Richard E.H. Wettenhall

Isolation and characterisation of cyclic AMP-dependent phosphorylation sites from rat liver ribosomal protein S6

Protein S6 of the small (40 S) ribosomal subunit is the major phosphoprotein of mammalian ribosomes [l-3], and can be phosphorylated at multiple sites both in vitro and in vivo [l-3]. The primary consequences of S6 phosphorylation have not yet been established, although a relationship between the extent of phosphorylation and protein synthetic activity is suggested by several observations [3-7; cf. 1981. the isolation of these sites and determination of their amino acid sequences. Surprisingly, the two major serine residues phosphorylated by cyclic AMP-dependent protein kinase were adjacent in the primary structure of S6.

Substrate specificity of wheat embryo calcium-dependent protein kinase

Wheat embryo Ca2+ ‐dependent protein kinase (CDPK) phosphorylates a variety of synthetic peptides having a Basic‐X‐X‐Ser sequence but peptides with a Basic‐Basic‐X‐Ser(Thr) sequence are relatively poor substrates. Wheat germ CDPK phosphorylates a variety of proteins of which histone H1 and bovine serum albumin are among the better substrates. A single phosphorylated tryptic peptide was purified from bovine histone H1 phosphorylated by wheat embryo CDPK and subjected to Edman degradation yielding the sequence Gly97‐Thr‐Gly‐Ala‐Ser‐Gly‐Ser(PO4)‐Phe‐Lys105. Ser103 on bovine histone H1 is also the residue phosphorylated by rat brain protein kinase C.

Isolation and characterization of a (1 → 3)-β-glucan endohydrolase from germinating barley (Hordeum vulgare): amino acid sequence similarity with barley (1 → 3, 1 → 4)-β-glucanases

A (1 → 3)‐β‐glucan 3‐glucanohydrolase (EC 3.2.1.39) has been purified approx. 190‐fold from extracts of germinating barley. The enzyme has an apparent M r 32 000, a pI of 8.6, and a pH optimum of 5.6. Analysis of hydrolysis products released from the (1 → 3)‐β‐glucan, laminarin, shows that the enzyme is an endohydrolase. Sequence analysis of the 46 NH2‐terminal amino acids of the (1 → 3)‐β‐glucanase reveals 54% positional identity with barley (1 → 3,1 → 4)‐β‐glucanases (EC 3.2.1.73) and suggests a common evolutionary origin for these two classes of β‐glucan endohydrolases. The barley (1 → 3)‐β‐glucanase also exhibits significant similarity with a (1 → 3)‐β‐glucanase from tobacco.

Differential phosphorylation of ribosomal protein S6 in isolated rat hepatocytes after incubation with insulin and glucagon.

Glucagon and insulin both stimulated the 32P‐labelling of ribosomal protein S6 in rat hepatocytes that had been incubated with 32Pi. Glucagon selectively enhanced the labelling of the tryptic peptide phosphorylated by cyclic AMP‐dependent protein kinase, demonstrating that 6 S is a physiological substrate for this enzyme. Insulin stimulated the phosphorylation of distinct tryptic peptides, at least one of which appears to be very close in the primary structure to the sites phosphorylated by cyclic AMP‐dependent protein kinase.

Phosphorylation of ribosomal protein S6 and a peptide analogue of S6 by a protease-activated kinase isolated from rat liver

A trypsin‐activated protein kinase has been isolated from rat liver using a peptide analogue of ribosomal protein S6 as a substrate in kinase assays. The structure of the peptide, Arg‐Arg‐Leu‐Ser‐Ser‐Leu‐Arg‐Ala, was based on a region of S6 containing both an insulin‐ and cyclic AMP‐regulated phosphorylation site. The trypsin‐activated protein kinase phosphorylated a corresponding site in the peptide analogue and ribosomal protein S6 that was distinct from the preferred site for cyclic AMP‐dependent protein kinase. Ribosomal S6 contained at least one other major site for the trypsin‐activated protein kinase.

Phosphorylation sites for ribosomal S6 protein kinases in mouse 3T3 fibroblasts stimulated with platelet-derived growth factor

Platelet release products and purified platelet‐derived growth factor stimulated the phosphorylation of ribosomal protein S6 in cultured mouse Balb/c 3T3 fibroblasts. The post‐nuclear fraction of the stimulated cells was enriched in S6 kinase activity specific for sites resembling those phosphorylated within intact cells in response to PDGF as determined by tryptic peptide mapping. 3T3‐S6 sites closely resembled those phosphorylated in S6 of rat hepatocytes stimulated with insulin and included sites for both cAMP‐dependent and independent kinases.

Evidence for a second phosphorylation site on eIF-2α from rabbit reticulocytes

Ser 51 in the NH2‐terminal sequence of the α‐subunit of eukaryotic peptide initiation factor 2 (eIF‐2) has been identified as a second phosphorylation site for the heme‐controlled eIF‐2α kinase from rabbit reticulocytes. Increased phosphorylation of this serine relative to the previously described phosphorylation site (Ser 48) is observed when the kinase reaction is carried out in the presence of the α‐subunit of spectrin. A synthetic peptide corresponding to eIF‐2α(41–54) is phosphorylated only in Ser 51 by the eIF‐2α kinase.

Isolation of protein S6 from rat liver ribosomes by reversed-phase high-performance liquid chromatography

A rapid procedure for the isolation of ribosomal protein S6 from rat liver ribosomes has been developed in which proteins were separated by reversed-phase HPLC using wide-pore n-butyl-, n-octyl-, or diphenyl-bonded silica phases. Rapid processing of whole ribosomal material was achieved by the extraction of proteins in 6 M guanidinium hydrochloride and subsequent precipitation of RNA by acidification. Highly purified S6 was obtained in two chromatographic steps as shown by sodium dodecyl sulfate-gel electrophoresis, amino acid analysis, and automated microsequencing. The purification of S6 was monitored using 32P-labeled S6 as a marker which cochromatographed with unphosphorylated S6 under the low-pH elution conditions employed. Other ribosomal proteins were also purified using these reversed-phase supports, although in the case of more hydrophobic proteins such as S4 and S10 further optimization of the gradient conditions was required.

Rate-limiting factors for lymphocyte protein synthesis Ribosome commitment and the capacity of lymphocyte cell-free systems to translate exogenous mRNAs

The properties of the protein synthesising systems of different lymphocyte preparations have been compared with those of non-lymphoid tissues. Polysome profiles from rat thymocytes, sheep mesenteric lymphocytes, rat liver and mouse ascites tumours showed that the commitment of ribosomes to protein synthesis in lymphocytes was relatively low. Initiation factor activities, assessed on the abilities of post-mitochondrial fractions to support exogenous mRNA translation, were limited or undetectable in lymphoid tissues. While the thymocyte system translated globin mRNA, the response was enhanced by ascites extracts rich in initiation factors. The mesenteric lymphocyte system responded only marginally to globin mRNA and poly(U) but the responses were not enhanced by ascites extracts. The activity of isolated mesenteric ribosomes was comparable with ribosomes from other tissues, indicating that extraribosomal factors were responsible for the poor overall activity of the mesenteric system. Finally, the effects of cycloheximide on the recruitment of polysomes in lymphocytes indicated that the commitment of ribosomes to protein synthesis might be restricted by both limited mRNA availability and limited capacity for initiation of mRNA translation.