and the glut1 glucose transporter. bandyopadhyay g*, sajan … · 2000-09-27 · sensitive pkcs....

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Glucose Activates MAP Kinase (ERK) Through Proline-Rich Tyrosine Kinase-2

and the Glut1 Glucose Transporter.

Bandyopadhyay G*, Sajan MP*, Kanoh Y*, Standaert ML*, Burke RT**, Jr., Quon M**,

Reed BC***, Dikic I****, Noel LE*****, Newgard CB*****, and Farese RV*

J. A. Haley Veterans' Hospital Research Service, and

Department of Internal Medicine, University of South Florida College of Medicine,

Tampa, Florida 33612*

Hypertension-Endocrine Branch, National Heart, Lung and Blood Institute,

National Institutes of Health, Bethesda, Maryland 20892**

Department of Biochemistry and Molecular Biology

Louisiana State University Health Science Center-Shreveport, Shreveport, LA 71130***

Ludwig Institute for Cancer Research, Uppsala, Sweden 75124****

And Departments of Biochemistry and Internal Medicine and Gifford Laboratories for

Diabetes Research, University of Texas Southwestern Medical Center,

Dallas, Texas 75235*****

*Send correspondence to: Robert V. Farese, M.D. Research Service (VAR 151) J.A. Haley Veterans' Hospital 13000 Bruce B. Downs Blvd. Tampa, FL 33612 (813)972-7662 (813)972-7623 fax

[email protected] August 29, 2000

Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on September 27, 2000 as Manuscript M007920200 by guest on M

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ABSTRACT

Glucose serves as both a nutrient and regulator of physiological and pathological

processes. Presently, we found that glucose and certain sugars rapidly activated

extracellular signal-regulated kinase (ERK) by a mechanism that was: (a) independent

of glucose uptake/metabolism and protein kinase C, but nevertheless cytochalasin B-

inhibitable; (b) dependent upon proline-rich tyrosine kinase-2 (PYK2), GRB2, SOS,

RAS, RAF and MEK1; and (d) amplified by overexpression of the Glut1, but not Glut2,

Glut3 or Glut4, glucose transporter. This amplifying effect was independent of glucose

uptake, but dependent on residues 463-468, IASGFR, in the Glut1 C-terminus.

Accordingly, glucose effects on ERK were amplified by expression of Glut4/Glut1 or

Glut2/Glut1 chimeras containing IASGFR, but not by Glut1/Glut4 or Glut1/Glut2

chimeras lacking these residues. Also, deletion of Glut1 residues 469-492 was without

effect, but mutations involving serine-465 or arginine-468 yielded dominant-negative

forms that inhibited glucose-dependent ERK activation. Glucose stimulated the

phosphorylation of tyrosine residues 402 and 881 in PYK2, and binding of PYK2 to Myc-

Glut1. Our findings suggest that: (a) glucose activates the

GRB2/SOS/RAS/RAF/MEK1/ERK pathway by a mechanism that requires PYK2 and

residues 463-468, IASGFR, in the Glut1 C-terminus; (b) Glut1 serves as a sensor,

transducer and amplifier for glucose signaling to PYK2 and ERK.

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INTRODUCTION

Glucose, the ubiquitous carbohydrate nutrient, activates intracellular signaling systems

and thereby alters physiological and pathological processes. Effects of glucose on

signaling are generally thought to be effected through the uptake and subsequent

intracellular metabolism of glucose. For example, in rat adipocytes, glucose is rapidly

converted, via glycolysis and generation of glycerol-3-PO4, to phosphatidic acid and

diacylglycerol (DAG) (1-3), which, in turn, activates DAG-sensitive protein kinase C

(PKC) isoforms (4,5). Also, in pancreatic islet β-cells, glucose, via glycolysis and ATP

generation, closes K+ channels, causing plasma membrane depolarization, Ca++-gating

via L-type channels (6-8), hydrolysis of phosphatidylinositol (PI)-4,5-(P04)2 (9),

mobilization of Ca++ by inositol-(PO4)3, generation of DAG and activation of DAG-

sensitive PKCs. Increases in PKC activity by either mechanism can activate RAS or

RAF and subsequent signaling to MEK1 and extracellular signal-regulated kinases,

ERK1 and 2 (10,11). Activation of either PKC or ERK by glucose can alter physiological

processes, such as gene expression and cellular proliferation, differentiation and

survival. Moreover, in diabetes mellitus, inordinate increases in glucose-dependent PKC

and ERK signaling play important roles in the pathogenesis of both diabetic

complications (12-14) and acquired insulin resistance (15-18). Such aberrant, diabetes-

related, glucose-stimulated signaling through PKC to ERK has generally been attributed

to increased de novo synthesis of phosphatidic acid and DAG. However, the latter

hypothesis, while plausible, is neither proven nor exclusive, and other signaling

mechanisms may also be operative. Presently, we found, in rat adipocytes, 3T3/L1

fibroblasts, L6 myotubes and several other cell types, that glucose activates ERK


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independently of both glucose metabolism and PKC activation, but dependent upon the

activation of non-receptor tyrosine kinase, proline-rich tyrosine kinase (PYK2). We also

found that the Glut1 glucose transporter, apparently through specific amino acid

residues in its C-terminus, binds PYK2 and amplifies ERK activation by glucose.

EXPERIMENTAL METHODS

Rat Adipocyte Preparation and Incubation Conditions. As described (4,5,19-21),

adipocytes were isolated by collagenase digestion of rat epididymal fat pads,

equilibrated in glucose-free Krebs Ringer phosphate (KRP) medium containing 1%

BSA, with or without inhibitors [PKC inhibitors, GF109203X and GO6976 (Alexis); MEK1

inhibitor, PD98059 (Alexis); PI 3-kinase inhibitor, wortmannin (Sigma); GRB2-SH2

domain inhibitor, L-20d, Na-oxalyl-tripeptide-phosphotyrosine mimetic (see 22); tyrosine

kinase inhibitor, genistein (Calbiochem); glucose transporter inhibitor, cytochalasin B

(Sigma); internal Ca++ pool and PYK2 inhibitor, dantrolene (Alexis)], and then treated

with or without glucose, other carbohydrates, insulin, or tetradecanoyl phorbol-13-

acetate (TPA;Sigma), in most cases for 10 min. In some experiments, uptake of [3H]-

labeled 2-deoxyglucose (2-DOG) or D-glucose (both from NEN) was measured as

described (21).

Cell Culture Preparations and Incubation Conditions. 3T3/L1 fibroblasts, 3T3/L1

adipocytes and L6 myotubes were cultured as described (23,24). Rat A-10 vascular

smooth muscle cells (VSMC) and mouse kidney mesangial cells were obtained from

American Type Culture Collection. After growth and differentiation as needed, all cells


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were incubated in KRP medium as in rat adipocyte experiments described above.

