Insulin-Receptor Tyrosine Kinase and Glucose Transport
We identified the earliest events in autophosphorylation of the insulin receptor after insulin addition. Insulinstimulated autophosphorylation at specific sites in the tyrosine kinase domain of the receptor's (5-subunit is correlated kinetically with activation of kinase-catalyzed phosphorylation of a model substrate (reduced and carboxyamidomethylated lysozyme; RCAM-lysozyme). To identify these sites, the deduced amino acid sequence of the 3T3-L1 adipocyte insulin receptor of the mouse was determined. Insulin-induced activation of substrate phosphorylation was shown to require autophosphorylation of three neighboring tyrosines (Tyr1148, Tyr1152, and Tyr1153) in the mouse receptor. A search for cellular substrates of the receptor kinase revealed that insulin causes accumulation of a 15,000-A4r phosphorylated (on tyrosine) cytosolic protein (pp15) in 3T3-L1 adipocytes treated with oxophenylarsine (PAO). PAO blocks turnover of the phosphoryl group of pp15, causing its accumulation, and thereby appears to interrupt signal transmission from the receptor to the glucose-transport system. Two membrane-bound protein phosphotyrosine phosphatases that are inhibited by PAO and are apparently responsible for the turnover of the pp15 phosphoryl group have been purified from 3T3-L1 adipocytes and characterized. These and other results support the hypothesis that turnover of the phosphoryl group of pp15, a product of insulin-receptor tyrosine kinase action, couples signal transmission to the glucose-transport system. [32P]pp15 was purified to homogeneity from 3T3-L1 adipocytes. Amino acid and radiochemical sequence analysis of the purified tryptic [32P]phosphopeptide revealed that pp15 is the phosphorylation product of 422(aP2) protein, a 15,000-/Vfr
From the Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland. Address correspondence and reprint requests to Dr. M. Daniel Lane, Department of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205.
DIABETES CARE, VOL. 13, NO. 6, JUNE 1990
M. Daniel Lane, PhD Jaime R. Flores-Riveros, PhD Richard C. Hresko, PhD Klaus H. Kaestner, MS Kan Liao, BS Michel Janicot, PhD Robert D. Hoffman, PhD John C. McLenithan, BS Tania Kastelic, MS Robert J. Christy, PhD
adipocyte protein whose cDNA we previously cloned and sequenced. 422(aP2) protein was found to bind fatty acids. When exposed to a free fatty acid, notably oleic acid, 422(aP2) protein becomes an excellent substrate of the isolated insulin-receptor tyrosine kinase. Compelling evidence indicates that on binding fatty acid, 422(aP2) protein undergoes a conformational change whereby Tyr19 becomes accessible to the receptor tyrosine kinase and undergoes O-phosphorylation. Adipose tissue and skeletal and heart muscle, which exhibit insulinstimulated glucose uptake, express a specific insulinresponsive glucose transporter. A cDNA (GT2) that encodes this protein was isolated from a mouse 3T3-L1 adipocyte library and sequenced. We also isolated and characterized the corresponding mouse gene GLUT4. DNase I footprinting with nuclear extracts from 3T3-L1 cells revealed that a differentiation-specific nuclear factor binds to the GLUT4 promoter. The purified transcription factor C/EBP binds at the same position. Transient cotransfection into 3T3-L1 preadipocytes of a chimeric GLUT4 promoter-chloramphenicol acetyltransferase gene and a C/EBP expression vector revealed that C/EBP frans-activates the GLUT4 promoter. We suggest that C/EBP plays an important role in tissue-specific and metabolic regulation of the insulinresponsive glucose-transporter gene. Diabetes Care 13:565-75, 1990
cute activation of glucose uptake by animal tissues is perhaps the most widely recognized function of insulin (1). This action of insulin is limited primarily to two major cell types, i.e., muscle cells and adipocytes. Thus, in both type I (insulin-dependent) and type II (non-insulin-dependent) diabetes mellitus, impaired glucose uptake by muscle and adipose tissue and accelerated hepatic gluconeogenesis
A
565
INSULIN-ACTIVATED GLUCOSE UPTAKE
