Biomedical subjects

G I Welsh

Publications and source records attributed to G I Welsh.

25 records · Page 2Linked to original sources

PubMed1994

Wortmannin inhibits the effects of insulin and serum on the activities of glycogen synthase kinase-3 and mitogen-activated protein kinase.

We have previously shown that insulin causes inactivation of glycogen synthase kinase-3 (GSK-3) in Chinese hamster ovary cells over-expressing the human insulin receptor (CHO.T cells). We now show that serum and phorbol ester also cause rapid inactivation of GSK-3, both in CHO.T cells and in the nontransfected parental cell line, CHO.K1 cells. Rapamycin was without effect on the inactivation of GSK-3 by insulin, serum or phorbol ester, indicating that the p70 S6 kinase pathway is not involved. In contrast, wortmannin, a potent inhibitor of phosphatidylinositol 3-kinase, blocked the effects of both insulin and serum on GSK-3 activity, and also substantially reduced the activation of both p90 S6 kinase (by insulin) and mitogen-activated protein (MAP) kinase (by insulin and serum). These findings imply (i) that GSK-3 activity is regulated by a cascade involving MAP kinase and p90 S6 kinase and (ii) that wortmannin affects an early step in the MAP kinase pathway. One can infer from this that GSK-3 may be an important regulatory enzyme for the control of several biosynthetic pathways, key enzymes in which are regulated by GSK-3-mediated phosphorylation. Wortmannin had a smaller effect on the activation of MAP kinase by phorbol ester, indicating that phorbol esters may stimulate MAP kinase by a different or additional mechanism to that employed by insulin or serum. Wortmannin had very little effect on the inactivation of GSK-3 by phorbol ester: possible reasons for this are discussed.

Amino Acid Sequence↗

PubMed1994

Identification of novel phosphorylation sites in the beta-subunit of translation initiation factor eIF-2.

Initiation factor eIF-2 (a trimer of subunits alpha, beta and gamma) attaches the initiator Met-tRNA to the ribosome during the initiation of translation in eukaryotic cells. Both the alpha and beta subunits can be phosphorylated although the sites in the beta-subunit have not previously been fully identified. Here we identify the sites at which eIF-2 beta is phosphorylated in vitro by three well-characterised protein kinases, casein kinase-2 (which phosphorylates serine residues-2 and -67), protein kinase C (serine-13) and cAMP-dependent protein kinase (serine-218). This constitutes an essential prerequisite for studying the phosphorylation of eIF-2 beta in vivo. Indeed, we present evidence that at least one of these sites (serine-67) is phosphorylated in reticulocytes. The major kinase activity against eIF-2 beta in reticulocyte lysates appears in CK-2 and protein phosphatase-2A is the principal enzyme responsible for dephosphorylation of eIF-2 beta phosphorylated by this kinase.

Amino Acid Sequence↗

PubMed1993

Glycogen synthase kinase-3 is rapidly inactivated in response to insulin and phosphorylates eukaryotic initiation factor eIF-2B.

We have studied the control of insulin-regulated protein kinases in Chinese hamster ovary cells transfected with the human insulin receptor (CHO.T cells). Among these enzymes is one that is obtained after chromatography of cell extracts on Mono-S, whose activity is decreased (7.3 +/- 1.9-fold) within 10 min of insulin treatment. This enzyme phosphorylates glycogen synthase and the largest subunit of protein synthesis eukaryotic initiation factor (eIF)-2B (the guanine nucleotide exchange factor). The kinase appears to be glycogen synthase kinase-3 (GSK-3), on the basis of: (1) its ability to phosphorylate a peptide based on the phosphorylation sites for GSK-3 in glycogen synthase, and (2) the finding that the fractions possessing this activity contain immunoreactive GSK-3, whose peak is coincident with that of kinase activity, as judged by immunoblotting using antibodies specific for the alpha- and beta-isoforms of GSK-3. The decrease in kinase activity induced by insulin was reversed by treatment of the column fractions with protein phosphatase-2A. These data indicate that insulin rapidly causes inactivation of GSK-3 and that this is due to phosphorylation of GSK-3. The implications of these findings for the control of glycogen and protein metabolism are discussed.

Amino Acid Sequence↗

PubMed1993

Evidence for a role for protein kinase C in the stimulation of protein synthesis by insulin in swiss 3T3 fibroblasts.

We have examined the role of protein kinase C (PKC) in the stimulation of protein synthesis by insulin using two complementary approaches. In the first, fibroblasts were pretreated with phorbol esters to down-regulate PKC. In these cells, the effects of insulin and of phorbol esters on protein synthesis were completely abolished, although serum still elicited an effect approaching that seen in control cells. Secondly, we used newly developed inhibitors of PKC which, again, blocked the effects of insulin and phorbol esters without greatly reducing the response to serum. Thus PKC apparently plays an important role in the stimulation of translation by insulin.

3T3 Cells↗

PubMed1992

Regulation of protein synthesis in Swiss 3T3 fibroblasts. Rapid activation of the guanine-nucleotide-exchange factor by insulin and growth factors.

Insulin, whole serum, phorbol esters and epidermal growth factor each rapidly stimulate protein synthesis in serum-depleted Swiss 3T3 fibroblasts. The activation of protein synthesis by each of these agents is associated with stimulation of the activity of the guanine-nucleotide-exchange factor (GEF). This protein recycles the initiation factor eIF-2 by promoting exchange of GDP bound to eIF-2 for GTP. Activation of GEF is rapid, becoming maximal within 15 min. The degree of activation of GEF by these stimuli (to greater than 170% of control for insulin, serum or epidermal growth factor; 120% for phorbol dibutyrate) is more than enough to account for their effects on the overall rate of translation. Stimulation of protein synthesis and GEF activity occurs at low nanomolar insulin concentrations, indicating they are mediated through the insulin receptor. The best-characterized mechanism for regulating GEF activity is through changes in the phosphorylation of the smallest subunit of eIF-2 (eIF-2 alpha); however, none of the stimuli studied altered the level of phosphorylation of eIF-2 alpha in Swiss fibroblasts. It seems that direct regulation of GEF activity may be occurring here, and possible mechanisms for this are discussed.

3T3 Cells↗

PubMed1991

Evidence that insulin activates casein kinase 2 in rat epididymal fat-cells and that this may result in the increased phosphorylation of an acid-soluble 22 kDa protein.

Casein kinase 2 activity as measured by phosphorylation of the peptide substrate Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu is increased by about 50% in extracts from insulin-treated epididymal fat-pads or isolated fat-cells after purification by Mono Q chromatography. Insulin acts to increase the Vmax. of the kinase. An acid-soluble protein with an apparent subunit molecular mass of about 22 kDa appears to be a substrate for casein kinase 2. The protein possesses a number of properties in common with the acid-soluble heat-stable 22 kDa protein which exhibits increased phosphorylation in rat adipose tissue exposed to insulin.

Adipose Tissue↗

PubMed1991

Phosphorylation of only serine-51 in protein synthesis initiation factor-2 is associated with inhibition of peptide-chain initiation in reticulocyte lysates.

We have examined the phosphorylation of the alpha-subunit of initiation factor-2 (eIF-2 alpha) in reticulocyte lysates in which translational shut-off was induced by haem-deficiency or by double-stranded RNA. To maximise the phosphorylation of eIF-2 alpha, lysates were supplemented with the broad spectrum phosphatase inhibitor microcystin. Under all conditions tested, serine-51 was the only residue to become labelled. This is consistent with the observation of only two species of eIF-2 alpha in isoelectric focusing/immunoblotting analyses of lysates treated as described above.

Adenosine Triphosphate↗