Regulation of FRTL-5 thyroid cell growth by phosphatidylinositol (OH) 3 kinase-dependent Akt-mediated signaling.

Thyrotropin (TSH)-initiated cell cycle progression from G1 to S phase in FRTL-5 thyroid cells requires serum, insulin, or insulin-like growth factor 1 (IGF-1) and involves activation of 3-hydroxy-3-methylglutaryl-CoA reductase, geranylgeranylation of RhoA, p27Kip1 degradation, and activation of cyclin-dependent kinase (cdk) 2. In the present report, we show that the serine-threonine kinase Akt is an important mediator of insulin/IGF-1/serum effects on cell cycle progression in FRTL-5 thyroid cells. The phosphoinositol (OH) 3 kinase inhibitors, Wortmannin (WM) and Ly294002 (LY), block the ability of insulin/IGF-1 to reduce p27 expression, to induce expression of cyclins E, D1, and A as well as cdk 2 and 4, and to phosphorylate retinoblastoma protein. They also inhibit insulin/IGF-1-increased DNA synthesis and cell cycle entrance (S+G2/M). Insulin/IGF-1 rapidly induced activation of Aktl in a PI3 kinase-dependent manner, and increased Aktl RNA levels. Most importantly, FRTL-5 cells transfected with a constitutively active form of Aktl have higher basal rates of DNA synthesis and no longer require exogenous insulin/IGF-1 or serum for TSH-induced growth. In sum, Aktl appears to have an important role in insulin/IGF-1 regulation of FRTL-5 thyroid cell growth and cell cycle progression.

Oncogenic transformation of cells by a conditionally active form of the protein kinase Akt/PKB.

The Akt/PKB protein kinase is implicated in the control of cell cycle progression and the suppression of apoptosis in cancer cells. Here we describe the use of a conditionally active form of Akt/PKB (M+ Akt:ER*) to study the ability of this protein to influence biological processes that are central to the process of oncogenic transformation of mammalian cells. Activation of M+ Akt:ER* in Rat1 cells elicited alterations in cell morphology and promoted anchorage-independent growth in agarose with high efficiency. Consistent with these observations, activation of M+ Akt:ER* suppressed the apoptosis of Rat1 cells that occurs after the detachment of these cells from extracellular matrix. Furthermore, activation of M+ Akt:ER* was sufficient to promote the progression of quiescent Rat1 cells into the S and G2-M phases of the cell cycle. In accord with this is the observation that activation of M+ Akt:ER* led to decreased expression of the cyclin-dependent kinase inhibitor p27Kip1 with a concomitant increase in cyclin-dependent kinase-2 activity. Perhaps surprisingly, activation of M+ Akt:ER* or expression of a constitutively active form of Akt led to rapid activation of MAP/ERK Kinase (MEK) and the extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinases in Rat1 cells. However, pharmacological inhibition of MEK by PD098059 did not inhibit the morphological alterations of Rat1 cells that occur after M+ Akt:ER* activation. These data suggest that M+ Akt:ER* can activate a number of pathways in Rat1 cells, leading to significant alterations in a number of biological processes. The conditional transformation system described here will allow further elucidation of the ability of Akt to contribute to both the normal response of cells to mitogenic stimulation and the aberrant proliferation observed in cancer cells.

The role of glycogen synthase kinase 3beta in insulin-stimulated glucose metabolism.