account for the accompanying hyperglycemia (2). Inefficient disposal of glucose by peripheral tissues can result either from an absolute deficiency of insulin (as in type I diabetes) or from the resistance of target cells to insulin (as in type II diabetes). Resistance of cells to insulin appears to have a complex etiology caused by a decrease in the number of functional cell-surface insulin receptors on target cells (3), by a defect in signal transduction by the receptor (4), or as recent evidence suggests, by impaired expression of the insulin-responsive glucose transporter (5-7). The ability to take up glucose is a fundamental property of all animal cells and is mediated by a family of eel I-type-specific glucose-transporter proteins (1,8). Recently, the cDNAs encoding several of these glucose transporters have been isolated, sequenced, and shown to be expressed in a tissue-specific manner (9-14). Another glucose-transporter cDNA, unique to cells that exhibit insulin-stimulated glucose uptake (adipocytes and skeletal and heart muscle cells), was recently cloned in our (10) and several other laboratories (15-18). The protein encoded by these cDNAs exhibits the properties of the insulin-responsive glucose transporter or GLUT4 by the nomenclature of Bell et al. (8) and Gould and Bell (19). More recently, the cloning and characterization of the mouse GLUT4 gene and the trans-activation of its promoter by nuclear transcription factor C/EBP have been reported (20). It is possible that expression of this gene is altered in certain insulin-resistant states. In this article, we review primarily the work done in our laboratory on insulin-receptor tyrosine kinase and the glucose-transport system of the mouse 3T3-L1 adipocyte. The 3T3-L1 adipocyte is an excellent model cell type that is extraordinarily responsive to insulin and with which insulin action can be studied under the controlled conditions of cell culture.
3T3-L1 ADIPOCYTE MODEL The role of adipose tissue is to store energy in the form of triacylglycerol during periods of nutritional abundance and to mobilize this store for use during periods of nutritional deficit. To perform these functions, the adipocyte is uniquely endowed with a complement of enzymes and transport systems that are exquisitely responsive to hormones such as insulin. Although adipocytes isolated from adipose tissue are suitable for certain types of acute experiments, they are generally unsuitable for longer-term experiments. An ideal model for the adipocyte and its development that has proved useful for both acute and long-term studies on insulin action is the 3T3-L1 preadipocyte/adipocyte system (10,21-23). When fully differentiated, the 3T3-L1 adipocyte is extraordinarily responsive to insulin (10,21). In the pathway of development leading to the permanently differentiated adipocyte, successive steps of determination narrow developmental options until a single pathway (i.e., differentiation of the preadipo-
566
cyte to an adipocyte) remains. Under appropriate conditions, confluent 3T3-L1 preadipocytes in culture can be induced to differentiate into cells that possess the morphological and biochemical characteristics of adipocytes (24-30). During differentiation of 3T3-L1 preadipocytes, there is specific activation of a family of genes that encodes proteins whose coordinate expression characterizes the adipocyte phenotype (10,23-30). Included among these proteins are the insulin receptor (23); the insulin-responsive glucose transporter (10,21); the 422(aP2) protein (29,30), which is a cellular target of insulin-receptor tyrosine kinase (31-33); and a large group of other proteins with other adipocyte-specific functions (24-28). Concomitant with expression of these adipocyte proteins, 3T3-L1 adipocytes acquire the capacity to carry out insulin-activated glucose uptake (10,21,31). In the fully differentiated 3T3-L1 adipocyte, insulin acutely activates hexose uptake 15- to 20-fold. Thus, the differentiated 3T3-L1 adipocyte is an ideal cell type with which to investigate insulin-activated glucose uptake.
