Insulin and Insulin-like Growth Factor-I Signal Transduction Requires ...

THEJOURNALOF BIOUXICAL CHEMISTRY

Vol. 269, No. 8, Issue of February 25, pp. 5699-5704, 1994 Printed in U S A

Insulin and Insulin-likeGrowth Factor-I Signal Transduction Requires p21ra8* (Received for publication, October 15, 1993)

Byung H. JhunS§, Judy L. Meinkotha J. Wayne Leitnerll, Boris Drazninll, and Jerrold M. OlefskyS§** From the mivision of Endocrinology and Metabolism and the ICuncer Center, Department of Medicine, University of California at SunDiego, La Jolla, California 92093, the §Veterans Administration Medical Center, Sun Diego, California 92161, and the lllepartment of Medicine, Veterans Administration Medical Center, Denver, Colorado 80220

We have investigated the role of cellular p21rW protein The protein product of the c-rus proto-oncogene, p21rQ", is associated with the plasma membraneand belongs tothe famin insulin and insulin-like growh factor-I (IGF-I) signalily of small GTP-binding proteinsthat cycle between an active ing pathways. Insulin stimulation increased Ras-GTP formationinRat-1fibroblastsoverexpressingnormal GTP-bound conformation and a n inactive GDP-bound state. human insulin receptors (HlRc-B), far greater than in Cellular rus protein is believed to participate in the transmisparental Rat-1 fibroblasts, indicating that competent sion in- of various stimuli to intracellular targets (see Refs. 8 a n d sulin receptors mediate this response. Cellular microin9 for reviews). Evidence exists suggesting a role for p21m8GTP jection of a dominant-negative mutant p21" protein in platelet-derivedgrowthfactor,epidermalgrowthfactor, (N17 ras) or anti-p21" monoclonal antibody (Y13-259) nerve growth factor, and serum-induced mitogenesis (10-13), into HIRc-B cells reduced insulin- and IGF-I-stimulated a n d i t has been shown that insulin stimulates and increases DNA synthesis by 7 6 " . Insulin-induced c-fos protein the GTP formof ~21"" (14-16). expression was also inhibitedby 74%. Microinjection of We have directly assessed the role of p21"" protein in insuoncogenic p21rm (T-24 ras) into HI&-B cells activated and IGF-]'-mediated mitogenic signaling by single-cell milinN17 ras and the mitogenic pathway, and coinjection of T-24 ras showed that oncogenicp21" rescued the cells croinjection of anti-palm8 monoclonal antibody as well as a from the N17 ras blockade. This later finding indicates recombinant dominant-negative mutantp21"" protein. These studies showed that inactivation of endogenous ~21'"" abrothat T-24ras acts downstream ofN17 ras. In conclusion, 1) microinjection of a dominant inter- gated insulin and IGF-I signaling, demonstratingthe essential role of p21""GTP in the transmembrane signaling cascade iniferring rus mutant into quiescent cells abrogated subsequent insulin and IGF-I mitogenic signaling; 2) onco- tiated by insulin and IGF-I. genic ras proteinrescuedcells fromthe N17 ras EXPERIMENTALPROCEDURES blockade, indicating that T24 m a action is downstream Cell Lines and Materials of the site of N17 inhibition; and3) p21" is an intermediatesignalingmoleculeintheinsulin/IGF-Isignal The Rat-1 fibroblasts expressing wild type human insulin receptors transduction pathway and is required for gene expres- (HI&-B) were maintained as previously described(4). Pork insulin and sion and DNA synthesis. IGF-I were generously providedby Lilly. Bromodeoxyuridine(BrdUrd)

Insulin stimulates a spectrum of physiologic effects, including transport of glucose and amino acids, increased synthesis of glycogen and certain metabolic enzymes, as well as enhanced synthesis of DNA a n d RNA ( 1). The initial stepin insulin action is the binding of insulin to its cell surface receptor (2). The insulin receptoris a heterotetrameric glycoprotein consisting of

and monoclonal anti-BrdUrd antibody were purchased from Amersham Corp. Polyclonal anti-c-fosantibody was from Oncogene Science. Monoclonal anti-pal"'" antibody (Y13-259) was obtained from Santa Cruz Biotechnology. Rat and rabbit immunoglobulin G (IgG), FITC- or rhodamine-conjugated anti-rat, -mouse, and -rabbit IgG antibodies were purchased from TheJackson Laboratories.All other reagents were purchased from Sigma.