Assays of Immunoprecipitable ERK. As described (20,25), after incubation, adipocytes

or other cells were sonicated in buffer containing 40mM β-glycerophosphate (pH, 7.3),

0.5mM DTT, 0.75mM EGTA, 0.15mM Na3VO4, 5µg/ml leupeptin, 5µg/ml aprotinin,

0.1mM phenylmethylsulfonyl fluoride and 5µg/ml trypsin inhibitor. After centrifugation at

700xg for 10 min, the fat cake, cell debris and nuclei were removed. Post-nuclear

supernatants were supplemented with 0.154M NaCl, 1% Triton- X100 and 0.5%

Nonidet, and equal amounts of lysate protein (200-500µg) were subjected to overnight

immunoprecipitation at 4ºC with mouse monoclonal anti-ERK2 antibodies (Santa Cruz

Biotechnologies) or with anti-epitope (HA, Myc) antibodies in co-transfection studies.

Precipitates were collected on Protein-AG Agarose beads, washed and incubated for 10

min at 30ºC in 50µl buffer containing 25mM β-glycerophosphate (pH, 7.3), 0.5mM DTT,

1.25mM EGTA, 0.5mM Na3VO4, 10mM MgCl2, 1mg/ml BSA, 1µM okadaic acid, 0.1mM

[γ−32P]ATP (NEN; 1,500,000dpm/nmol) and 50µg myelin basic protein (MBP; Sigma).

After incubation, aliquots were spotted on p81 filter paper, washed and counted for 32P

radioactivity, as described (20,25). Blank values were obtained by substituting a non-

immune antibody preparation for anti-ERK2 antibodies, or by omitting MBP substrate

(results were similar). As noted previously (25), the Santa Cruz anti-ERK2 mouse

monoclonal antibodies, although recognizing only ERK2 in Western analyses,

precipitated ERK1, as well as ERK2, and the ratio of ERK2 to ERK1 in

immunoprecipitates was approximately 1.4-1.5/1, as measured by Western analysis,

using a Santa Cruz rabbit polyclonal antiserum that recognizes both ERK1 and ERK2.


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Treatment with glucose and other carbohydrates did not alter the levels of either ERK2

or ERK1, or the ratio of ERK2 to ERK1, in ERK2, immunoprecipitates (also see 25).

Transfection Studies. Rat adipocytes and 3T3/L1 fibroblasts were transiently co-

transfected as described (21,23,25,26). In brief, for transient co-transfection

experiments with rat adipocytes, 0.4ml cells were suspended and electroporated in an

equal volume of Dulbecco’s Modified Eagle’s Medium (DMEM) containing 5% BSA and

3.3µg pCEP4 encoding Hemagglutinin (HA)-tagged ERK2 or 3.3 µg pCMV5 encoding

Myc-tagged ERK2 (both kindly supplied by Dr. Melanie Cobb) along with 6.6µg of

indicated amounts of: (a) pRSV encoding N17 dominant-negative RAS (gift of Dr Jane

Reusch); (b) pCEP4 encoding dominant-negative c-RAF-1 (gift of Dr Melanie Cobb; this

truncated mutant contains the N-terminal RAS-binding, but not the catalytic, domain, of

c-RAF-1); (c) pCDNA3 encoding kinase-inactive PKC-ζ (see 21,26); (d) pSRα encoding

wild-type or dominant-negative SOS (gifts of Dr. Masato Kasuga; see 27); (e) pCGN

encoding wild-type or dominant-negative (deletion of C-terminal or N-terminal SH3

domain) GRB2 (see 28); (f) pSGT encoding wild-type or kinase-inactive pp60SRC (see

28); (g) pRK5 encoding wild-type or dominant-negative [kinase-inactive; or PYK2-

Related Non-Kinase (PRNK), the non-catalytic C-terminal domain] forms of proline-rich

tyrosine kinase-2 (PYK2) (see 28); (h) pCDNA3 encoding mouse Glut1 glucose

transporter; (i) pCIS2 encoding human HA-tagged Glut4 glucose transporter (see

21,26); (j) pCMV5 encoding either HA-tagged Glut1/Glut4 chimera containing a human

Glut1 N-terminus (equivalent to residues 1-462 in mouse Glut1) and the 29 C-terminal

residues of rat Glut4 (residues 481-509, QISATFRRTPSLLEQEVKPSTELEYLGPD), or


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HA-tagged Glut4/Glut1 chimera containing residues 1-478 of the rat Glut4 N-terminus

and the 30 C-terminal residues of human Glut1 (identical to mouse residues 463-492,

IASGFRQGGASQSDKTPEELFHPLGADSQV) (both HA-tagged chimeras were kindly

supplied by Dr. Michael Czech-see 29) (note that these HA-tags are in exofacial loops

and, when present in the plasma membrane, are accessible with extracellular

antibodies); (k) pCDNA3 encoding human Glut1; (m) pCB6 encoding mouse Myc-Glut1;

(l) pCB6 encoding mouse Myc-∆489-492-Glut1 (missing the last C-terminal 4 amino

acids); (m) pCB6 encoding mouse Myc-∆469-492-Glut1 (missing the last C-terminal 24

amino acids); and (n) pCB6 encoding mouse Myc-Glut1 mutants, Myc-S465A-Glut1,

Myc-A464S/S465A/G466A-Glut1 (note that Glut1 and Glut4 contain ASG and SAA,

respectively, in these corresponding positions) or Myc-R468G-Glut1. 3T3/L1 fibroblasts

were transfected with pCB6 encoding mouse Myc-tagged Glut1 using Lipofectamine

(Life Technologies, Inc.) as described (23). After overnight (rat adipocytes) of 48-hour

(3T3/L1 cells) incubation to allow time for expression, cells were washed and incubated,

in most cases for 10 min, in glucose-free KRP medium, without or with addition of

glucose, insulin or TPA, as described in the text. After incubation, HA-ERK2 or Myc-

ERK2 was immunoprecipitated with anti-HA mouse monoclonal antibody (Covance) or

rabbit polyclonal anti-Myc antiserum or mouse monoclonal anti-Myc antibodies (Upstate

Biotechnologies Inc; UBI), and assayed for MBP phosphorylation, as described above.

In some experiments, plasma membrane levels of HA-tagged or Myc-tagged glucose

transporters were quantified by measuring the cell surface content of HA or Myc

epitopes, using anti-HA or anti-Myc mouse monoclonal primary antibodies (Covance or

UBI) and 125I-labeled rabbit anti-mouse IGG second antibody, as described (21,26). In


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some experiments, Myc-Glut1 was immunoprecipitated and examined for associated

immunoreactive PYK2.