SIGNAL TRANSMISSION FROM INSULIN RECEPTOR All of the pleiotropic cellular responses to insulin, including activation of glucose uptake, are mediated by a specific cell-surface insulin receptor (34). Understanding the mechanism(s) by which the insulin signal is transmitted across the plasma membrane by the receptor requires detailed analysis of the molecular properties of purified insulin receptor. The insulin receptor is a transmembrane allosteric enzyme composed of two types of subunits (i.e., a- and fi-subunits) that are stabilized in a p-a-a-P tetrameric structure by intersubunit disulfide bonds (34). Thus, the a-subunit, which lies external to the plasma membrane, contains the insulin-binding site, whereas the (3-subunit, which spans the plasma membrane, contains a cytoplasmic tyrosine-specific protein kinase catalytic domain (34,35). The mechanism by which the insulin-induced allosteric signal is transmitted across the membrane is unknown. Nevertheless, there is substantial evidence that insulin induces a conformational change that activates autophosphorylation of the (3-subunit (34-37). Autophosphorylation in turn causes activation of receptor kinase-catalyzed phosphorylation of model protein substrates and, presumably, cellular protein substrates (36; Fig. 1). Thus, Rosen (35) first demonstrated that the lag in insulin-activated substrate phosphorylation (initiated with ATP) is eliminated by prior incubation of the receptor with insulin and ATP. Because the receptor underwent autophosphorylation on tyrosine during preliminary incubation, it appeared that insulin-stimulated autophosphorylation might be an essential step in the activation process. Compelling evidence that insulin-stimulated autophosphorylation is required for activation of substrate phosphorylation has been obtained by use of high-
DIABETES CARE, VOL. 13, NO. 6, JUNE 1990
M.D. LANE AND ASSOCIATES
Protein substrate "active" receptor tyrosine kinase-P
Insulin
>+\
Kinase
Phosphorylated protein substrate-P
Phosphatase
"inactive" receptor tyrosine kinase FIG. 1. Insulin-stimulated autophosphorylation and protein substrate phosphorylation carried out by insulin receptor.
affinity model protein substrates as inhibitors of insulinstimulated autophosphorylation. It was shown, for example, that reduced and carboxyamidomethylated lysozyme (RCAM-lysozyme), which is an excellent model substrate of mouse insulin-receptor tyrosine kinase {Km = 10 |xM), is also a potent inhibitor of insulin-stimulated autophosphorylation (/(, = 2 |xM) (36,37). Thus, RCAM-lysozyme added before ATP and insulin blocks insulin-stimulated autophosphorylation and, as a consequence, blocks insulin-stimulated substrate phosphorylation (36,37). By taking advantage of these properties of RCAM-lysozyme, the dependence of substrate-phosphorylation capacity on fractional autophosphorylation (stimulated by insulin) of the receptor was demonstrated. It became evident, however, that the maximal rate of substrate phosphorylation was achieved when only - 5 0 % of maximal autophosphorylation had occurred (36). This finding indicated that not all sites of autophosphorylation are required for activation of substrate phosphorylation and led us to identify the responsible sites in the mouse insulin receptor. To identify the critical sites of insulin-activated autophosphorylation within the cytoplasmic domain of the receptor from mouse 3T3-L1 adipocytes, it was necessary to determine the amino acid sequence of the mouse receptor, because only the human receptor had been sequenced. Therefore, cDNAs that encode the mouse insulin proreceptor were isolated from a cDNA library prepared with mRNA from differentiated 3T3-L1 adipocytes (36). The amino acid sequence deduced from the nucleotide sequence of proreceptor cDNA revealed that it possessed 95% amino acid sequence identity to human insulin receptor, with the a- and (3-subunits exhibiting 97 and 94% identity, respectively (36). With this sequence information in hand, it was possible to proceed with studies to identify the critical autophosphorylation sites (36). The kinetics of insulin-stimulated autophosphorylation (by |>y-32P]ATP) of specific