Analysis of Guanine Nucleotide Bound to ~21""

Cells g r o w n to confluency were serum-starved for 16 h and phostwo extracellular a-subunits and two transmembrane P-subphate-starved for an additional 1h. The serum- and phosphate-starved the a-subunits and activates the cells were incubated for 4 h with [32Plorthophosphateand then chalunits (3). Insulin binds to tyrosine kinaseof the p-subunit resulting in receptor autophos- lenged with insulin for 10 min at 37 "C.~21"'"was immunoprecipitated phorylation and phosphorylationof endogenous substrates.Al- with anti-p21"" monoclonal antibody (Y13-259) fromthe cell lysates as described elsewhere (17, 18). GTP- and GDP-bound ~21"" were sepathough many studies suggest that receptor kinase activity is rated by thin layer chromatography and analyzed by autoradiography. central to insulin signal transduction (4-7), the post-receptor Quantitation of the nucleotides was determined by cutting out and processes mediating the signal from the receptor tyrosine ki- counting the labeled nucleotides in a liquid scintillation counter. The nase to intracellular target(s)are largely unknown. results were expressed as GTPMGTP + GDP) x loo%, reflecting the amount of GTP-bound ras before and after exposure to insulin (14).

Microinjection

* This work was supported by National Institutes of Health Grant DK33651, the Sankyo Diabetes Research Foundation, and theVeterans Administration Research Service. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely toindicate this fact, ** To whom correspondence should be addressed: Dept. of Medicine (9111G), University of California at San Diego, 9500 Gilman Dr., La Jolla, CA 92093. Tel.: 619-534-6651; Fax: 619-534-6653.

The HI&-B cells were plated on scored 12-mm coverslipsand incubated for 24 h. The cells wererendered quiescent by starvation for 48h The abbreviations used are: IGF-I, insulin-like growth factor-I; BrdUrd, bromodeoxyuridine; GNRF, guanine nucleotide-releasingfactor; N17 ras, dominant-negative mutant p21"" protein; PBS, phosphate-buffered saline; T-24 rus, oncogenic ~21""protein; FITC, fluorescein isothiocyanate; HI&, human insulin receptor.

5699

5700

Pathway Dansduction Signal Insulin

in serum-free Dulbecco'smodified Eagle's medium containing 2 m~ L-glutamine, 100 units of penicillirdml, and 100pg of streptomycin sulfate/ml. On the day of injection, the coverslips were transferred to 35-mm tissue culture dishes. Cells were microinjected by using glass capillary needles as previously described (19) except that the needles were made by using a Kopf vertical pipette puller (model 720). Dominant-negative mutant ~21"" protein (N17)(a generous gift of Dr. Feramisco, La Jolla, CA) was microinjected at a concentration of 3.0 mg/ml in 5 m~ NaPO,, 100 nm KCI,pH7.4. To facilitate identification of microinjected cells, nonspecific rat or rabbit IgG was coinjectedwith the N17 protein at a concentration of 3.0 mg/ml. Withan average injection liters, a molecular size of 21 kDa, and a concentravolume of 1 x tion of 3.0 mg/ml, approximately 1 x IO6 molecules of N17 protein were injected per cell. In addition, the injection included approximately 4 x lo5 molecules of IgG. Oncogenic rus (T-24 rus) protein was also used in microinjection studies at the indicated concentrations. Immunofluorescence Microscopy c-fos Protein Staining--Two hours after injection, the cellswere stimulated with either insulin (100 ng/ml), IGF-I (100 ng/ml), or 20% fetal calf serum for 1.5 h. The cells were washed with PBS, fixed in 4% formaldehyde in serum-free Dulbecco's modified Eagle's medium containing 2 n m L-glutamine, 100 units of penicillidml, and 100 pg of streptomycin sulfatelml for 20 min at 22 "C, washed with PBS, and permeabilized with 0.3%Triton X-100 in PBS for 5 min. The cells were incubated with 100mM glycine in PBS for10 min a t 22 "C,washed with PBS, and again incubated with 10 m~ ethanolamine in PBS for 15 min at 22 "C. After washing with PBS, the coverslips were then incubated with 10%horse serum in PBS for 40min at 22 "C to reduce nonspecific staining. An anti-c-fos antibody (1:lOO dilution) in 1%horse serum in PBS was applied for 90 min at 37 "C. The cells were washed with PBS and incubated with rhodamine-conjugated anti-rabbit IgG antibody (1:lOO dilution) in 1%horse serum in PBS for 60 min at 22 "C. &r washing with PBS, the cells were stained with FITC-labeled anti-rat IgG antibody (1:lOO dilution in 1%horse serum in PBS) to identify the microinjected cells. The coverslips were extensively washed with PBS, rinsed with water, and mounted in PBS containing 15%GeIvatol (polyvinyl alcohol),33% glycerol, and 0.1% sodium azide. DNA Synthesis-Two hours following injection, BrdUrd (1:lOOO dilution) was added and the cells were incubated at 37 "C for 16 h with either insulin (100 ng/ml), IGF-I (100 ng/ml), or 20% fetal calf serum supplemented medium. Afterfixation in 90% EtOH, 5% acetic acid, 5% distilled HzO for 25min a t 22 "C, the cells were washedin TPBS (PBS containing 0.1% -Tween-201, then incubated with mouse anti-BrdUrd antibody for 60 min at 22 "C, followed by incubation with rhodamineconjugated anti-mouse IgG antibody (1:lOO dilution) in TPBS for 60 min at 22 "C. The cells were then stained for co-injected rabbit IgG with FITC-labeled anti-rabbit IgG antibody (1:lOO dilution) in TPBS for 60 min a t 22 "C. The coverslips were washed and mounted as described above. The cells were inspected and photographed with a Zeiss Axiophot fluorescence microscope under a x 63 (1.4 numerical aperture) oil-immersion lens forc-fos staining and an x 40 oil-immersion lens for BrdUrd staining. Phase-contrast photographs were made with Kodak Tech Pan film and fluorescence photomicrographs with Kodak T-Max film.

