Effect of 3T3 plasma membranes on cells exposed to epidermal growth factor

Epidermal growth factor (EGF) induced DNA synthesis in non-confluent, G₀-arrested Swiss 3T3 fibroblasts is partially blocked by plasma membranes isolated from the EGF receptor deficient NR-6 Swiss 3T3 cell line. This inhibition could be due to either a steric block of the receptor by the membranes, a membrane induced down regulation of the EGF receptor, or a signal generated by membrane binding which is antagonistic towards the mitogenic signal generated by EGF. Binding measurements utilizing ¹²⁵I-labeled EGF demonstrated that membranes do not block either the EGF induced down regulation of the receptor or alter the number of receptors on the surface. These results suggest that the membranes exert their inhibitory effect via generation of a signal which is antagonistic to the EGF induced mitogenic signal, with the result expressed as a reduced mitogenic response.     

Protein folding pathways of adenylate kinase from E. coli: hydrostatic pressure and stopped-flow studies.

Adenylate kinase (AKe) from E. coli is a small, single-chain, monomeric enzyme with no tryptophan and a single cysteine residue. We have constructed six single-Trp mutants of AKe to facilitate optical studies of these proteins and to specifically examine the interrelationship between their structure, function, dynamics, and folding reactions. In this study, the effects of hydrostatic pressure on the folding reactions of AKe were studied. The native structure of AKe was transformed to a non-native, yet pressure stable, conformation by hydrostatic pressure of about 300 MPa. This pressure lability of AKe is rather low for a monomeric protein and presumably may be attributed to substantial conformational flexibility and a correspondingly large volume change. The refolding of AKe after pressure-induced denaturation was reversible under ambient conditions. At low temperature (near 0 degrees C), the refolding process of pressure-exposed AKe mutants displayed a significant hysteresis. The observation of a slow refolding rate in the 193 region and a faster folding rate around the active site (86, 41, 73 regions) leads us to suggest that in the folding process, priority is afforded to functional regions. The slow structural return of the 193 region apparently does not hinder the more rapid return of enzymatic activity of AKe. Circular dichroism studies on the pressure-denatured Y193W mutant show that the secondary structure (calculated from far-UV spectra) returned at a rapid rate, but the tertiary structure alignment (calculated from near-UV spectra) around the 193 region occurred more slowly at rates comparable to those detected by fluorescence intensity. Denaturation of AKe mutants by guanidine hydrochloride and subsequent refolding experiments were also consistent with a much slower refolding process around the 193 region than near the active site. Fast refolding kinetic traces were observed in F86W, S41W, and A73W mutants using a fluorescence detection stopped-flow rapid mixing device, while only a slow kinetic trace was observed for Y193W. The results suggest that the differences in regional folding rates of AKe are not derived from the specific denaturation methods, but rather are inherent in the structural organization of the protein.     

Amino-terminal processing of proteins by N-myristoylation. Substrate specificity of N-myristoyl transferase

Using synthetic octapeptides, we examined the amino-terminal sequence requirements for substrate recognition by myristoyl-CoA:protein N-myristoyl transferase (NMT). NMT is absolutely specific for peptides with amino-terminal Gly residues. Peptides with Asn, Gln, Ser, Val, or Leu penultimate to the amino-terminal Gly were substrates, whereas peptides with Asp, D-Asn, Phe, or Tyr at this position were not myristoylated. Peptides with aromatic residues at this position competitively inhibited myristoylation of substrates, introducing the possibility of developing specific in vivo inhibitors of NMT. Peptides having sequences which correspond to those of known N-myristoyl proteins, including p60src, appear to be recognized by a single enzyme, and yeast and murine NMT have identical substrate specificities. The catalytic selectivity of NMT for myristoyl transfer accounts for the remarkable acyl chain specificity of this enzyme.     

Control of muscle differentiation in BC3H1 cells by fibroblast growth factor and vanadate

We have examined the control of actin isoform synthesis by pituitary-derived fibroblast growth factor and serum in BC3H1 cells, a tumor-derived nonfusing muscle cell line. Under differentiating conditions in BC3H1 cells, the synthesis of beta- and gamma-actin ceases, and the rate of alpha-actin synthesis is increased concomitant with cessation of cell growth. Addition of fetal calf serum to differentiated cells reverses the process, whereas the addition of pituitary-derived fibroblast growth factor inhibits synthesis of alpha-actin but fails to induce the synthesis of beta- and gamma-actin. Analysis of RNA from differentiated BC3H1 cells after the addition of fetal calf serum indicated that the serum-induced increase in beta- and gamma-actin synthesis reflected an increase in their mRNA levels. In contrast, the repression of alpha-actin synthesis by fetal calf serum or fibroblast growth factor appears to reflect the translation efficiency of alpha-actin mRNA. Fibroblast growth factor is a competence factor for BC3H1 cells which allows them to progress from G0 4 h into the G1 phase of the cell cycle. In order to understand the nature of the intracellular signals responsible for the effect of fibroblast growth factor, we treated cells with vanadate, a known inhibitor of tyrosine-specific protein phosphatases. Vanadate fully mimics the action of fibroblast growth on actin synthesis and creatine phosphokinase synthesis and causes BC3H1 cells to exit the G0 portion of the cell cycle, as demonstrated by the induction of the c-fos proto-oncogene following addition of serum, vanadate, or bovine pituitary-derived fibroblast growth factor to these cells. We conclude that repression of alpha-actin synthesis and induction of the synthesis of beta- and gamma-actin are under independent control and that the induction of beta- and gamma-nonmuscle actin synthesis following serum addition is independent from movement into the cell cycle, and dependent on as yet unidentified serum components. The rate of synthesis of alpha-actin can be controlled by a defined mitogenic polypeptide fibroblast growth factor, which in short term experiments primarily affects the rate of translation of alpha-actin mRNA. The repression by fibroblast growth factor is most likely due to activation of a tyrosine specific protein kinase(s).