To characterize the contribution of glycogen synthase kinase 3beta (GSK3beta) inactivation to insulin-stimulated glucose metabolism, wild-type (WT-GSK), catalytically inactive (KM-GSK), and uninhibitable (S9A-GSK) forms of GSK3beta were expressed in insulin-responsive 3T3-L1 adipocytes using adenovirus technology. WT-GSK, but not KM-GSK, reduced basal and insulin-stimulated glycogen synthase activity without affecting the -fold stimulation of the enzyme by insulin. S9A-GSK similarly decreased cellular glycogen synthase activity, but also partially blocked insulin stimulation of the enzyme. S9A-GSK expression also markedly inhibited insulin stimulation of IRS-1-associated phosphatidylinositol 3-kinase activity, but only weakly inhibited insulin-stimulated Akt/PKB phosphorylation and glucose uptake, with no effect on GLUT4 translocation. To further evaluate the role of GSK3beta in insulin signaling, the GSK3beta inhibitor lithium was used to mimic the consequences of insulin-stimulated GSK3beta inactivation. Although lithium stimulated the incorporation of glucose into glycogen and glycogen synthase enzyme activity, the inhibitor was without effect on GLUT4 translocation and pp70 S6 kinase. Lithium stimulation of glycogen synthesis was insensitive to wortmannin, which is consistent with its acting directly on GSK3beta downstream of phosphatidylinositol 3-kinase. These data support the hypothesis that GSK3beta contributes to insulin regulation of glycogen synthesis, but is not responsible for the increase in glucose transport.

Protein kinase B/akt and rab5 mediate Ras activation of endocytosis.

Transient expression of oncogenic Ha-Ras (Ras:V12) stimulates endocytosis. Using NIH3T3 cells expressing constitutively active protein kinase B/akt (PKB/akt) or kinase-dead PKB/akt, we show that PKB/akt mediates the stimulatory effect of Ras on endocytosis. Fluid phase endocytosis of horseradish peroxidase in cells expressing the constitutively active form of PKB/akt was elevated and insensitive to phosphatidylinositol 3-kinase inhibitors. However, expression of dominant negative Rab5:N34 blocked endocytosis in cells expressing the constitutively active form of PKB/akt. Transient expression of either Rab5:wt or Rab5:L79, a GTPase deficient mutant of Rab5, in cells expressing constitutively activated PKB/akt further increased endocytic rate. However, in cells expressing kinase-dead PKB/akt, endocytic rate was not affected by transient expression of Rab5:wt. Rab5:L79, on the other hand, increased endocytosis in cells expressing kinase-dead PKB/akt. Similar results were obtained using an in vitro endosome fusion reconstitution assay with cytosol prepared from cells expressing the activated PKB/akt or kinase-dead PKB/akt. Both Rab5:wt and Rab5:L79 stimulated endosome fusion when assayed in cytosol containing the activated PKB/akt, whereas only Rab5:L79 activated fusion when the assay utilized cytosol from kinase-dead expressing cells. We conclude that Ras activation of endocytosis requires both PKB/akt and Rab5 and that active kinase is required for activation Rab5.

Construction and characterization of a conditionally active version of the serine/threonine kinase Akt.

Akt is a serine/threonine kinase that requires a functional phosphatidylinositol 3-kinase to be stimulated by insulin and other growth factors. When directed to membranes by the addition of a src myristoylation sequence, Akt becomes constitutively active. In the present study, a conditionally active version of Akt was constructed by fusing the Akt containing the myristoylation sequence to the hormone binding domain of a mutant murine estrogen receptor that selectively binds 4-hydroxytamoxifen. The chimeric protein was expressed in NIH3T3 cells and was shown to be stimulated by hormone treatment 17-fold after only a 20-min treatment. This hormone treatment also stimulated an approximate 3-fold increase in the phosphorylation of the chimeric protein and a shift in its migration on SDS gels. Activation of this conditionally active Akt resulted in the rapid stimulation of the 70-kDa S6 kinase. This conditionally active Akt was also found to rapidly stimulate in these cells the phosphorylation of properties of PHAS-I, a key protein in the regulation of protein synthesis. The conditionally active Akt, when expressed in 3T3-L1 adipocytes, was also stimulated, although its rate and extent of activation was less then in the NIH3T3 cells. Its stimulation was shown to be capable of inducing glucose uptake into adipocytes by stimulating translocation of the insulin-responsive glucose transporter GLUT4 to the plasma membrane.

Evidence of insulin-stimulated phosphorylation and activation of the mammalian target of rapamycin mediated by a protein kinase B signaling pathway.