Insulin lOOnM lOnM lOOnM Rat I HIRc-3 FIG.1.Effect ofinsulin on p2P"TP formation inHIRc-B and Rat-1 cells. Cells were serum-starved for 16 h and subsequently phosphate-starved for 1h. The cells werethen incubated for 4 hwith [32P]orthophosphate and then stimulated with insulin for 10 min at 37 "C. Insulin-stimulated p2lmSGTPformation in Rat-1 (n = 12), HI&-B a t 100 nM insulin (n = 12), and at 10 n~ insulin (n = 4) was measured as described under "Experimental Procedures." The percentages of GTPbound ~ 2 1 were ~ " determined (GTPRGTP + GDP) x loo%), and the percentage of GTP-bound ~21"" in the absence of insulin was subtracted. The results are expressed as mean + S.E. In HIRc-B cells,p < 0.01 at 100 n~ insulin and -4.05 at 10 n~ insulin.

quiescent HI%-B cells were microinjected withN17 rus (3 mg/ ml). Two hours after microinjection, t h e cells were stimulated with insulin (100 ng/ml), IGF-I (100 ng/ml), or serum (20%). Earlier control studies indicated that these concentrations of hormones or serum elicited a maximal stimulatory effect on DNA synthesis andc-fos induction (data not shown). Following stimulation with insulin, IGF-I, or serum, the cells were labeled with BrdUrd for 20 h a n d DNA synthesis in individual cells was assessed using indirect immunofluorescence. Fig. 2 depicts insulin-, IGF-I-, and serum-stimulated cells rabbit IgG or N17 rus protein. microinjected with either control Approximately 75-92% of the uninjected cells underwentDNA synthesis (contained labeled nuclei),and t h e coinjected rabbit I g G had no effect on DNA synthesis compared to uninjected cells (Panels M, P , a n d R ) . In contrast, injection of N17 ras (Panels 0, Q , a n d S) markedly reduced the number of cells or sestimulated to undergo DNA synthesis by insulin, IGF-I, rum. The results of three independent experiments are summarized inFig. 3. In the absenceof stimulatory agents, about10% of the cells synthesizedDNA. In uninjected cells,insulin, IGF-I, or serum stimulated 922 3, 75 2 3, a n d 9 2 2 2%, respectively, of the cells to incorporate BrdUrd, and microinjection of the control rabbit IgG had no effect on these values. In contrast, microinjection of N17 ras (3 mg/ml) reduced theeffect of insulin by 6 2 8 , of IGF-I by 57%, and of serum by 4 0 8 . The concentration of N17 ras used here was sufficient to abolish DNA synRESULTS thesis in response to co-injected cellular ras protein in a rasEffect of Insulin on p2l""GTP-The GTP bound form of sensitive cell line.2 These results provide direct evidencethat ~ 2 1 ' ~functions " in mitogen-stimulated growth responses and endogenous p2lraS protein isa critical link in the signal transinsulin can increase p21""GTP (15,201. To demonstrate thisin duction pathway of insulin and IGF-I to stimulate DNA synHIRcB cells, the effect of insulin on cellularp21'""GTP content thesis and presumably cell proliferation. was measured. Fig. 1 shows that insulin increases p21m"GTP Znhibition of e-fos Protein Induction by Cellular Microinjeccontent in HIRc-B cells to a much greater extent than t h e tion of N17 ras Protein-The role of p21m" protein in insulin negligible effects observedin Rat-1cells. These results indicate stimulated fos protein expression was also examined. As can be that expression of competent insulin receptors confers insulin- seen i n Fig. 4, microinjection of cells with control IgG had no induced stimulation of p21""GTP production. effect on insulin and serum stimulated fos expression, clearly Znhibition of DNA Synthesis by Cellular Microinjection of demonstrating that the microinjection process itself was withDominant-negative p2lms Protein (N17 r a s b T o evaluate the out adverse effects. In contrast, microinjection of N17 ras rerole of p2lTas in insulin signaling, a recombinant dominantduced insulin or serum induced c-fos protein expression by 74 interfering ~21'"s protein (N17 ras),known to inhibit the func- and 87%, respectively (Table I). These results demonstrate that tion of endogenous ~21"" protein (21, 221, was microinjected ~21"" protein is required for insulin-stimulated c-fos expresinto HIRc-B cells and insulin stimulated c-fos expression or sion. DNA synthesis was assessed. 