Phosphorylation of ribosomal protein S6 in the aquatic fungus Blastocladiella emersonii

Abstract

The changes in the degree of phosphorylation of ribosomal protein S6 during the life cycle of the aquatic fungus Blastocladiella emersonii were analyzed by two-dimensional gel electrophoresis. Three phosphorylated derivatives of S6 are present throughout the kntire life cycle. However, under certain germination conditions, more highly phosphorylated derivatives of S6 appear. Nonetheless, the resumption of protein synthesis that occurs during germination is not dependent on those highly phosphorylated derivatives of S6.The pattern and sites of phosphorylation of S6 labelled in uivo with [32P]orthophosphate have been compared with those of 40s ribosomal subunit labelled in uitro by partially purified protein kinases. Three major phosphopeptides were found in S6 isolated from the zoospore, while six phosphopeptides were found after zoospore germination (in germling cells). The phosphopeptide patterns of S6 phosphorylated by the CAMP-dependent protein kinase and by casein kinases I and I1 were completely distinct. Only the CAMP-dependent protein kinase gives rise to a phosphopeptide found in 32P-labelled cells, indicating that one of sites phosphorylated in vivo is also phoshorylated in vitro by the CAMP-dependent protein kinase.Protein S6 of the small (40s) ribosomal subunit, the major phosphoprotein of eukaryotic ribosomes, can be phosphorylated at multiple sites (at least five) in viuo [I -41. Although S6 phosphorylation has been observed under different physiological conditions [3], its role is not clear. Correlations have often been observed between an increase in the phosphorylation of protein S6 and elevated levels of protein synthesis [3,4], whereas dephosphorylation is generally associated with decreased protein synthesis [5,6]. However, an increasing number of exceptions to this rule have appeared and phosphorylation of S6 can be stimulated under conditions in which total protein synthesis is either increased, inhibited, or unaltered [3,4,7]. The dephosphorylation of S6 which accompanies the heat-shock response in cultured Drosophila cells also may not be causally associated with either the synthesis of heat-shock proteins or the turn-off of normal protein synthesis that results from heat shock [S]. As demonstrated by Ward et al. [9], increased phosphorylation of S6 does not seem to be universal event in the activation of protein synthesis at fertilization of sea urchin eggs. For hepatocytes and HeLa cells, it has been shown that different agonists lead to significantly different S6-derived phosphopeptide patterns [lo, 111, suggesting that two seperate pathways (CAMPdependent or CAMP-independent) are involved in the process. In quiescent 3T3 cells stimulated with serum, an ordered phosphorylation of S6 was observed and ten major phosphopeptides were found [12].The life cycle of Blastocladiella emersonii is characterized by certain features which make this aquatic fungus an experimentally convenient organism for the study of the (EC 2.7.1.37).significance of S6 phosphorylation. The life cycle consists of a...

Changes in the pattern of protein synthesis during zoospore germination in Blastocladiella emersonii

“…Cultures of B. emersonji were maintained on plates containing 0.13% peptone, 0.13% yeast extract, 0.3% glucose, and 1% agar. Zoospores (3 x 105/ml) were inoculated into DM3 medium (3) Fig. 1.…”

Fibroblast growth factor treatment of Swiss 3T3 cells activates a subunit S6 kinase that phosphorylates a synthetic peptide substrate.

“…Soluble extracts from insulin, epidermal growth factor, and fetal calf serum-treated cells exhibit elevated kinase activity towards reconstituted S6 (7)(8)(9)(10)(11). It is unclear whether one or more S6 kinases are involved, since S6 can be phosphorylated by as many as nine distinct protein-serine kinases in vitro (11)(12)(13)(14)(15)(16)(17)(18). However, ion-exchange chromatography studies with extracts from chicken embryo fibroblasts infected with Rous sarcoma virus or exposed to phorbol 12-myristate 13-acetate (PMA) or 10% calf serum have implied that a unique S6 kinase may be activated by these diverse treatments (10).…”

Regulation of adenylyl cyclase from Blastocladiella emersonii by guanine nucleotides

“…Changes in adenylyl cyclase activity during sporulation. Cells previously grown for 14 h at 19°C in nutrient medium [10] were harvested, resuspended in sporulation solution (1 mM Tris-maleate, pH 6.8, 1 mM CaC12) and incubated at 27°C. Zoospores were fully released after 4 h. At the indicated times, samples were harvested and the adenylyl cyclase activity of the P15 fraction was assayed in the presence of 3 mM MnCI2 (dashed boxes), 3 mM MgCI: (closed boxes) and 3 mM MgCI: plus 100 jIM GTPyS (open boxes).…”