Ora M. Rosen

Purification of a bovine liver S6 kinase

A bovine liver protein serine kinase that catalyzes the multisite phosphorylation of ribosomal protein S6 has been purified to near homogeneity. The enzyme has an Mr of 67,000 on SDS-polyacrylamide gel electrophoresis and an apparent molecular weight of 55,000 on glycerol gradient sedimentation. Its enzymic properties, substrate specificity, molecular size and chromatographic behaviour are similar to those of the principal growth factor--and phorbol 12-myristate 13-acetate-stimulated S6 kinase of cultured cells.

Hydrolysis of Glycosyl-Phosphatidylinositol in Response to Insulin is Reduced in Cells Bearing Kinase-Deficient Insulin Receptors

A glycosyl-phosphatidylinositol (GPI) has been previously identified that serves as a precursor of the polar head group that mimics and may mediate some of the intracellular actions of insulin. Since many of the biological activities of insulin may depend upon the activity of the insulin receptor kinase, we evaluated the requirement for this activity in insulin-dependent GPI hydrolysis. For the analysis we used stably transfected CHO cell lines, expressing either the wild-type human insulin receptor or a mutant receptor that lacks tyrosine kinase activity (Chou et al., 1987) and a stably transfected CHO cell line, expressing the wild-type human insulin-like growth factor I (IGF-1) receptor (Steele-Perkins et al., 1988). A GPI was identified in both types of transfected cells and in both sets of parental cells by metabolic labeling with [3H]glucosamine or [3H]galactose. The isolated glycolipid was sensitive to hydrolysis by phospholipase C and to deamination by nitrous acid. Insulin induced a time- and dose-dependent hydrolysis of the GPI in the parental line and in the transfected cell types. Cells bearing normal human receptors hydrolyzed up to 70% of their radiolabeled GPI within 2 min of the addition of 0.1 nM insulin, whereas parental cells and cells expressing the mutant receptor hydrolyzed only 20-30% in response to 100 nM insulin. IGF-1 (5-50 nM) had little effect on GPI hydrolysis in these cells as well as in CHO cells expressing the human IGF-1 receptor. It is concluded that insulin-dependent GPI hydrolysis is mediated by the normal but not by a kinase-deficient insulin receptor.

Polylysine activates and alters the divalent cation requirements of the insulin receptor protein tyrosine kinase

Protamine and poly(Lys) activate the protein tyrosine kinase of both the human placental insulin receptor and its purified recombinant cytoplasmic domain. Spermidine, poly(Arg) (average molecular mass 15 kDa), poly(Glu), Arg or Lys are not effective. Activation is stable, reversible, and optimal when the enzyme is preincubated with activator, divalent cation and ATP prior to the addition of exogenous protein substrates. The most striking feature of the activation is that it results in 20–30‐fold stimulation of the kinase in the presence of 0.2–0.4 mM Mn2+ and induces equivalent activity in the presence of Mg2+ alone (0.4–4.0 mM). The activated protein tyrosine kinase has a specific activity (0.25–0.5 μmol/mg protein) that approaches that of well characterized protein serine kinases.

A possible role for a mammalian facilitative hexose transporter in the development of resistance to drugs.

We show that D- but not L-hexoses modulate the accumulation of radioactive vinblastine in injected Xenopus laevis oocytes expressing the murine Mdr1b P-glycoprotein. We also show that X. laevis oocytes injected with RNA encoding the rat erythroid/brain glucose transport protein (GLUT1) and expressing the corresponding functional transporter exhibit a lower accumulation of [3H]vinblastine and show a greater capacity to extrude the drug than do control oocytes not expressing the rat GLUT1 protein. Cytochalasin B and phloretin, two inhibitors of the mammalian facilitative glucose transporters, can overcome the reduced drug accumulation conferred by expression of the rat GLUT1 protein in Xenopus oocytes but have no significant effect on the accumulation of drug by Xenopus oocytes expressing the mouse Mdr1b P-glycoprotein. These drugs also increase the accumulation of [3H]vinblastine in multidrug-resistant Chinese hamster ovary cells. Cytochalasin E, an analog of cytochalasin B that does not affect the activity of the facilitative glucose transporter, has no effect on the accumulation of vinblastine by multidrug-resistant Chinese hamster cells or by oocytes expressing either the mouse Mdr1b P-glycoprotein or the GLUT1 protein. In all three cases, the drug verapamil produces a profound effect on the cellular accumulation of vinblastine. Interestingly, although immunological analysis indicated the presence of massive amounts of P-glycoprotein in the multidrug-resistant cells, immunological and functional studies revealed only a minor increase in the expression of a hexose transporter-like protein in resistant versus drug-sensitive cells. Taken together, these results suggest the participation of the mammalian facilitative glucose transporter in the development of drug resistance.

Insulin-Receptor Approaches to Studying Protein Kinase Domain

Protein tyrosine kinase activity found in the β-subunit of the insulin receptor provides a mechanism by which insulin binding on the outside of the cell transmits its signal across the plasma membrane into the cytosol. The autophosphorylation of the insulin receptor on tyrosyl residues activates the intrinsic tyrosine kinase of the receptor; rendering its ligand independent. Evidence suggests that phosphorylation of tyrosyl residues 1146, 1150, and 1151 in the kinase domain of the β-subunit play a role in activation. Point mutations in the cytoplasmic portion of the β-subunit confirm the above suggestions and indicate that additional sites are important for receptor function. We present methodology for overproducing the cytoplasmic domain of the receptor in the Baculovirus expression system. The protein, produced in insect cells and larvae, is soluble and fully active on autophosphorylation. Like the intact receptor, its autophosphorylation is intramolecular. Because >10 mg of pure protein can be isolated from 1010 insect cells infected with the recombinant Baculovirus encoding the human insulinreceptor kinase domain, sufficient enzyme is available for various studies, including physicochemical analysis. Isolation of β-subunit defects found in the receptors of patients with various forms of diabetes mellitus also implicates the insulin-receptor kinase in insulin action. Finally, α potential model system for the genetic analysis of the insulin-insulin-receptor system with Drosophila melanogaster is noted. Conservation of the deduced amino acid sequence for both α- and β-subunit sequences between humans and insects highlights the significance of this manner of signal transduction throughout nearly 1 billion years of evolution.