2 B. Jhun and J. Olefsky, unpublished observation. Cells were plated on glass coverslipsand groups of 150-200

- - Pathway Insulin Dansduction Signal

ICF-I

Insulin

IgC

N17

5701

Serum

N17

IFC

N17

FIG. 2. Effect of microinjected dominant-negative p21" mutant (N17 ras) protein on DNA synthesis in HI&-B cells. Cells were serum-starved for 48 h and theninjected with eithernonspecific rabbit IgG (3 mg/ml) orN17 m s protein (3 mg/ml) containing rabbitIgG (3 mdml). After stabilization for 2 h, cells were stimulated with BrdUrd plus either insulin(100 ng/ml), IGF-I (100 ng/ml), or fetal calf serum (20%)for 20 h a t 37 "C. They were then processed for double-label indirect immunofluorescence by sequential incubation with mouse anti-BrdUrd antibody, biotinylated anti-mouse I g G antibody, TexasRed-conjugated streptoavidin, and FITC-conjugated anti-rabbit IgG antibody. The injectedcells were identified by cytoplasmic FITC staining (middle panel, G-L) and the BrdUrd-incorporatedcells were identifiedby the nuclearTexas Red staining (bottom panel, M S ) .Phase-contrast photomicrographs (A-F) are shown in thetop panel. stimulated DNA synthesis ina dose-responsive manner, with a maximal effect equal to that of insulin. When cells microinjected with T-24 ras were subsequently stimulated with insulin, the effects were additive to submaximal concentrations of T-24 ras, while a t maximally effective levels ofT-24 ras no additivity with insulin was observed. Co-injection of Inhibitory (N17) and Oncogenic (T-24) ras Proteins-Having demonstrated thepositive phenotype induced by T-24 ras, we next asked whether T-24 ras could rescue cells from the inhibition induced by the dominant interfering mutant N17 ras. Since N17 ras inhibits quanine nucleotide exchange by inhibiting GNRF activity, and since T-24 ras is active independent of GNRF activity, one could reason that the site of activity of T-24 ras is downstream of the N17 ras blockade, and, Bawl Insulin IGF-I Serum therefore, T-24 ras should restore DNA synthesis. ConseFIG.3. Inhibition of DNA synthesis by microiqiection of dominant-negative p21" mutant (N17 ras) protein in HI&-B cells. quently, N17 ras and T-24 ras were coinjected into quiescent Following microinjection of N17 ras (filled bar) or control IgG (open cells and the results are seen in Fig. 6. When injected alone, bar) into quiescent HI&-B cells, DNA synthesis in the injected cells N17 ras inhibited mitogen-stimulated DNA synthesis, whereas (open and filled bar) and uninjected cells (hatched bar) on the same T-24 ras was stimulatory.When coinjected, T-24 ras fully stimucoverslip was determined as described in the legend of Fig. 2. An average of 178 N17ras-injected (range 159-198) and 180 uninjected (range lated DNA synthesis, despite the presence of N17 ras, demon158-198) cells per coverslip were counted. Similarly,an average of 176 strating rescue of the N17 ras blockade, consistent with the formulation that the siteof action of T-24 ras is distal to N17 control IgG-injected (range 162-192) and 176 uninjected (range 154195)cells were counteda s controls. The results presented represent theras and independent of GNRF activity. mean (&.E.) of three experiments.

