Polyisoprenylation was detected first in fungal and yeast mating factors (18,19) and subsequently in regulatory proteins of eukaryotic cells (14, 20-22). In animal cells only three types of proteins are known to be farnesylated and only four to be geranylgeranylated, even though many more proteins contain a carboxyl-terminal CAjA2X motif (reviewed in ref. 23). The specificity of the protein polyisoprenyltransferase appears to MATERIALS AND METHODS Endoproteinase Lys-C was from Boehringer Mannheim, ethanethiol from Ja...

Identification of O-phosphorylated amino acids within the primary structure of regulatory proteins is important in understanding the mechanisms by which their functions are regulated. In many cases radioactive labeling with [32P]phosphate is tedious or sometimes impossible. Therefore, we have established a series of new non-radioactive methods that permit the localization of phosphoserine, phosphothreonine, and phosphotyrosine. After partial hydrolysis of a phosphopeptide or phosphoprotein, phosphoserine, phosphothreonine, or phosphotyrosine are determined by capillary electrophoresis as their dabsyl-derivatives. Chemical modification transforms phosphoserine or phosphothreonine to S-ethyl-cysteine or beta-methyl-S-ethyl-cysteine, respectively, allowing their localization during sequence analysis. We apply solid-phase sequencing to overcome the limitations of the gas-phase sequenator in the case of phosphotyrosine-containing peptides. Liquid chromatography on-line connected to an electrospray mass spectrometer is a powerful new method of increasing importance in the protein chemistry field. It is especially well suited for identification of phosphoserine- or phosphothreonine-containing peptides in a proteolytic digest of a phosphoprotein. In this article we will describe how to work with these new methods practically.