Generation of Myc-Tagged Glut 1 Mutants. Mutant forms of Myc-taggedGlut1 were

generated by site-directed mutagenesis using a Gene Editor kit from Promega and the

following primers: S465A mutation,

5’-CCTGCCGGAAGCCGGCCGCGATCTCATCGAAG-OH-3’, which includes a

CGGCCG BstZ-1 site; A464S/S465A/G466A mutation,

5’-GCACCCCCCTGCCGGAAGGCTGCAGAGATCTCATCGAAGGTTCG-OH-3’, which

includes a GAGATCT Bgl-II site; R468G mutation,

5’-GCTGGCACCCCCCTGGCCAAAGCCGGAAGCGATC-OH-3’, which includes a

TGGCCA Bal1 site. These mutations were confirmed by restriction enzyme analysis

and sequencing.

Adenoviral Gene Transfer Studies. In initial studies, we found that rat adipocytes were

damaged (i.e., leaky with respect to glucose transport) by adenoviral infection and were

considered to be unsuitable for use in the present study. We therefore used 3T3/L1

fibroblasts, as they have only Glut1 glucose transporters, are not damaged significantly

by adenoviral infection, and are highly responsive to glucose and other saccharides in

terms of increasing ERK activity independently of PKC and subsequent metabolism

(see below). Fully confluent 3T3/L1 fibroblasts were infected with adenovirus alone or

adenovirus encoding Glut1, Glut2, or chimeras in which their C-termini were

transposed, viz., N(1-457)Glut1/C(490-525)Glut2 and N(1-489)Glut2/C(458-492)Glut1;


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as described previously (30). Each of these viral constructs encode glucose

transporters that are functional with respect to hexose transport (see ref 30; also

documented presently - see below). After 48 hours to allow time for expression, cells

were placed into serum-free DMEM medium for 3 hours, and then equilibrated in

glucose-free KRP medium for 20 min, and subsequently treated for10 min with or

without 20mM glucose. After incubation, cell lysates were examined for total

immunoprecipitable ERK activity. Cellular uptake of [3H]-labeled D-glucose over 10 min

was also monitored to compare the effects of expressed glucose transporters on

cytochalasin B-inhibitable glucose uptake to effects on ERK.

RESULTS

I Studies in Rat Adipocytes

Effects of Glucose and other Monosaccharides on ERK. Glucose, over a range of 2.5

to 30mM, provoked rapid progressive increases in immunoprecipitable ERK activity (Fig

1A and B). Effects of 10 and 20mM glucose on ERK activity were comparable and non-

additive to those of submaximal and maximally effective insulin (Fig 1A and B). The

latter was surprising, as glucose uptake in rat adipocytes, which increases steadily over

the concentration range of 2.5-20mM in the absence of insulin (19), is increased further

by insulin uptake throughout this glucose concentration range (19).

The failure of insulin to augment glucose effects suggested that the uptake and

intracellular metabolism of glucose may not be the major determinant for ERK

activation. Interestingly, transported, but non-metabolizable, sugars, 3-0-methyl-


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glucose (30MG), which is not phosphorylated, and 2-deoxyglucose (2-DOG), which is

converted only to glucose-6-PO4, were as effective as glucose in activating ERK (Fig

1D). Also, both mannose, which is transported and metabolized like glucose, and

glucosamine, which is transported similarly to, but metabolized differently from, glucose

[i.e., is primarily used to synthesize glycolated products (31)], activated

immunoprecipitable ERK as potently as glucose (Fig 1D). In contrast, L-glucose and

mannitol had little or no effect on ERK activity (Fig 1B and D). Thus, glucose-stimulated

ERK activation was not due to changes in medium osmolality and required specific

structural features. The ability of each of these carbohydrates to activate ERK was

paralleled by their ability to inhibit 2-DOG uptake (not shown), through isotopic dilution

or competitive interaction at the level of the glucose transporter (see below).

Requirements for Downstream Signaling Factors in Glucose-Induced Activation of ERK.

Glucose effects on ERK, unlike those of phorbol esters, were not influenced by PKC

inhibitors, GF109203X and GO6976 (Fig 2A), or by downregulation of DAG-sensitive

PKCs by prolonged phorbol ester treatment (Fig 2B). Also, glucose effects on ERK,

unlike those of insulin, were not inhibited by the PI 3-kinase inhibitor, wortmannin (Fig

2A), or expression of kinase-inactive PKC-ζ (Fig 3D), both of which inhibit insulin-

induced activation of ERK2 in rat adipocytes (Figs 2 and 3 and ref 25). On the other

hand, glucose effects on ERK, like those of insulin (25), were inhibited by: (a) the

tyrosine kinase inhibitor, genistein (Fig 2A); (b) a GRB2-SH2 domain inhibitor (Fig 2A);

(c) dominant-negative forms of GRB2, SOS, RAS and c-RAF-1 (Fig 3A,B and C); and

(d) the MEK1 inhibitor, PD98059 (Fig 2A). As with glucose, effects of 3OMG and 2DOG


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on ERK were independent of PKC in rat adipocytes (not shown).

Requirement for the Glut1 Glucose Transporter in Glucose-Induced Activation of ERK.

The activation of ERK by transported sugars, glucose, mannose, glucosamine, 30MG

and 2-DOG, suggested that one or more glucose transporters is required for ERK

activation. Accordingly, cytochalasin B, which binds to Glut1 and Glut4 glucose

transporters and non-competitively blocks the binding and subsequent uptake of

glucose and other saccharides (32-34), inhibited both ERK activation by glucose (Fig

2C) and other sugars (see below), and cellular uptake of [3H]-labeled D-glucose (Fig

2D). Half-maximal and maximal inhibitory effects of cytochalasin B on both processes

were observed at approximately 1 and 30-100µM, respectively. Note that (a) 20mM

glucose was used in experiments shown in Fig 2C and D, and (b) the same

concentrations of cytochalasin B were required to inhibit the uptake of 20mM glucose

(Fig 2D) and 50µM [3H]-labeled 2-DOG (not shown). Thus, assuming that glucose

uptake is dependent on binding to glucose transporters, the Kds for binding of

cytochalasin B and glucose to glucose transporters (see below - Fig 6) are markedly

different. Also note that cytochalasin B did not inhibit insulin-induced ERK activation

(Fig 2C).

Overexpression of increasing amounts of the Glut1 glucose transporter (Fig 4D) led to

progressive increases in (a) plasma membrane levels of such expressed (epitope-

tagged) Glut1 (Fig 4C), and (b) glucose-induced (Fig 4A), but not phorbol ester-induced

(not shown), activation of Myc-ERK2. In contrast, overexpression of the Glut4 glucose


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transporter inhibited glucose-dependent Myc-ERK2 activation (Figs 4B, 5C, and 6A),

even when insulin was added to increase plasma membrane Glut4 content and

subsequent glucose transport (Figs 5C and 5C). Thus, since Glut4 expression and

insulin treatment did not amplify glucose effects on ERK, and, since plasma membrane

levels of epitope-tagged Glut1 and Glut4 were similar (not shown), simple changes in

glucose uptake did not account for observed effects of glucose transporters on ERK.