Inhibition ofDNA Synthesis by Microinjection of Inactivating Anti-p21raS antibody (Y13-259)-To further demonstrate the specificity of this effect, we attempted to inhibitp21ra" activity through an independentmechanism, namely by microinjecting inactivating antibody. Monoclonal antibody Y13-259 is known to interfere with p2lraS function (lo), and when we microinjected this antibody into cells, we found that it inhibited the subsequent effects of insulin and IGF-I to stimulateDNA synthesis (Fig. 5). These results demonstrate the specificity of the findings observed with the ras mutant, and further highlight the importance ofp21""GTP for efficient insulin and IGF-I mitogenic signaling. Stimulation of DNA Synthesis by Microinjection of Oncogenic p21"" (T-24) Protein into Quiescent HZRc-B Cells-To further explore the importance of activated p21ms in mitogenic signaling, we utilized a mutated, oncogenic form of p21"" (T-24 ras) which is constitutively active (23). Table I1 presents a dose response in which increasing amounts of T-24 ras were microinjected into quiescent HI&-B cells. As can be seen, T-24 rus

DISCUSSION

Ample evidence exists indicating that p21"" is an important intermediate component in the growth factor-signaling pathway leading to DNA synthesis andcell proliferation. The p21"" protein exists in anactive GTP-bound form as well as an inactive GDP-bound form, and severalgrowthfactorsrapidly stimulate production ofp21""GTP (12-15, 20). In addition, transfection or microinjection of oncogenically activated p21"" mimics growth responses (24-26), whereas blockade of ~ 2 1 ~ " " function inhibits growth factor action (9, 11, 12) (for recent review, see Ref. 27). With respect to insulin signaling, it has been demonstrated that insulin treatment leads to arapid stimulation of p21m"GTP in a variety of cell lines (14-16, 20). This stimulation correlates with insulin's long term effects to stimulate DNA synthesis andcell replication. In addition, overexpression of wild type p2lraS inrat fibroblasts enhanced the effect of insulin to stimulate cell growth (28).Moreover, transfection of oncogenically activated p21"" into 3T3-Ll cells leads to differentiation in a manner analogous to insulin and IGF-I

Insulin Signal ll-ansduction Pathway

5702

Serum

Insulin

I

r

kG

7-

N17

FIG.4. Effect of microinjected dominant-negative ~21'"" mutant (N17 ras) p r o t e i n o n c-fos expression in HI&-B cells. Serumdeprived cells were injected with N17 ras protein ( 3 mgiml) containing rat I& ( 3 mg/ml) or rat IgG ( 3 mg/ml) alone.After stabilization for 2 h,cells were stimulated with either insulin (100 ng/ml) or fetal calf serum (20%) for 1.5 h a t 37 "C, and were then processed for double-label indirect immunofluorescence.Thisassay employed the following incubationorder:rabbit anti-c-fos antibody, rhodamine-conjugatedanti-rabbit I@ antibody, and FITC-conjugated anti-rat IgG antibody. The injected cells were identified by the cytoplasmic FITC staining ( F J ) and the C-fos Phase-contrast photomicrographs (A-E) are shown in the top protein-expressing cells were identified by the nuclear rhodamine staining(K-0). panel.

