L. Alberghina

Phosphorylation of Cdc28 and regulation of cell size by the protein kinase CKII in Saccharomyces cerevisiae.

The CDK (cyclin-dependent kinase) family of enzymes is required for the G(1)-to-S-phase and G(2)-to-M-phase transitions during the cell-division cycle of eukaryotes. We have shown previously that the protein kinase CKII catalyses the phosphorylation of Ser-39 in Cdc2 during the G(1) phase of the HeLa cell-division cycle [Russo, Vandenberg, Yu, Bae, Franza and Marshak (1992) J. Biol. Chem. 267, 20317-20325]. To identify a functional role for this phosphorylation, we have studied the homologous enzymes in the budding yeast Saccharomyces cerevisiae. The S. cerevisiae homologue of Cdc2, Cdc28, contains a consensus CKII site (Ser-46), which is homologous with that of human Cdc2. Using in vitro kinase assays, metabolic labelling, peptide mapping and phosphoamino acid analysis, we demonstrate that this site is phosphorylated in Cdc28 in vivo as well in vitro. In addition, S. cerevisiae cells in which Ser-46 has been mutated to alanine show a decrease in both cell volume and protein content of 33%, and this effect is most pronounced in the stationary phase. Because cell size in S. cerevisiae is regulated primarily at the G(1) stage, we suggest that CKII contributes to the regulation of the cell cycle in budding yeast by phosphorylation of Cdc28 as a checkpoint for G(1) progression.

Effect of the different dimeric forms of the platelet‐derived growth factor on cellular responses in mouse Swiss 3T3 fibroblasts

PDGF consists of two polypeptide chains, A and B, and all three possible dimers have been isolated from different sources. Human PDGF, essentially AB, porcine PDGF (BB) and recombinant PDGF‐AA were tested on Swiss 3T3 fibroblasts for their ability to stimulate mitogenesis, phosphoinositide turnover and tyrosine phosphorylation of the PDGF receptor. When used in saturating amounts, the three isoforms were equally active in inducing mitogenesis. However, PDGF‐AA was less active than AB and BB to induce the phosphorylation of the receptor and the turnover of phosphoinositides (30% and 50%, respectively). These findings suggest that, in Swiss 3T3 fibroblasts, PDGF receptors of the α‐type are present in a slightly lower amount than β‐type. In addition, the two types of receptor appear to have similar physiological functions.

G1 phase heterogeneity in exponentially growing Swiss 3T3 mouse fibroblasts

The growth rate of normal cultured Swiss 3T3 fibroblasts is function of serum concentration and the fraction of G1 cells, and hence the average residence time in G1, increases with the generation time. Serum contains two sets of factors: competence factors, essentially platelet-derived growth factor (PDGF), which induces competence in quiescent fibroblasts and prevents replicating cells from entering G0, and plasma, which allows progression. The increase in the duplication time and the duration of G1 at low serum concentration could hence be due to the fact that competence factors become limiting. The fraction of non-competent cells, operationally defined as those G1 cells unable to leave G1 in the presence of plasma alone, was determined in populations exponentially growing at serum concentrations between 5 and 20%. To do so exponentially growing cultures were shifted to plasma plus colcemid: one part of the cell population progressed through the cycle and accumulated with a G2 DNA content, whereas non-competent cells remained in G1. Analysis of the DNA distributions performed 24 h after the shift showed that as serum concentration was lowered more cells were found in the non-competent state: they were less than 5% in 20% serum and almost 50% in 5% serum. The non-competent cells constitute a dynamic fraction of the population, since in the presence of serum they can leave G1 and progress in the cycle. These data indicate that one of the steps limiting exponential growth is the acquisition of competence and that this event gives rise to heterogeneity within the G1 population.

Dissociation of the ligand and dephosphorylation of the platelet-derived growth factor receptor

The ligand‐induced phosphorylation of the platelet‐derived growth factor (PDGF) receptor was followed at 37°C by a rapid dephosphorylation which was roughly parallel to the down regulation of the 125I‐PDGF binding sites. At 4°C, when the ligand‐receptor complexes remain associated with the cell surface, the phosphorylated form of the receptor was more stable. However if the ligand was dissociated from the receptor by means of a mild acid wash or a treatment with suramin, the dephosphorylation of the receptor also occurred at a low temperature. These data suggest that, due to the dissociation of the ligand, the kinase activity of the receptor is switched off so that the phosphotyrosine‐containing receptors remain exposed to the action of phosphatases that rapidly dephosphorylate them.