[Note- Glut4 translocation in transfected cells is increased only 2-3-fold by insulin, as

compared to 5-10-fold increases in 2-DOG uptake observed in freshly incubated rat

adipocytes. As discussed (21,26), this reflects an artifactual increase in basal

translocation/transport caused by electroporation and overnight culture of rat

adipocytes.]

Molecular Requirements for Amplifying effects of Glut1 on Glucose-Dependent ERK

Activation. Expression of a Glut1/Glut4 glucose transporter chimera that contained a

Glut4 C-terminus, like wild-type Glut4, inhibited glucose effects on Myc-ERK2 (Fig 5A).

In contrast, a Glut4/Glut1 glucose transporter chimera that contained a Glut1 C-

terminus, like wild-type Glut1, amplified glucose effects on Myc-ERK2 (Fig 5A). This

difference in ERK amplification was apparent, despite the fact that plasma membrane

levels of these chimeras were only slightly different, and both chimeras, like Glut4, were

present in agonist-responsive pools (Fig 5B). Of further note, expression of Glut3, unlike

Glut1, but like Glut4, did not amplify glucose effects on ERK (Fig 6A).

Findings with chimeras suggested that the last 30 C-terminal amino acids of Glut 1


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(residues 463 through 492) were required for amplifying glucose effects on ERK.

However, deletion of the last 4 or 24 amino acids of the Glut 1 C-terminus did not

compromise the amplifying effects of Glut 1 on glucose-dependent ERK (Fig 6B). Thus,

residues 463 through 468, viz., IASGFR, were required for amplifying effects of Glut1 on

glucose-dependent ERK activation (note that this sequence is identical in human,

mouse and rat Glut1 glucose transporters). Since the corresponding human, mouse,

and rat Glut4 sequences are ISAAFH, ISAAFR and ISATFR, respectively, we

speculated that AS may be required for amplifying effects of Glut1, and, contrariwise,

SAA may be required for inhibitory effects of GLUT4, on glucose-dependent ERK

activation. We also questioned whether the basic arginine-468 residue was required for

amplifying effects of Glut1 on glucose-dependent ERK. Indeed, mutation of Glut1

residues 464-468, IASGFR, to produce IAAGFR, ISAAFR and IASGFG sequences, not

only led to a loss of amplifying effects of Glut1, but yielded dominant-negative forms of

Glut1 that, like Glut4, inhibited glucose effects on ERK (Fig 6C). As the dominant-

negative effects of these Glut1 mutants suggested that they inhibited endogenous Glut1

that resided in the plasma membrane, it was of interest to find that plasma membrane

levels (as per exofacial Myc epitopes) of Myc-S465A-Glut1, Myc-A464S/S465A/G466A-

Glut1 and Myc-R468G-Glut1 mutants were sizeable as comparable to that of Myc-WT-

Glut1 (Fig 6D), which potentiated glucose effects on HA-ERK (Fig 6C). We also

documented that wild-type and each of these mutant forms of Glut1 were expressed to

about the same extent in transfected adipocytes, as measured by blotting for Myc

immunoreactivity migrating at the level of the Glut1 glucose transporter on SDS-PAGE

(not shown). In view of the plasma membrane levels of these GLUT1 mutants, and


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since substitution of corresponding Glut4 residues in the Glut1 IASGFR sequence

would not be expected to impair transport activities, and, moreover, since arginine-468

is not required for transport activity of Glut1 (35), it seems clear that effects of the

presently used Glut1 mutants on glucose-dependent ERK activation can not be

attributed to alterations in glucose uptake.

Effects of Disaccharides on ERK Activity and 2-DOG Uptake. The above findings were

compatible with two possibilities, viz., saccharides that activate ERK must either enter

the cell or interact with a cell surface protein, such as Glut1. To evaluate these

possibilities, we used maltose and lactose, which contain galactose or glucose

connected to glucose through a 1:4 glycosidic linkage, and which, like bis-mannose,

should interact with Glut1 or other proteins, but not be transported. Indeed, both

disaccharides, like the more potent D-glucose, activated ERK by a cytochalasin B-

dependent mechanism (Fig 7B and C) and concomitantly inhibited 2-DOG uptake (Fig

7A and D). Moreover, the Ki for inhibition of 2-DOG uptake and the Km for activation of

ERK by both non-transportable disaccharides were only slightly different, viz., 20mM

versus 26mM, respectively (Fig 7C and D). This small difference in Ki and Km values

was similar to that observed with glucose, viz., 4mM and 10mM (Fig 7C and D), and

may reflect the fact that 2-DOG uptake and ERK activation are decidedly different

processes and ERK activation may require a higher level of ligand binding than glucose

transport through Glut1.

II Activation of ERK by Glucose and Other Sugars in 3T3/L1 Fibroblasts, 3T3/L1


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Adipocytes, L6 Myotubes, and Rat A-10 Vascular Smooth Muscle Cells and Other

Cell Types.

In addition to rat adipocytes, ERK was activated by glucose, 3OMG and 2-DOG in

3T3/L1 fibroblasts and rat A-10 vascular smooth muscle (VSM) cells, which contain only

Glut1 glucose transporters, and 3T3/L1 adipocytes and L6 myotubes, which contain

both Glut1 and Glut4 glucose transporters (Fig 8A-D). In 3T3/L1 fibroblasts and L6

myotubes, glucose effects on ERK were largely independent of PKC, as evidenced by

the failure of GF109203X to inhibit these effects. In rat A-10 VSM cells, glucose effects

on ERK were partly independent, and partly dependent on PKC. In mouse kidney

mesangial cells (which contain Glut1, but not Glut4, glucose transporters), glucose

effects on ERK were largely PKC-dependent, and 3OMG had no effect on ERK (data

not shown). Thus, there is considerable hetereogeneity of mechanisms used by

glucose to activate ERK in various cell types, and the simple presence of Glut1 glucose

transporters does not necessarily imply the presence of metabolism/ PKC-independent

effects of glucose on ERK.

Interestingly, 3T3/L1 fibroblasts appeared to be more responsive to glucose, but less

responsive to insulin, for ERK activation, as compared to 3T3/L1 adipocytes (Fig 8C).

Also, 3OMG, 2-DOG and maltose, as well as glucose, activated ERK by a cytochalasin

B-sensitive mechanism and concomitantly inhibited 2-DOG uptake in 3T3/L1 fibroblasts

(Fig 8E and F). Thus, the Glut1 glucose transporter, operating in the absence of the

Glut4 glucose transporter, as in 3T3/L1 fibroblasts, is sufficient to mediate the effects of

metabolizable and non-metabolizable, and transported and non-transported sugars on


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both hexose uptake and ERK activation.

III Effects of Adenoviral Gene Transfer of Glut 1 and Glut 2 and Their Chimeras

on Glucose-Dependent Activation of ERK and Glucose Uptake in L6 Myotubes

and 3T3/L1 Fibroblasts.