DIABETES CARE, VOL. 13, NO. 6, JUNE 1990
tyrosines in the (3-subunit of the mouse receptor were compared with the kinetics of activation of receptor kinase-catalyzed phosphorylation of substrate (i.e., RCAM-lysozyme). Sequence analysis of each 32P-labeled tryptic peptide generated during autophosphorylation was accomplished by Edman radiosequencing (36). It was established that, whereas the maximal rate of receptor kinase-catalyzed substrate phosphorylation is achieved within a few minutes, autophosphorylation reached maximum only after —30 min. From detailed kinetic and autophosphorylation site analyses, it was determined that insulin stimulates sequential autophosphorylation of adjacent tyrosines at positions 1148, 1152, and 1153. Although autophosphorylation also occurs at other sites in the p-subunit, it is the transition from the doubly to triply phosphorylated forms of the receptor within this domain that is responsible for activation of catalysis of substrate phosphorylation (Fig. 2). The doubly phosphorylated forms were found to contain phosphotyrosines either at positions 1148 and 1152 or 1153 or at positions 1152 and 1153 (36). We suggest that the development of a center of high negative charge, caused by phosphorylation of the three adjacent tyrosyl residues, induces a conformational change at the active site of the tyrosine kinase, which activates catalysis of substrate phosphorylation and perhaps another mode of signal transduction. 1148
1152-3
—-DIYETDYYR(K)—"inactive" INSULIN )
DIYETD(YY)R(K) + DIYETDYYR(K) "inactive" INSULIN- +
DIYETDYYR(K) active FIG. 2. Stepwise autophosphorylation of specific tyrosines within cytoplasmic domain of mouse insulin receptor that correlate with activation of substrate phosphorylation catalyzed by receptor kinase. Amino acid sequence of relevant tryptic phosphopeptide (positions 1146-1155) is given in single-letter amino acid code. Phosphorylation sites are denoted (•). Results on which schema is based are from ref. 36.
567
INSULIN-ACTIVATED GLUCOSE UPTAKE
Several lines of evidence indicate that a similar insulin-activated autophosphorylation of insulin-receptor kinase occurs in the intact cell (22). Thus, when 3T3L1 adipocytes were incubated with 32Pj, after which the insulin receptor was isolated and analyzed, the (3-subunitof the receptor was found to contain a small amount of 32P-labeled phosphoserine but little phosphorylation of tyrosine (22). On addition of insulin to the cells, however, there was immediate {hA ^10 s) further phosphorylation of the P-subunit of the receptor, which occurred exclusively on tyrosine (see below). The insulin-activated increase in labeling of tyrosine residues was seven- to eightfold (22). Evidence was also obtained that the same sites of autophosphorylation detected in vitro (see above) undergo phosphorylation in the intact cell (22). It was found that the extent of the autophosphorylation that could be accomplished in vitro (i.e., after isolation of the receptor from broken cell preparations) was markedly reduced by first treating the cells with insulin. These sites of tyrosine autophosphorylation occupied during insulin stimulation of the intact cell were no longer available for subsequent autophosphorylation by ATP in vitro in the presence of insulin. Furthermore, insulin receptor isolated from cells stimulated with insulin exhibited a higher rate of substrate (RCAM-lysozyme) phosphorylation than receptor isolated from unstimulated cells (22). These findings indicate that in the intact cell insulin activates autophosphorylation of its receptor and thus its capacity to catalyze tyrosine-specific substrate phosphorylation (in vitro). These phosphorylation events precede the activation of hexose uptake by insulin as discussed below.
