Phosphopeptide patterns of the ribosomal protein S6 following stimulation of guineapig parotid glands by secretagogues involving either cAMP or calcium as second messenger

The ribosomal protein S6 in exocrine cells is phosphorylated during stimulated during stimulation of exocytosis by cAMP‐dependent or calcium‐dependent agonists. Under both conditions the same tryptic S6 phosphopeptides (termed A, B, and C) were found [Padel, Kruppa, Jahn & Söling (1983) FEBS Lett. 159, 112–118]. Studies have now been made of the phosphorylation pattern of protein S6 from purified guinea pig parotid ribosomes following in vitro phosphorylation with calmodulin‐dependent, phospholipid‐dependent, and cAMP‐dependent protein kinases. Only the phospholipid‐dependent enzyme led to the phosphorylation of peptides A, B, and C, while the cAMP‐dependent enzyme phosphorylated only peptides A and C, and the calmodulin‐dependent enzyme did not phosphorylate any of the phosphopeptides found in S6 from unstimulated or stimulated intact cells. Guinea pig parotid microsomes contain substantial phospholipid‐dependent protein kinase activity. Stimulation of intact parotid glands with tetradecanolyphorbol acetate led to a significant phosphorylation of S6 and a similar tryptic S6 phosphopeptide pattern as seen with carbamoylcholine. It is concluded that activation of phospholipid‐dependent protein kinase is responsible for the phosphorylation of protein S6 during stimulation with calcium‐dependent and cAMP‐dependent secretagogues.

mentioning

confidence: 59%

Phosphorylation of the ribosomal protein S6 during agonist‐induced exocytosis in exocrine glands is catalyzed by calcium‐phospholipid‐dependent protein kinase (protein kinase C)

Padel¹,

Söling²

1985

“…Although in several cases protein kinases can phosphorylate proteins in vitro at different and/or additional sites (e. g. [62] and [28]), protein 111 was phosphorylated in vitro by CAMP-dependent protein kinase only at the same site which was phosphorylated in the intact cell.…”

Section: Discussionmentioning

confidence: 99%

“…The peptides that were obtained after trypsin or chymotrypsin digestion of protein I11 were separated by twodimensional thin-layer separation as described by Padel et al [28] and by reversed-phase HPLC employing either a Waters pBondapak c 1 8 (2.9x300mm) or a Nova Pak c 1 8 (3.9 x 150 mm) column equilibrated with 0.1 YO trifluoroacetic acid. After injection of the sample, the peptides were eluted with a linear gradient of acetonitrile (0-70%) in 0.1% trifluoroacetic acid over 60 min.…”

Section: Proteolytic Digestion and Peptide Mappingmentioning

confidence: 99%

Purification and characterization of a 22‐kDa microsomal protein from rat parotid gland which is phosphorylated following stimulation by agonists involving cAMP as second messenger

Thiel

Schmidt

Meyer

et al. 1988

Stimulation of secretion in exocrine glands by agonists involving cAMP as second messenger leads to the phosphorylation of the ribosomal protein S6 ( It could be extracted from the endoplasmic reticulum membrane only with Triton X-100, SDS or concentrated formic or acetic acid. The purification of this protein involved extraction of the microsomes with Triton X-100, removal of the detergent by acetone precipitation, extraction of water-soluble proteins, lipids and lipoproteins, and preparative SDS polyacrylamide gel electrophoresis. The protein has a basic PI (> 8.7). For determination of the amino acid composition of protein 111 and for sequencing of its amino-terminal portion, the protein was electroeluted out off the gel, the detergent removed and the protein finally purified by reversed-phase HPLC. Protein 111 could be phosphorylated in vitro by the catalytic subunit of the CAMP-dependent protein kinase to a degree of approximately 0.14 mol phosphate/mol protein. The only phosphopeptide obtained after in vitro phosphorylation and subsequent tryptic or chymotryptic digestion was identical with the phosphopeptide obtained after stimulation of intact rat parotid gland lobules with isoproterenol. The sequence of this peptide was Lys-Leu-Ser(P)-Glu-AlaAsp-Asn-Arg. It was confirmed by an analysis of the synthetic peptide following in vitro phosphorylation with CAMP-dependent protein kinase. The first 41 N-terminal residues of protein I11 were sequenced. So far no sequence homology with other known peptides or proteins could be found.

“…After a 1-h incubation at 37'-C, cell extracts were prepared and chromatographed on DEAE-Sephacel, and the eluate rractiona were probed for S6-phosphorylating protein kinase activity under standard assay conditions. After gel electrophoresis followed by autoradiography, thc S6 protein hands wcrc excised from the gels and their radioactivity determined lyophilization, the digest was finally dissolved in 6 PI H 2 0 , and usually half of it was subjected to two-dimensional separation on cellulose thin-layer sheets (Merck, Darmstadt, FRG) using conditions for electrophoresis and chromatography exactly as described in [34].…”

Section: Protein Kinuse Ussuys Electrophoresis Pept Ide Mappingmentioning

confidence: 99%

“…3). The mapping technique and the phosphopeptide code were essentially as used by several other groups [23,26,34,37,381. As shown in Fig.…”

Section: Peptide Mapsmentioning

confidence: 99%

Insulin‐induced S6 kinase activation in HeLa cells and its reversal by hyperthermic stress

Burger¹,

Lawen

Martini

1989

Insulin treatment of HeLa S3 cells activates an S6-phosphorylating protein kinase. Although this enzyme has chromatographic properties resembling those of described proteolytic fragments of other protein kinases, namely protein kinase C, protease-activated kinase 11 and histone-4 protein kinase, and although insulin has been proposed by others to cause S6 phosphorylation via proteolytic protein kinase activation, the insulin-induced increase in S6-kinase activity described here is probably not due to proteolysis. Rather, the activity indicates the existence, in HeLa cells, of an interconvertible S6 kinase, since the insulin-induced activity increase was rapidly reversed under hyperthermic stress, and since this effect of hyperthermia was itself reversible. The S6-kinase activities from serum-and from insulin-stimulated HeLa cells resemble each other closely and are likely to represent the same enzyme. The enzyme may therefore mediate both signals delivered by mitogens and the insulin signal. Analysed at an in vitvo transfer of 1 mol phosphate/mol S6, this S6 kinase activity does not phosphoryhte the (principal) S6 site recognized by the CAMP-dependent protein kinase.A characteristic facet of the very early phase of the response of cells to serum or growth factors, including insulin, is the multiple phosphorylation of the ribosomal protein S6 (for serum: see references in [I, 21; for growth factors [3-71; for insulin [8 -131). We have described an S6-phosphorylating protein kinase which is activated in serum-treated cells and therefore a candidate mediator of serum-induced S6 phosphorylation [l, 141. As noted previously [14], the serum effect could be mimicked with a high dose (5 x M) of insulin. We have since observed S6 kinase activation at lower insulin doses. This suggests that insulin-responsive S6 kinase from 3T3-Ll cells [15, 161 and other systems [17-211 may be homologues of the human mitogen-responsive S6 kinase, and furthermore, that both insulin and a non-insulin signal(s) contained in serum (the insulin content of sera was low) may activate the same or a very closely related S6-phosphorylating enzyme(s).As shown in [l], chromatographic criteria allowed us to distinguish the S6 kinase from other S6-phosphorylating enzymes implicated in the transfer of anabolic signals [22 -261.