To more definitively evaluate the question of whether or not the amplifying effects of the

Glut1 glucose transporter on glucose-dependent ERK activation are caused by glucose

uptake, we compared the effects of expression of wild-type forms of Glut1 and Glut2,

and chimeric forms in which the C-termini of Glut1 and Glut2 had been transposed, on

both glucose-stimulated ERK activation and glucose uptake, using adenoviral gene

transfer methods in L6 myotubes and 3T3/L1 fibroblasts. Whereas viral-mediated

expression of Glut1 in L6 myotubes amplified glucose-dependent increases in ERK

activity, expression of Glut2 had little or no effect on glucose-dependent ERK activation,

despite the fact that glucose uptake was increased in cells expressing Glut2 to an

extent slightly greater than that observed in cells expressing Glut1 (Fig 9). Similarly, in

3T3/L1 fibroblasts, viral-mediated expression of either wild-type Glut1, or, particularly at

the higher viral MOI, the chimera containing the Glut1 C-terminus led to an amplification

of glucose-dependent effects on ERK (Fig 10). In contrast, wild-type Glut2 and the

chimera containing the Glut2 C-terminus had little or no effect on glucose-dependent

ERK activation in 3T3/L1 fibroblasts, despite the fact that glucose uptake was increased

more effectively in cells expressing Glut2 or the chimera containing the Glut2 C-

terminus (Fig 10). These findings provided further evidence that (a) glucose uptake is


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not, of itself, sufficient to increase ERK activity in either L6 myotubes or 3T3/L1

fibroblasts, (b) amplifying effects of Glut1 and chimeras containing the Glut1 C-terminus

on glucose-stimulated ERK are not explicable on the basis of alterations in glucose

uptake, and (c) the Glut1 C-terminus is required for amplifying effects on glucose-

dependent ERK activation.

IV Linkage of Glut 1 to the ERK pathway via Non-Receptor Tyrosine Kinase,

PYK2.

The above findings suggested that a tyrosine kinase was required for glucose-induced

activation of ERK. Initial examination of cell lysates for phosphotyrosine(pY)-containing

proteins revealed increases in the pY-content of proteins migrating at approximately

125kDa following acute glucose treatment of rat adipocytes and 3T3/L1 fibroblasts (not

shown). Since the non-receptor tyrosine kinase, PYK2, migrates at 125kDa, and, since

PYK2 is activated by pY-dependent mechanisms, autophosphorylates and signals to

GRB2 (28), it was of interest to find that, in both rat adipocytes and 3T3/L1 fibroblasts,

glucose provoked acute increases in the phosphorylation of specific pY residues in

PYK2, viz., Y402, the autophosphorylation site, and Y881, the site of interaction with the

SH2 domain of GRB2 (Fig 11).

Of further note, expression of wild-type PYK2 provoked increases in glucose-dependent

ERK activation (Fig 11). In contrast, two dominant-negative forms of PYK2 inhibited

glucose-dependent ERK activation (Fig 11). Although PYK2 may function in

conjunction with pp60SRC family members, wild-type and kinase-inactive pp60SRC did


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not alter glucose-dependent ERK activation (not shown). In keeping with a requirement

for PYK2, dantrolene, which inhibits an internal Ca++ pool required by PYK2 (28),

inhibited glucose effects on ERK in rat adipocytes: mean±SE cpm for ERK activity =

control, 4064±773; 20mM glucose, 17409±797; 25µM dantrolene control, 3753±975;

25µM dantrolene plus 20mM glucose, 3740±1448. Similar inhibitory effects of

dantrolene were observed in 3T3/L1 fibroblasts (not shown).

Of further interest, PYK2 co-immunoprecipitated with Myc-tagged Glut 1 obtained from

extracts of 3T3/L1 fibroblasts transiently co-transfected with plasmids encoding wild-

type Myc-tagged Glut1 and wild-type PYK2 (the latter used to augment PYK2

availability for co-immunoprecipitation, and 3T3/L1 cells used because of relatively high

transient transfection rates). Most interesting, glucose acutely increased the interaction

between Myc-Glut1 and PYK2 (Fig 11). No such co-immunoprecipitation was seen in

cells in which S465A or R468G mutant forms of Myc-tagged Glut1 were used instead of

wild-type Myc-tagged Glut1.

DISCUSSION

It was surprising to find that, like glucose, non-metabolizable saccharides, 3OMG, 2-

DOG, lactose and maltose activated ERK in several cell types. This suggested that

certain non-metabolizable saccharides can, presumably like D-glucose, act as ligands

for a receptor that activates the ERK pathway. Moreover, since these non-

metabolizable saccharides activated ERK by a cytochalasin B-sensitive mechanism and

concomitantly inhibited 2-DOG uptake through Glut1, the Glut1 glucose transporter itself


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may serve as a receptor for these and other saccharides. However, further studies are

needed to verify that: (a) non-metabolizable and non-transported saccharides activate

ERK by the same mechanism used by glucose; and (b) inhibitory effects of cytochalasin

B on ERK activation are due to inhibition of binding of these saccharides to the Glut1

glucose transporter, rather to than another protein.

In keeping with the suggestion that the cellular uptake of glucose was not required for

glucose-dependent activation of ERK in the indicated cell types, the overexpression of

apparently functional Glut4, even in the presence of insulin, failed to augment glucose

effects on epitope-tagged ERK. In fact, Glut4 overexpression inhibited glucose-

dependent ERK activation. The reason for this inhibitory effect of Glut4 is uncertain, but

findings with dominant-negative Glut1 mutants suggested that specific Glut4 C-terminal

sequences, perhaps via competition with endogenous wild-type Glut1 C-terminal

sequences, can impair the activation of upstream components of the ERK signaling

pathway.

More convincing support for the notion that glucose uptake could not account for

amplifying effects of overexpression of Glut1 on glucose-stimulated ERK activation was

derived from adenoviral gene transfer studies in L6 myotubes and 3T3/L1 fibroblasts. In

these studies, the expression of Glut2 was, if anything, more effective than Glut1

overexpression for promoting glucose uptake, but, unlike Glut1 overexpression, Glut2

expression did not enhance glucose-dependent ERK activation.


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Taken together, findings with truncated forms and chimeric forms of Glut1 suggested

that C-terminal residues 463 to 468, viz., IASGFR, were responsible for amplifying

effects of Glut1 on glucose-dependent ERK activation. Of these amino acids, the

presence and position of serine-465 proved to be important, as simple replacement with

alanine, or swapping the positions of alanine-464 and serine-465 in the ISAAFR mutant

(i.e., assuming that the concomitant replacement of glycine-466 with alanine was

without effect), caused not only a loss of amplifying effects of Glut1, but generated

dominant-negative forms of GLUT1 that inhibited glucose effects on ERK. In addition to

serine-465, arginine-468 was important, as replacement with glycine also yielded a

dominant-negative form of Glut 1. This finding with the R468G mutant is of particular

interest, since mutation of arginine-468 to leucine does not alter glucose transporting

properties of Glut1 (35). Of further note, the ISAAFR sequence in the

A464S/A465S/G466A-Glut1 triple mutant is identical to that found in mouse Glut4, and

there is no reason to think that this mutant would be deficient in transport. On the other

hand, as discussed above, just as alterations in transport did not appear to account for

the stimulatory effects of Glut1 on ERK, it is unlikely that alterations in transport could

account for inhibitory effects of the mutant forms of Glut1 on glucose-dependent ERK

activation. In any event, the dominant-negative inhibitory effects of S465A,

A464S/S465A/G466A and R468G Glut1 mutants suggested that these mutants inhibited

the ability of endogenous wild-type Glut1 to either transmit and/or amplify signals from

glucose to the ERK pathway.