INSULIN-ACTIVATED GLUCOSE TRANSPORT IN 3T3-L1 ADIPOCYTES Hexose uptake by fully differentiated 3T3-L1 adipocytes is markedly activated by insulin (21). On addition of insulin, there is a short lag followed by a 15- to 20-fold increase in the rate of labeled hexose 2-deoxyglucose (or 3-O-methylglucose; results not shown) uptake (Fig. 3). That this hexose uptake occurs via a classic glucose transporter is indicated by the nearly complete inhibition of hexose uptake when cytochalasin B is added. Activation of hexose transport by insulin is due entirely to an increase in the maximal rate (Vmax) of uptake with no effect on the apparent affinity {KJ for hexose (21). To characterize the molecular events that occur after insulin binds to its receptor and that lead to activation of glucose uptake, it was first important to compare the kinetics of insulin-activated autophosphorylation of the receptor with the kinetics of insulin-activated hexose uptake in the intact cell (22). Therefore, 3T3-L1 adipocytes were first incubated with 32Pi to label cellular ATP, after which cellular insulin receptors were isolated for analysis. Concurrently, the glucose uptake rate of the cells was measured. As shown in Fig. 4, 32P activity was
568
12.0 - A
LU
f
We identified the earliest events in autophosphorylation of the insulin receptor after insulin addition. Insulinstimulated autophosphorylation at specific sites in the tyrosine kinase domain of the receptor's (5-subunit is correlated kinetically with activation of kinase-catalyzed phosphorylation of a model substrate (reduced and carboxyamidomethylated lysozyme; RCAM-lysozyme). To identify these sites, the deduced amino acid sequence of the 3T3-L1 adipocyte insulin receptor of the mouse was determined. Insulin-induced activation of substrate phosphorylation was shown to require autophosphorylation of three neighboring tyrosines (Tyr1148, Tyr1152, and Tyr1153) in the mouse receptor. A search for cellular substrates of the receptor kinase revealed that insulin causes accumulation of a 15,000-A4r phosphorylated (on tyrosine) cytosolic protein (pp15) in 3T3-L1 adipocytes treated with oxophenylarsine (PAO). PAO blocks turnover of the phosphoryl group of pp15, causing its accumulation, and thereby appears to interrupt signal transmission from the receptor to the glucose-transport system. Two membrane-bound protein phosphotyrosine phosphatases that are inhibited by PAO and are apparently responsible for the turnover of the pp15 phosphoryl group have been purified from 3T3-L1 adipocytes and characterized. These and other results support the hypothesis that turnover of the phosphoryl group of pp15, a product of insulin-receptor tyrosine kinase action, couples signal transmission to the glucose-transport system. [32P]pp15 was purified to homogeneity from 3T3-L1 adipocytes. Amino acid and radiochemical sequence analysis of the purified tryptic [32P]phosphopeptide revealed that pp15 is the phosphorylation product of 422(aP2) protein, a 15,000-/Vfr
From the Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland. Address correspondence and reprint requests to Dr. M. Daniel Lane, Department of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205.
DIABETES CARE, VOL. 13, NO. 6, JUNE 1990
M. Daniel Lane, PhD Jaime R. Flores-Riveros, PhD Richard C. Hresko, PhD Klaus H. Kaestner, MS Kan Liao, BS Michel Janicot, PhD Robert D. Hoffman, PhD John C. McLenithan, BS Tania Kastelic, MS Robert J. Christy, PhD
adipocyte protein whose cDNA we previously cloned and sequenced. 422(aP2) protein was found to bind fatty acids. When exposed to a free fatty acid, notably oleic acid, 422(aP2) protein becomes an excellent substrate of the isolated insulin-receptor tyrosine kinase. Compelling evidence indicates that on binding fatty acid, 422(aP2) protein undergoes a conformational change whereby Tyr19 becomes accessible to the receptor tyrosine kinase and undergoes O-phosphorylation. Adipose tissue and skeletal and heart muscle, which exhibit insulinstimulated glucose uptake, express a specific insulinresponsive glucose transporter. A cDNA (GT2) that encodes this protein was isolated from a mouse 3T3-L1 adipocyte library and sequenced. We also isolated and characterized the corresponding mouse gene GLUT4. DNase I footprinting with nuclear extracts from 3T3-L1 cells revealed that a differentiation-specific nuclear factor binds to the GLUT4 promoter. The purified transcription factor C/EBP binds at the same position. Transient cotransfection into 3T3-L1 preadipocytes of a chimeric GLUT4 promoter-chloramphenicol acetyltransferase gene and a C/EBP expression vector revealed that C/EBP frans-activates the GLUT4 promoter. We suggest that C/EBP plays an important role in tissue-specific and metabolic regulation of the insulinresponsive glucose-transporter gene. Diabetes Care 13:565-75, 1990