Reduction of ribosome activity and synthesis of stable RNA in Neurospora crassa

The addition of cycloheximide (0.02 micrograms/ml) to exponentially growing cultures of Neurospora crassa causes a reduction in growth rate and a decrease in the rate of protein accumulation, due to a partial inhibition of protein synthesis, while RNA accumulation is unaffected for about 1 h. Thus, an increased RNA:protein ratio is established in the presence of the inhibitor. RNA that accumulates during treatment with cycloheximide has the same characteristics as that of the control cultures and this, together with the enhancement of the relative rate of synthesis of ribosomal proteins induced by cycloheximide, seems to indicate that more mature ribosomes are present in cycloheximide-treated cultures. The endocellular level of several amino acids begins to increase significantly only 60 min after cycloheximide addition. A possible explanation of the stimulation of ribosome production induced by cycloheximide is given in terms of the existence of a feed-back mechanism controlling ribosome synthesis.

Effects of caffeine on RNA and protein synthesis in Neurospora crassa

The addition of caffeine (6 m m ) to exponential cultures of Neurospora crassa brings about an immediate strong inhibition of RNA and protein accumulation that is partially reduced after about 2 h, when exponential growth resumes at a lower growth rate. The rate of protein synthesis per unit of culture is initially inhibited by caffeine, which also causes a transient drop in the level of polysomes. However, with longer treatments, the average ribosomal efficiency reverts to the normal value. At all times of treatment the rates of methylation of both ribosomal RNA and soluble RNA are inhibited by caffeine, the inhibition of the methylation of ribosomal RNA being greater. The rate of uridine incorporation into total RNA (transcription) is strongly depressed by caffeine, but the relative rate of synthesis of polyadenylate-containing RNA [poly(A)+ RNA] is much less inhibited than is that of poly(A)− RNA. This causes a reduction in the level of ribosomes that persists when exponential growth is resumed.

Kinetics of tyrosine phosphorylation and internalization of human EGF receptors overexpressed in NIH 3T3 fibroblasts

Binding of epidermal growth factor (EGF) to cells rapidly induces tyrosine phosphorylation of its receptor which is followed by its internalization and dephosphorylation. The kinetics of these processes differs widely in time from minutes to hours according to cell types. In this paper we analyzed EGF receptor phosphorylation and down-regulation in NIH 3T3 cells transfected with the recombinant hEGF-R cDNA which express 4 X 10(5) receptors/cell. In the presence of EGF receptor phosphorylation reached a maximum after 1 min and was then maintained for about 1 h, while during this time the number of EGF-binding sites was reduced to 40% of the initial number. Detailed analysis of the fate of a population of receptors previously activated and autophosphorylated at 4 degrees C, after warming to 37 degrees C in the absence of the ligand, showed that internalization of the cell surface-associated EGF and dephosphorylation of the receptor were rapid (t1/2 15 min) and followed a similar kinetics. Our data indicate that at any given time only a fraction of the total cell surface receptors is phosphorylated on tyrosine and that dephosphorylation occurs at the cell surface or very rapidly after internalization. In addition the data also suggest that a certain recycling of previously internalized receptors may occur in these cells during EGF treatment.

Further Characterization of CDC25 Mm , a Mammalian Activator of p21ras

By using a cotransfection assay with a ras-responsive fos-luciferase reporter gene, we present evidence that the catalytic domain of CDC25Mm constitutively activates p21ras in mammalian cells in vivo. Using immunopurified CDC25Mm-specific antibodies we show that different protein species are recognized in mouse tissues.

Bombesin stimulates a high affinity GTPase activity in membranes of Swiss 3T3 fibroblasts

Peptides of the bombesin family are mitogenic for Swiss 3T3 fibroblasts and in these cells stimulate the turnover of polyphosphoinositides. Recent studies have suggested that G protein(s) may be involved in the signal transduction pathway triggered by bombesin. In this study we have found and characterized a high affinity GTPase activity stimulated by bombesin in membranes of Swiss 3T3 fibroblasts. Our results support the involvement of a G protein in the response of Swiss 3T3 cells to bombesin.