Our findings suggested that PYK2 functioned downstream of glucose and in conjunction


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with Glut1 during ERK activation. PYK2 was tyrosine-phosphorylated during acute

glucose treatment and, moreover, co-immunoprecipitated with Myc-labeled Glut1 in a

glucose-dependent manner. In addition, overexpression of wild-type PYK2 potentiated,

and dominant-negative forms of PYK2 inhibited, glucose effects on ERK. The inhibitory

effects of dantrolene also suggested that PYK2 was required for glucose effects on ERK

(see 28).

The mechanism whereby glucose provoked increases in the pY content of PYK2 is

uncertain. Phosphorylation of the autophosphorylation site in PYK2, Y402, implied that

PYK2 was itself activated. However, it is uncertain if PYK2 was activated as a result of

binding to Glut1, or if it was activated by other factors, including SRC family tyrosine

kinases, e.g., FYN and YES. In any event, pY residues in PYK2 are capable of

interacting with and activating upstream components of the ERK pathway, e.g., GRB2

via pY881-PYK2 and/or SHC via pY402-PYK2 (28).

With respect to dantrolene sensitivity, we have previously reported that glucose does

not activate phospholipase C-dependent hydrolysis of PI-4,5-(PO4)2 in rat adipocytes

(4), and we presently ruled out the involvement, not only of DAG-dependent and

atypical PKCs, but also of Ca++ influx via L-type channels, as 10µM nifedipine had no

effect on glucose stimulation of ERK (data not shown). In addition, glucose does not

increase cytosolic Ca++ in rat adipocytes (36) and the Ca++ ionophore A32187 was

without effect on ERK (not shown). Accordingly, although simple increases in cytosolic

Ca++ cannot explain glucose effects on ERK, the inhibitory effects of dantrolene suggest


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a requirement for Ca++ during PYK2 activation.

The presently observed effects of glucose on ERK in the rat adipocyte are different from

those seen in pancreatic β-cells, where glucose effects are dependent on both

glycolysis and Ca++ influx via nifedipine-sensitive L-type channels (37-39), and those

seen in kidney mesangial cells, where glucose effects are largely PKC-dependent (40

and present findings). We are presently examining various cell types to see if glucose

activates ERK by a mechanism that is independent of metabolism and PKC. To date, as

reported here, such activation has been seen in rat adipocytes, 3T3/L1 fibroblasts,

3T3/L1 adipocytes, L6 myotubes and rat A-10 vascular smooth muscle cells, and

preliminary studies suggest that glucose activates ERK independently of metabolism

and PKC in rat soleus muscles.

From our findings, we speculate that glucose interacts with the Glut1 glucose

transporter and/or other cell surface proteins that recruit/phosphorylate/activate PYK2 at

the inner surface of the plasma membrane. PYK2 in turn activates GRB2 and/or SHC

and other components of the ERK pathway. The recruitment of PYK2 and the amplifying

effect of Glut1 on ERK activation apparently require, and may be mediated through,

residues 463 to 468, IASGFR, in the Glut1 C-terminus. Accordingly, Glut 1 may act as a

sensor, transducer, and amplifier during glucose signaling to PYK2 and the ERK

pathway. Although further studies are needed to test this hypothesis, our findings are

likely to be important for understanding the physiological and pathological actions of

glucose.


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ACKNOWLEDGMENTS

This work was supported by funds from the Department of Veterans Affairs Merit

Review Program, a National Institutes of Health Research Grant #2R01DK38079-09A1

and a Research Award from the American Diabetes Association. We thank Sara M.

Busquets for her invaluable secretarial assistance.


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LEGENDS TO FIGURES

Fig 1 Effects of Glucose, Insulin and Various Saccharides on Immunoprecipitable ERK

Activity in Rat Adipocytes. Adipocytes were equilibrated in glucose-free KRP medium

containing 1% BSA, and then treated with or without 20mM D-glucose alone (GLU,

closed circles), 20mM mannitol alone (MAN, open circles), 10nM insulin (INS) plus

20mM mannitol (closed squares), or 20mM D-glucose plus 10nM insulin (open

triangles) for indicated times in Panel A, or with indicated concentrations of glucose

(GLU, closed circles) or mannitol (MAN, open circles) for 10 min in Panel B, or for 10

min with indicated concentrations of glucose, mannitol and insulin in Panel C, or for 10

min with indicated concentrations of D-glucose, 2-deoxyglucose (2-DOG), 3-O-methyl-

glucose (3-O-MG), D-mannose, glucosamine (GLU-NH2), mannitol, and L-glucose in

Panel D. Note that osmolality was kept constant in all groups in Panels A and C. After

incubation, immunoprecipitable ERK activity was measured. Values are mean ± SE of

4 determinations.

Fig 2 Effects of Inhibitors of PKC, PI 3-Kinase, MEK1, GRB2 and Tyrosine Kinase

(Panel A) and Downregulation of Diacylglycerol-Sensitive PKCs (Panel B) and

Cytochalasin B (Panels C and D) on the Activation of ERK by Glucose, Insulin, and

Phorbol Esters in Rat Adipocytes. In Panel A, adipocytes were incubated in glucose-

free KRP medium for 15 min without (0) or with 20µM GF109203X (GFX), 10µM

GO6976 (GO), 100nM wortmannin (WM), 10µM PD98059 or 10µM genistein (GEN), or

for 180 min without or with the GRB2-SH2 domain inhibitor (see Methods and 22), and


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then treated for 10 min without (NONE) or with agonists, 20mM glucose, 1µM

tetradecanoyl phorbol-13-acetate (TPA) or 10nM insulin. Note that, although not

shown, these inhibitors did not alter basal ERK activity, and glucose effects on ERK

were virtually identical in cells pre-incubated for either 15 or 180 min. In Panel B,

adipocytes were incubated overnight in DMEM containing 1µM TPA to deplete DAG-

sensitive PKCs (see 20,21), following which, the cells were washed and incubated in

glucose-free KRP medium for 10 min without (CON) or with 10nM insulin (INS), 1µM

TPA, or 20mM glucose (GLU). After incubation, immunoprecipitable ERK activity was

measured. Values in Panels A and B are mean ± SE of 4-16 determinations. In Panels

C and D, adipocytes were incubated first for 15 min in glucose-free KRP medium

without or with indicated concentrations of cytochalasin B. In Panel C, the cells were

then treated for 10 min without (CONTROL) or with 20mM glucose or 10nM insulin

before measuring immunoprecipitable ERK activity. In Panel D, 20mM D-glucose and

10µCi [6-3H]D-glucose were added and uptake of label over 30 min was measured.