cute activation of glucose uptake by animal tissues is perhaps the most widely recognized function of insulin (1). This action of insulin is limited primarily to two major cell types, i.e., muscle cells and adipocytes. Thus, in both type I (insulin-dependent) and type II (non-insulin-dependent) diabetes mellitus, impaired glucose uptake by muscle and adipose tissue and accelerated hepatic gluconeogenesis
A
565
INSULIN-ACTIVATED GLUCOSE UPTAKE
account for the accompanying hyperglycemia (2). Inefficient disposal of glucose by peripheral tissues can result either from an absolute deficiency of insulin (as in type I diabetes) or from the resistance of target cells to insulin (as in type II diabetes). Resistance of cells to insulin appears to have a complex etiology caused by a decrease in the number of functional cell-surface insulin receptors on target cells (3), by a defect in signal transduction by the receptor (4), or as recent evidence suggests, by impaired expression of the insulin-responsive glucose transporter (5-7). The ability to take up glucose is a fundamental property of all animal cells and is mediated by a family of eel I-type-specific glucose-transporter proteins (1,8). Recently, the cDNAs encoding several of these glucose transporters have been isolated, sequenced, and shown to be expressed in a tissue-specific manner (9-14). Another glucose-transporter cDNA, unique to cells that exhibit insulin-stimulated glucose uptake (adipocytes and skeletal and heart muscle cells), was recently cloned in our (10) and several other laboratories (15-18). The protein encoded by these cDNAs exhibits the properties of the insulin-responsive glucose transporter or GLUT4 by the nomenclature of Bell et al. (8) and Gould and Bell (19). More recently, the cloning and characterization of the mouse GLUT4 gene and the trans-activation of its promoter by nuclear transcription factor C/EBP have been reported (20). It is possible that expression of this gene is altered in certain insulin-resistant states. In this article, we review primarily the work done in our laboratory on insulin-receptor tyrosine kinase and the glucose-transport system of the mouse 3T3-L1 adipocyte. The 3T3-L1 adipocyte is an excellent model cell type that is extraordinarily responsive to insulin and with which insulin action can be studied under the controlled conditions of cell culture.
3T3-L1 ADIPOCYTE MODEL The role of adipose tissue is to store energy in the form of triacylglycerol during periods of nutritional abundance and to mobilize this store for use during periods of nutritional deficit. To perform these functions, the adipocyte is uniquely endowed with a complement of enzymes and transport systems that are exquisitely responsive to hormones such as insulin. Although adipocytes isolated from adipose tissue are suitable for certain types of acute experiments, they are generally unsuitable for longer-term experiments. An ideal model for the adipocyte and its development that has proved useful for both acute and long-term studies on insulin action is the 3T3-L1 preadipocyte/adipocyte system (10,21-23). When fully differentiated, the 3T3-L1 adipocyte is extraordinarily responsive to insulin (10,21). In the pathway of development leading to the permanently differentiated adipocyte, successive steps of determination narrow developmental options until a single pathway (i.e., differentiation of the preadipo-
566
cyte to an adipocyte) remains. Under appropriate conditions, confluent 3T3-L1 preadipocytes in culture can be induced to differentiate into cells that possess the morphological and biochemical characteristics of adipocytes (24-30). During differentiation of 3T3-L1 preadipocytes, there is specific activation of a family of genes that encodes proteins whose coordinate expression characterizes the adipocyte phenotype (10,23-30). Included among these proteins are the insulin receptor (23); the insulin-responsive glucose transporter (10,21); the 422(aP2) protein (29,30), which is a cellular target of insulin-receptor tyrosine kinase (31-33); and a large group of other proteins with other adipocyte-specific functions (24-28). Concomitant with expression of these adipocyte proteins, 3T3-L1 adipocytes acquire the capacity to carry out insulin-activated glucose uptake (10,21,31). In the fully differentiated 3T3-L1 adipocyte, insulin acutely activates hexose uptake 15- to 20-fold. Thus, the differentiated 3T3-L1 adipocyte is an ideal cell type with which to investigate insulin-activated glucose uptake.