The effects of insulin on the mammalian target of rapamycin, mTOR, were investigated in 3T3-L1 adipocytes. mTOR protein kinase activity was measured in immune complex assays with recombinant PHAS-I as substrate. Insulin-stimulated kinase activity was clearly observed when immunoprecipitations were conducted with the mTOR antibody, mTAb2. Insulin also increased by severalfold the 32P content of mTOR that was determined after purifying the protein from 32P-labeled adipocytes with rapamycin.FKBP12 agarose beads. Insulin affected neither the amount of mTOR immunoprecipitated nor the amount of mTOR detected by immunoblotting with mTAb2. However, the hormone markedly decreased the reactivity of mTOR with mTAb1, an antibody that activates the mTOR protein kinase. The effects of insulin on increasing mTOR protein kinase activity and on decreasing mTAb1 reactivity were abolished by incubating mTOR with protein phosphatase 1. Interestingly, the epitope for mTAb1 is located near the COOH terminus of mTOR in a 20-amino acid region that includes consensus sites for phosphorylation by protein kinase B (PKB). Experiments were performed in MER-Akt cells to investigate the role of PKB in controlling mTOR. These cells express a PKB-mutant estrogen receptor fusion protein that is activated when the cells are exposed to 4-hydroxytamoxifen. Activating PKB with 4-hydroxytamoxifen mimicked insulin by decreasing mTOR reactivity with mTAb1 and by increasing the PHAS-I kinase activity of mTOR. Our findings support the conclusion that insulin activates mTOR by promoting phosphorylation of the protein via a signaling pathway that contains PKB.

A constitutively active version of the Ser/Thr kinase Akt induces production of the ob gene product, leptin, in 3T3-L1 adipocytes.

The expression of the ob gene product leptin in adipose tissues has been previously described to be regulated by insulin in vivo and vitro. Akt, a ser/thr kinase with a pleckstrin homology domain, has recently been identified to function in the insulin receptor signaling cascade. The aim of this study was to investigate the role of Akt in the production of leptin by adipocytes. Therefore, we examined leptin production by 3T3-L1 adipocytes stably expressing a myristoylated version of Akt which is constitutively active. Leptin levels in the supernatants of serum starved, nonstimulated 3T3-L1 adipocytes were determined by radioimmunoassay (RIA). Expression of the constitutively active Akt was found to induce a more than 20-fold increase in leptin levels whereas a control non-myristoylated Akt had no effect. Leptin mRNA levels as determined by either RNase protection assay or reverse transcriptase (RT)-polymerase chain reaction (PCR) were not elevated by the constitutively active Akt. These results indicate that Akt can induce leptin production in 3T3-L1 adipocytes via a non-transcriptional mechanism.

Expression of a constitutively active Akt Ser/Thr kinase in 3T3-L1 adipocytes stimulates glucose uptake and glucose transporter 4 translocation.

Akt is a serine/threonine kinase that requires a functional phosphatidylinositol 3-kinase to be stimulated by insulin and other growth factors. When directed to membranes by the addition of a src myristoylation sequence, Akt becomes constitutively active. In the present studies, the constitutively active Akt and a nonmyristoylated control mutant were expressed in 3T3-L1 cells that can be induced to differentiate into adipocytes. The constitutively active Akt induced glucose uptake into adipocytes in the absence of insulin by stimulating translocation of the insulin-responsive glucose transporter 4 to the plasma membrane. The constitutively active Akt also increased the synthesis of the ubiquitously expressed glucose transporter 1. The increased glucose influx in the 3T3-L1 adipocytes directed lipid but not glycogen synthesis. These results indicate that Akt can regulate glucose uptake and metabolism.

Akt, a pleckstrin homology domain containing kinase, is activated primarily by phosphorylation.