Values in Panels C and D are mean ± SE of 4 determinations.

Fig 3 Effects of Expression of Wild-Type (WT) or Dominant-Negative (DN) Forms of

GRB2 (Panel A), SOS (Panel B), RAS or RAF (Panel C), or Kinase-Inactive (KI) PKC-ζ

(Panel D) on Glucose-, Insulin- or Phorbol Ester-Induced Activation of Epitope-Tagged

ERK2 in Rat Adipocytes. Adipocytes were co-transfected with plasmids encoding

epitope-tagged ERK2 and indicated signaling proteins as described in Methods. After

overnight incubation to allow time for expression, cells were washed and incubated for

10 min in glucose-free KRP medium without (C) or with 20mM glucose (G), 10nM


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insulin (I), or 1µM tetradecanoyl phorbol-13-acetate (T). After incubation, HA- or Myc-

tagged ERK2 was immunoprecipitated and assayed. Values are mean ± SE of 4

determinations.

Fig 4 Effects of Overexpression of Glut1 (Panels A, C and D) and Glut4 (Panel B)

Glucose Transporters on Glucose-Induced Activation of Epitope-Tagged ERK2 in Rat

Adipocytes. Adipocytes were co-transfected with: (a) indicated amounts (µg DNA/0.8ml

50% adipocyte suspension) of pCDNA3 encoding Glut1 (Panels A and D), or pCIS2

encoding Glut4 (Panel B), or pCB6 encoding Myc-Glut1 (Panel C); along with (b) 3.3µg

pCMV5 encoding Myc-ERK2. Total DNA was kept constant by varying the amount of

vector. After overnight incubation in DMEM to allow time for expression, cells were

washed and equilibrated for 20 min in glucose-free KRP medium, and then treated for

10 min with or without 20mM glucose, as indicated. After incubation, in Panels A and B,

Myc-ERK2 was immunoprecipitated and assayed. In Panel C, cell surface level of Myc-

Glut1 was measured as described in Methods. Panel D shows the dose-dependent

expression of Glut1 (as employed in Panel A and measured with rabbit polyclonal

antiserum kindly provided by Dr. Ian Simpson) and levels of immunoprecipitated Myc-

ERK2 (as measured with anti-ERK antiserum): note that vector and Glut1 expression

did not influence expression or immunoprecipitability of Myc-ERK2. See refs 21 and 26

for expression data and evidence of functionality of transfected Glut4. We have also

documented that transfected Glut1 is translocated in response to insulin (not shown)

and therefore appears to be functional. All values are mean ± SE of 4 determinations.


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Fig 5 Effects of Wild-Type and Chimeric Forms of Glut1 and Glut4 Glucose

Transporters on Activation of Epitope-Tagged ERK2 in Rat Adipocytes. Adipocytes

(0.8ml 50% adipocyte suspension) were co-transfected with: (a) 6.6 µg of pCDNA3

encoding Glut1, or pCIS2 encoding wild-type HA-Glut4 (N-Glut4/C-Glut4), or pCMV5

encoding HA-Glut1/Glut4 chimera containing a Glut4 C-terminus (N-Glut1/C-Glut4), or

HA-Glut4/Glut1 chimera containing a Glut1 C-terminus (N-Glut4/C-Glut4): and (b) 3.3µg

pCMV5 encoding Myc-ERK2. Total DNA was kept constant by varying the amount of

vector (VEC). After overnight incubation in DMEM to allow time for expression, cells

were washed and equilibrated for 20 min in glucose-free KRP medium, and then treated

for 10 min with or without 20mM glucose or 10nM insulin in Panels A and C, or for 30

min with or without 10nM insulin in Panel B, as indicated. After incubation, in Panels A

and C, Myc-ERK2 was immunoprecipitated and assayed. In Panel B, cell surface levels

of HA-Gluts were measured as described in Methods. Panel B shows comparison of

plasma membrane levels of wild-type N-Glut4/C-Glut4, chimeric N-Glut4/C-Glut1 and

chimeric N-Glut1/C-Glut4 glucose transporters in transiently transfected rat adipocytes

treated with or without insulin. Panel C shows the effects of overexpression of the Glut4

glucose transporter and subsequent insulin (INS) treatment on basal and glucose(GLU)-

stimulated Myc-ERK2 activation. All values are mean ± SE of 4 determinations.

Fig 6 Effects of Expression of Wild-Type Forms of Glut1 or Glut4 or Glut3 (Panel A), or

Wild-Type (WT) Glut1 or C-Terminal Truncated (∆) Forms of Glut1 (missing amino acids

489-492 and 469-492) or the N-Glut1/C-Glut4 Chimera (in which the last 30 amino acids


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of Glut1 were replaced by corresponding 29 amino acids of Glut4) (Panel B), or Wild-

Type or S465A Single Mutant or A464S/S465A/G466A Triple Mutant or R468G Single

Mutant Forms of Myc-Tagged Glut1 (Panels C and D) on Glucose-Induced Activation of

HA-ERK2 (Panels A,B and C) or Plasma Membrane Levels of Expressed Myc-Tagged

Glut1 Transporters (Panel D) in Rat Adipocytes. Cells were transiently co-transfected

with PCMV5 encoding HA-ERK2 and pCB6 vector or vector encoding indicated forms of

glucose transporters, as described in Methods. After overnight incubation in DMEM to

allow time for expression, cells were washed, equilibrated, and incubated for 10 min in

glucose-free KRP medium with or without 20mM glucose, as indicated, after which, HA-

ERK2 was immunoprecipitated and assayed (Panels A, B and C). Panel D shows the

plasma membrane levels (as per quantitation of the exofacial Myc epitopes – see

Methods) of wild-type and mutant forms of Myc-Glut1 employed in Panel C. Although

not shown, Myc-tagged wild-type and mutant forms of GLUT1 were expressed to similar

extents, i.e., as per total cellular 50kDa Myc immunoreactivity, as would be expected in

view of the similar levels of plasma membrane-associated exofacial Myc

immunoreactivity depicted in Panel D. Values are mean ± SE of 4 determinations in

Panels A, B and D, and 6-8 determinations in Panel C.

Fig 7 Effects of Glucose, 3OMG, Maltose and Lactose on [3H]2-Deoxyglucose (2-DOG)

Uptake (Panels A and D) and Cytochalasin B-Dependent Activation of ERK (Panels B

and C) in Rat Adipocytes. In Panel A, [3H]2-DOG uptake over 5 min was determined in

cells incubated in KRP medium containing 0, 10 or 20mM carbohydrate as indicated.