SIGNAL TRANSMISSION FROM INSULIN RECEPTOR All of the pleiotropic cellular responses to insulin, including activation of glucose uptake, are mediated by a specific cell-surface insulin receptor (34). Understanding the mechanism(s) by which the insulin signal is transmitted across the plasma membrane by the receptor requires detailed analysis of the molecular properties of purified insulin receptor. The insulin receptor is a transmembrane allosteric enzyme composed of two types of subunits (i.e., a- and fi-subunits) that are stabilized in a p-a-a-P tetrameric structure by intersubunit disulfide bonds (34). Thus, the a-subunit, which lies external to the plasma membrane, contains the insulin-binding site, whereas the (3-subunit, which spans the plasma membrane, contains a cytoplasmic tyrosine-specific protein kinase catalytic domain (34,35). The mechanism by which the insulin-induced allosteric signal is transmitted across the membrane is unknown. Nevertheless, there is substantial evidence that insulin induces a conformational change that activates autophosphorylation of the (3-subunit (34-37). Autophosphorylation in turn causes activation of receptor kinase-catalyzed phosphorylation of model protein substrates and, presumably, cellular protein substrates (36; Fig. 1). Thus, Rosen (35) first demonstrated that the lag in insulin-activated substrate phosphorylation (initiated with ATP) is eliminated by prior incubation of the receptor with insulin and ATP. Because the receptor underwent autophosphorylation on tyrosine during preliminary incubation, it appeared that insulin-stimulated autophosphorylation might be an essential step in the activation process. Compelling evidence that insulin-stimulated autophosphorylation is required for activation of substrate phosphorylation has been obtained by use of high-
DIABETES CARE, VOL. 13, NO. 6, JUNE 1990
M.D. LANE AND ASSOCIATES
Protein substrate "active" receptor tyrosine kinase-P
Insulin
>+\
Kinase
Phosphorylated protein substrate-P
Phosphatase
"inactive" receptor tyrosine kinase FIG. 1. Insulin-stimulated autophosphorylation and protein substrate phosphorylation carried out by insulin receptor.
affinity model protein substrates as inhibitors of insulinstimulated autophosphorylation. It was shown, for example, that reduced and carboxyamidomethylated lysozyme (RCAM-lysozyme), which is an excellent model substrate of mouse insulin-receptor tyrosine kinase {Km = 10 |xM), is also a potent inhibitor of insulin-stimulated autophosphorylation (/(, = 2 |xM) (36,37). Thus, RCAM-lysozyme added before ATP and insulin blocks insulin-stimulated autophosphorylation and, as a consequence, blocks insulin-stimulated substrate phosphorylation (36,37). By taking advantage of these properties of RCAM-lysozyme, the dependence of substrate-phosphorylation capacity on fractional autophosphorylation (stimulated by insulin) of the receptor was demonstrated. It became evident, however, that the maximal rate of substrate phosphorylation was achieved when only - 5 0 % of maximal autophosphorylation had occurred (36). This finding indicated that not all sites of autophosphorylation are required for activation of substrate phosphorylation and led us to identify the responsible sites in the mouse insulin receptor. To identify the critical sites of insulin-activated autophosphorylation within the cytoplasmic domain of the receptor from mouse 3T3-L1 adipocytes, it was necessary to determine the amino acid sequence of the mouse receptor, because only the human receptor had been sequenced. Therefore, cDNAs that encode the mouse insulin proreceptor were isolated from a cDNA library prepared with mRNA from differentiated 3T3-L1 adipocytes (36). The amino acid sequence deduced from the nucleotide sequence of proreceptor cDNA revealed that it possessed 95% amino acid sequence identity to human insulin receptor, with the a- and (3-subunits exhibiting 97 and 94% identity, respectively (36). With this sequence information in hand, it was possible to proceed with studies to identify the critical autophosphorylation sites (36). The kinetics of insulin-stimulated autophosphorylation (by |>y-32P]ATP) of specific
DIABETES CARE, VOL. 13, NO. 6, JUNE 1990