Akt is a serine/threonine kinase that is stimulated by receptor tyrosine kinases and contains a pleckstrin homology domain. One model proposed to explain this activation suggests that receptor tyrosine kinases stimulate a phosphatidylinositol 3-kinase whose lipid products directly activate Akt kinase by interacting with its pleckstrin homology domain. In the present study, we show, in three cell types, that Akt does not require its pleckstrin homology domain to respond to either insulin or platelet-derived growth factor. Moreover, attachment of the src myristoylation signal to target Akt, without its pleckstrin homology domain, to the membrane constitutively activates Akt by causing an increase in its basal level of phosphorylation. This constitutively active form of Akt can also activate p70(S6K), indicating that the pleckstrin homology domain is not necessary for downstream interactions. Fusion of the inter src homology 2 domain from the p85 regulatory subunit of the phosphatidylinositol 3-kinase to Akt also constitutively activated Akt and induced an association with the lipid kinase. Phosphorylation of this fusion protein still critically contributes toward its increased activity. The sum of these results indicates that the primary mechanism of Akt activation is via protein phosphorylation.

Insulin stimulates the kinase activity of RAC-PK, a pleckstrin homology domain containing ser/thr kinase.

In the present study, insulin is shown to rapidly stimulate by 8- to 12-fold the enzymatic activity of RAC-PK alpha, a pleckstrin homology domain containing ser/thr kinase. In contrast, activation of protein kinase C by phorbol esters had almost no effect on the enzymatic activity of RAC-PK alpha. Insulin activation was accompanied by a shift in molecular weight of the RAC-PK alpha protein, and the activated kinase was deactivated by treatment with a phosphatase, indicating that insulin activated the enzyme by stimulating its phosphorylation. This insulin-induced shift in RAC-PK was also observed in primary rat epididymal adipocytes, as well as in a muscle cell line called C2C12 cells. The insulin-stimulated increase in RAC-PK alpha activity was inhibited by wortmannin (an inhibitor of phosphatidylinositol 3-kinase) in a dose-dependent manner with a half-maximal inhibition of 10 nM, but not by 20 ng/ml of rapamycin. Activation of RAC-PK alpha activity was also observed in a variant RAC lacking the pleckstrin homology domain. These results indicate that RAC-PK alpha activity can be regulated by the insulin receptor. RAC-PK alpha may therefore play a general role in intracellular signaling mediated by receptor tyrosine kinases.

Photoaffinity labeling of lysosomal membrane proteins with [125I]diiodotyrosine, a system h ligand.

Percoll-purified rat thyroid FRTL-5 cell lysosomes were photoaffinity-labeled with [125I]diiodotyrosine to identify proteins which bind diiodotyrosine, a ligand for lysosomal transport system h. SDS-PAGE and autoradiography of these membranes showed specific labeling of a 70-kDa protein and weak labeling of three smaller proteins. [125I]Diiodotyrosine photolabeling of the 70-kDa protein was specifically competed against by ligands of lysosomal transport system h ligands. The 70-kDa protein was photolabeled more strongly in lysosomal membranes isolated from thyrotropin-stimulated cells when compared with those grown in the absence of thyrotropin, consistent with previous demonstrations that thyrotropin stimulates system h transport. The 70-kDa protein may represent some portion of the system h carrier protein.

Thyrotropin regulation of malic enzyme in FRTL-5 rat thyroid cells.

TSH-induced increases in malic enzyme mRNA levels in FRTL-5 rat thyroid cells are paralleled by increases in malic enzyme activity and are mimicked by 8-bromo-cAMP. Apparent approximately 4 h after TSH challenge and maximal after 16 h, they decline by 24 h and are at basal levels by 48 h. The increase occurs in the absence of a measurable effect of TSH on DNA synthesis related to cell growth, since [3H] thymidine incorporation into DNA is still at basal levels 24 h after TSH challenge and is maximal only at 48 h. A protein(s) whose formation is inhibited by cycloheximide appears to be critical to the ability of TSH to increase malic enzyme mRNA levels. Thus, cycloheximide given 30 min before TSH prevents the hormone-induced increase in malic enzyme mRNA; also, when given 24 h after TSH, cycloheximide accelerates the loss of the TSH-induced increase in malic enzyme mRNA. In neither case does cycloheximide affect beta-actin mRNA levels. A second factor(s) whose formation is prevented by actinomycin-D appears to be important for the decrease in malic enzyme mRNA levels seen 24 and 48 h after TSH challenge. Thus, in experiments in which it is given 24 h after TSH, actinomycin-D preserves the hormone-induced increase in malic enzyme mRNA levels rather than accelerating the decrease, as does cycloheximide. In the same experiment, beta-actin mRNA levels decrease to less than 10-20% of control values over the same period; this factor also, therefore, appears to exhibit some degree of specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyrotropin increases malic enzyme messenger ribonucleic acid levels in rat FRTL-5 thyroid cells.