Values are mean ± SE of 4 determinations. In Panel B, cells were incubated first for 15


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min without or with 100µM cytochalasin B, as indicated, and then incubated for 10 min

without (NONE) or with 20mM carbohydrate, as indicated. Values are mean ± SE of (n)

determinations. In Panels C and D, cells were incubated for 10 min in KRP medium with

or without added [3H]2-DOG and indicated concentrations of carbohydrates, after which,

cytochalasin B-dependent uptake of [3H]2-DOG (Panel C) or activity of

immunoprecipitable ERK (Panel D) was measured. Values are mean ± SE of 4

determinations.

Fig 8 Effects of Glucose, other Carbohydrates and Insulin (i) on ERK Activation in

3T3/L1 Fibroblasts (Panels A,B and E), 3T3/L1 Adipocytes (Panel B), L6 Myotubes

(Panel C), and Rat A-10 Vascular Smooth Muscle Cells (Panel D), and (ii) on [3H]2-

Deoxyglucose (2-DOG) Uptake in 3T3/L1 Fibroblasts (Panel F). In Panels A,B,C,D, and

E, cells were first incubated for 15 min in glucose-free KRP medium with or without

10µM GF109203X or 100µM cytochalasin B, as indicated, and then treated for 10 min

without (NONE, CON) or with 20mM D-glucose (GLU), 3-O-methyl-glucose (3OMG), 2-

deoxyglucose (2-DOG), maltose (MAL) or 100nM insulin (INS), as indicated, following

which ERK was immunoprecipitated and assayed as described in Methods. In Panel F,

cells (in 24-well plates) were incubated in glucose-free KRP medium and [3H]2-DOG

uptake over 5 min was determined in the absence (NONE) or presence of 20mM

carbohydrate as indicated. Values are mean ± SE of 4 determinations.

Fig 9 Effects of adenovirally-transferred Glut1 and Glut 2 on glucose-dependent

activation of ERK and glucose uptake in L6 myotubes. Confluent, fully


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differentiated/fused myotubes in 24-well and 100mm plates were use for studies of

glucose uptake and ERK activation, respectively. The cells were infected with 4MOI

(multiplicity of infection) adenovirus alone or adenovirus encoding Glut 1 or Glut2 as

indicated. After 48 hours of incubation to allow time for expression, cells were

equilibrated and incubated for 10 min in KRP medium containing indicated

concentrations of D-glucose, along with, in Panel B, 2µCi [3H]-labeled D-glucose for

determination of glucose uptake. After incubation, cell lysates were examined for

immunoprecipitable ERK activity, and cytochalasin B-inhibitable glucose uptake

(calculated by dividing cpm in cells by the specific activity of medium glucose).

Bargrams and brackets reflect mean ±SE of 4 determinations. Shown below are

immunoblots reflecting changes in contents of immunoreactive Glut1 and Glut2 (both

blotted with rabbit polyclonal antisera) in virus-infected cells (note-Glut1 levels were not

reproducibly altered by expression of Glut2). Although not shown, levels of ERK 1 and 2

were not altered by expression of viral constructs.

Fig 10 Effects of adenovirally-transferred Glut1, Glut2 and chimeric forms of Glut1 and

Glut2 on glucose-dependent activation of ERK and glucose uptake in 3T3/L1

fibroblasts. As in Fig 8, fully confluent cells were infected with 3 (left) or 12 (right) MOI

(multiplicity of infection) adenovirus alone or adenovirus encoding wild-type Glut1, wild-

type Glut2, chimeric N-Glut1/C-Glut2 or chimeric N-Glut2/C-Glut1. After 48 hours to

allow time for expression, cells were washed, equilibrated and incubated for 10 min in

glucose-free KRP medium without or with 20mM D-glucose and, in Panels B and D,

2µCi [3H]-labeled D-glucose to measure glucose uptake. After incubation, cell lysates


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were examined for glucose-dependent ERK activity and cytochalasin B-inhibitable

glucose uptake (see Fig 8 for other details). Values are mean ± SE of 4 determinations.

As shown by the representative immunoblots, expression of ERK 1 and 2 was not

altered by infections with adenoviral constructs.

Fig 11 Effects of Glucose on Phosphorylation of Specific Tyrosine Residues in Proline-

Rich Tyrosine Kinase-2 (PYK2) in Rat Adipocytes and 3T3/L1 Fibroblasts (Panel A),

Effects of Expression of Wild-Type (WT) and Dominant-Negative (DN) Forms of Proline-

Rich Tyrosine Kinase-2 (PYK2) on Glucose-Induced Activation of HA-ERK2 in Rat

Adipocytes (Panel B), and Effects of Glucose on Co-Immunoprecipitation of PYK2 with

Myc-Tagged Glut1 in 3T3/L1 Fibroblasts (Panel C). . In Panel A, cells were incubated

in glucose-free KRP medium for indicated times with or without 20mM glucose, and

lysates were resolved by SDS-PAGE and blotted for immunoreactivity with phospho-

specific anti-pY-PYK2 antibodies (using rabbit polyclonal antisera obtained from

BioSource) as indicated. Representative blots are shown here. Similar results were

observed in at least 4 experiments. In Panel B, adipocytes were transiently co-

transfected with plasmids encoding HA-ERK2 and WT or DN forms of PYK2 [KD=

kinase-defective; PRNK = PYK2-Related Non-Catalytic Kinase, i.e., C-terminal domain

that lacks ability to phosphorylate substrate and therefore serves as effective dominant-

negative] and subsequently incubated for 10 min in KRP medium with or without 20mM

glucose, following which, HA-ERK2 was immunoprecipitated and assayed. Insets show

expression-related increases in contents of immunoreactive WT and KD forms of PYK2

in total cellular lysates (note that approx 5-10 % of adipocytes are successfully


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transfected and relative increases in these transfected cell are greater than those seen

in total cell extracts). Values are mean±SE of (n) determinations. In Panel C, 3T3/L1

fibroblasts were transiently co-transfected with plasmids encoding Myc-tagged Glut 1

and PYK2, and subsequently incubated in KRP medium for 10 min with or without

20mM glucose as indicated. After incubation, lysates were immunoprecipitated with

anti-Myc mouse monoclonal antibodies (from UBI), and, after resolution by SDS-PAGE,

blotted for immunoreactivity with anti-PYK2 antibodies (28). Shown here are

representative blots from 2 experiments. Similar results were obtained in 4 experiments.


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and Robert V. FareseNewgardBurke, Jr, Michael Quon, Brent C. Reed, Ivan Dikic, Laura E Noel, Christopher B

Gautam Bandyopadhyay, Mini P. Sajan, Yoshinori Kanoh, Mary L. Standaert, Terrance R.Glut1 glucose transporter

Glucose activates MAP kinase (ERK) through proline-rich tyrosine kinase-2 and the

published online September 27, 2000J. Biol. Chem.

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and the glut1 glucose transporter. bandyopadhyay g*, sajan … · 2000-09-27 · sensitive pkcs....

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