tyrosines in the (3-subunit of the mouse receptor were compared with the kinetics of activation of receptor kinase-catalyzed phosphorylation of substrate (i.e., RCAM-lysozyme). Sequence analysis of each 32P-labeled tryptic peptide generated during autophosphorylation was accomplished by Edman radiosequencing (36). It was established that, whereas the maximal rate of receptor kinase-catalyzed substrate phosphorylation is achieved within a few minutes, autophosphorylation reached maximum only after —30 min. From detailed kinetic and autophosphorylation site analyses, it was determined that insulin stimulates sequential autophosphorylation of adjacent tyrosines at positions 1148, 1152, and 1153. Although autophosphorylation also occurs at other sites in the p-subunit, it is the transition from the doubly to triply phosphorylated forms of the receptor within this domain that is responsible for activation of catalysis of substrate phosphorylation (Fig. 2). The doubly phosphorylated forms were found to contain phosphotyrosines either at positions 1148 and 1152 or 1153 or at positions 1152 and 1153 (36). We suggest that the development of a center of high negative charge, caused by phosphorylation of the three adjacent tyrosyl residues, induces a conformational change at the active site of the tyrosine kinase, which activates catalysis of substrate phosphorylation and perhaps another mode of signal transduction. 1148
1152-3
—-DIYETDYYR(K)—"inactive" INSULIN )
DIYETD(YY)R(K) + DIYETDYYR(K) "inactive" INSULIN- +
DIYETDYYR(K) active FIG. 2. Stepwise autophosphorylation of specific tyrosines within cytoplasmic domain of mouse insulin receptor that correlate with activation of substrate phosphorylation catalyzed by receptor kinase. Amino acid sequence of relevant tryptic phosphopeptide (positions 1146-1155) is given in single-letter amino acid code. Phosphorylation sites are denoted (•). Results on which schema is based are from ref. 36.
567
INSULIN-ACTIVATED GLUCOSE UPTAKE
Several lines of evidence indicate that a similar insulin-activated autophosphorylation of insulin-receptor kinase occurs in the intact cell (22). Thus, when 3T3L1 adipocytes were incubated with 32Pj, after which the insulin receptor was isolated and analyzed, the (3-subunitof the receptor was found to contain a small amount of 32P-labeled phosphoserine but little phosphorylation of tyrosine (22). On addition of insulin to the cells, however, there was immediate {hA ^10 s) further phosphorylation of the P-subunit of the receptor, which occurred exclusively on tyrosine (see below). The insulin-activated increase in labeling of tyrosine residues was seven- to eightfold (22). Evidence was also obtained that the same sites of autophosphorylation detected in vitro (see above) undergo phosphorylation in the intact cell (22). It was found that the extent of the autophosphorylation that could be accomplished in vitro (i.e., after isolation of the receptor from broken cell preparations) was markedly reduced by first treating the cells with insulin. These sites of tyrosine autophosphorylation occupied during insulin stimulation of the intact cell were no longer available for subsequent autophosphorylation by ATP in vitro in the presence of insulin. Furthermore, insulin receptor isolated from cells stimulated with insulin exhibited a higher rate of substrate (RCAM-lysozyme) phosphorylation than receptor isolated from unstimulated cells (22). These findings indicate that in the intact cell insulin activates autophosphorylation of its receptor and thus its capacity to catalyze tyrosine-specific substrate phosphorylation (in vitro). These phosphorylation events precede the activation of hexose uptake by insulin as discussed below.
INSULIN-ACTIVATED GLUCOSE TRANSPORT IN 3T3-L1 ADIPOCYTES Hexose uptake by fully differentiated 3T3-L1 adipocytes is markedly activated by insulin (21). On addition of insulin, there is a short lag followed by a 15- to 20-fold increase in the rate of labeled hexose 2-deoxyglucose (or 3-O-methylglucose; results not shown) uptake (Fig. 3). That this hexose uptake occurs via a classic glucose transporter is indicated by the nearly complete inhibition of hexose uptake when cytochalasin B is added. Activation of hexose transport by insulin is due entirely to an increase in the maximal rate (Vmax) of uptake with no effect on the apparent affinity {KJ for hexose (21). To characterize the molecular events that occur after insulin binds to its receptor and that lead to activation of glucose uptake, it was first important to compare the kinetics of insulin-activated autophosphorylation of the receptor with the kinetics of insulin-activated hexose uptake in the intact cell (22). Therefore, 3T3-L1 adipocytes were first incubated with 32Pi to label cellular ATP, after which cellular insulin receptors were isolated for analysis. Concurrently, the glucose uptake rate of the cells was measured. As shown in Fig. 4, 32P activity was
568
12.0 - A
LU
f