The addition of TSH to FRTL-5 thyroid cells induces a 7- to 8-fold increase in the steady state level of malic enzyme [L-malate-NADP+ oxidoreductase (decarboxylating); EC 1.1.1.40] mRNA, but does not alter beta-actin mRNA levels. Insulin alone or together with TSH has no effect on malic enzyme mRNA. The effect of TSH is not the result of thyroid hormone formation, since the addition of T3 in the presence or in the absence of TSH and the addition of 5% serum (which includes T3 and T4) have no effect. Forskolin (10(-6) M) reproduces the TSH effect, suggesting that cAMP is involved.

Carrier-mediated transport of monoiodotyrosine out of thyroid cell lysosomes.

Monoiodotyrosine (MIT) crosses the lysosomal membrane of rat FRTL-5 thyroid cells by a carrier-mediated process. In egress studies, MIT lost from inside lysosomes was quantitatively recovered outside lysosomes as MIT, indicating that the compound was transported intact across the lysosomal membrane. In uptake studies, [125I]MIT entry required intact lysosomes and exhibited saturation kinetics. The apparent Km for MIT was approximately 1.5 microM and the Vmax was approximately 0.24 pmol/unit hexosaminidase/min. Countertransport of MIT was demonstrated, with an initial velocity of [125I]MIT uptake which reached a maximum at high intralysosomal MIT loading. Nonradioactive MIT and diiodotyrosine competed to approximately equivalent extents with [125I]MIT for uptake in countertransport experiments. The existence of a lysosomal MIT carrier in thyroid cells may explain how this product of thyroglobulin catabolism is transported to the cytosol for iodine salvage and reutilization.

Receptors of the thyroid: the thyrotropin receptor is only the first violinist of a symphony orchestra.

A basic reason for undertaking these studies was to further our knowledge of the structure and function of the TSH receptor as well as its interaction with other receptors on thyroid cells. The multiplicity of observations suggests the approach is bearing fruit, does not provide a simple answer, and can have pitfalls. We hope they may also contribute to understanding the structure and function of autoantigens in Graves' disease and glycoprotein hormone receptors in general. The authors are grateful to their collaborators in the National Dental Institute, particularly Drs. Bellur Prabhakar, Edward Oates, and Abner Notkins, in the National Cancer Institute, Drs. W. O. McBride and M. Lerman for their contributions to the cloning studies.

Monoclonal antibody studies defining the origin and properties of autoantibodies in Graves' disease.

The present report summarizes experiments with monoclonal antibodies to the TSH receptor. The data provide further insight into the TSH receptor structure and into the basis of autoimmune antibodies implicated in the pathogenesis of Graves' disease. They resolve many clinical questions and provide new approaches to enhance our understanding of autoimmune disease. In one new approach, it has been noted that the 11E8 TBIAb can precipitate the phosphorylated beta subunit of the insulin and IGF1 receptor. This cross-reactivity or recognition of determinants adjacent to the TSH receptor may not be random. Insulin, IGF1, alpha 1 adrenergic, and TSH receptors have been linked to a synergistic cascade response system of the thyroid involving growth, thyroglobulin biosynthesis, iodination of thyroglobulin, and thyroid hormone formation. Future studies with the monoclonals may help unravel this cascade system and its regulatory relationships, along with the relationships between autoimmune thyroid disease and autoimmune diseases of other organs.