TARI.E I Inhibition of insulin- or serum-inducedc-fos expression by microinjection of dominant negative p21"" (N17rasl protein into HIRc-B cells N17 ras protein (3 mg/ml) was injected into quiescent HIRc-B cells which were then stimulated with either insulin (100 ng/ml) or fetal calf serum (20%) for 1.5 h at 37 "C. An average of 161 N17 ras-injected (range 162-187) cells and179uninjected(range 157-198) cells per coverslip were countedin each of three separate experiments. Similarly, an average of 175 control IgG-injected (range 158-189) and 179 uninjected (range 158-198) cells in three separate experiments was counted a s controls. Unstimulated quiescentcells showed undetectable nuclear c-fos immunofluorescence. Results are expressed as the percentage of stained cells exhibiting nuclearfluorescence. Each value is presented as the mean & S.E. of three experiments. Cells

N17 ras-injected IgG-injected Uninjected 86.84

Percent of c-fos-stained cells Insulin

Serum

14.34 -c 1.58 88.46 -c 2.18 2 2.97

6.36 2 1.32 93.46 -c 3.37 91.57 -c 2.11

stimulation, and transfectionof dominant negative p21""" mutants blocks insulin's differentiation effects in these cells (15, 26) andalso blocks insulin stimulation of early gene transcription (29). Taken together, these studies areindicative of a role for ~21"" in thelong term biologic effects of insulin related to differentiation and cell replication. Single cell microinjection provides a direct means to determine whether anendogenous signaling molecule is requiredfor a particular phenotype (in this case insulin bioeffects). In the current studies, we have shown that microinjection of a dominant-negative ~21""" mutant (N17 rus), or anti-p21"" monoclonal antibody, abrogates the effects of insulin and IGF-I to promote DNA synthesis. This clearly represents a long term growth-promoting effect of insulidIGF-I, and our datademonstrate that activated ~21"" isa critical and necessary component of this signalingcascade. We have also shown that microinjection of N17 rus protein blocks insulin stimulation of c-fos protein. Sinceinsulin leads to relatively rapid induction of c-fos protein (90 min), our results demonstrate thatp21"" is also a necessary signaling component for this acute effect of insulin. Other studies have indicated that p2lra" lies upstream of the serinelthreonine kinasessuch as ruf-1 and the membersof the MAP or extracellular signal-regulated kinasefamily which are

"

Basal

lnsulm

IGI:-I

FIG.5. Inhibition of DNA synthesis b y microinjection of inactivating anti-p21r"" antibody (Y13-259). Serum-deprived HIRc-B cells were injected with monoclonal anti-p21"" antibody (Y13-259) (2.5 mg/ml). After stabilization for 2 h, cells were stimulated with insulin (100 ng/ml) for 16 h a t 37 "C, and DNA synthesis was determined as described in the legend of Fig. 2. An average of 155 anti-p21"" I&injected (range133-167) cells and 188 uninjected (range177-192) cells per coverslip were counted. The results represent the mean (=S.E.) of three experiments.

components of the signaling pathways for insulin as well as other growth factors (15, 30-32). For example, transfection of oncogenic ras into 3T3cells leads to activation of MAP kinase ( E ) , and transfection of dominant negative p2lraS inhibitsinsulin stimulationof ERK2 (33). Furthermore, single cell microinjection studies have shown that in the presence of an intact rus signaling system, insulin stimulation ofc-fos protein requires competent raf kinase a ~ t i v i t ySince .~ it is thought that MAP kinase kinase and MAP kinase are distal to raf kinase (34), when taken together, the above observations indicate that p21""GTP lies downstream of the insulin receptor kinase but upstream of the serinelthreonine kinase signalingcomponents and, therefore, ~21"" provides alinkbetween the tyrosine phosphorylation events initiated by the insulin and IGF-I receptors and the downstream serinelthreonine phosphorylation cascade. The mechanism by which ~21'"" is converted into the GTPbound form by insulin stimulation is unknown. The level of p21""GTP within cells is determinedby a balance between the D. Rose and J. Olefsky, unpublished observations.

5703

Insulin Signal ll-ansductionPathway TABLE I1 Stimulation of DNA synthesis by microinjection of oncogenic p21"" (T-24ras) protein into quiescentHIRc-B cells The indicated amounts of T-24 rus protein were injected into quiescent HI&-B cells which werethen incubated in the absence or presence of insulin (100 ng/ml) for 16 h at 37 "C. DNA synthesis was determined as described inthe legend of Fig. 6. An average of 148 T-24 rus-injected (range 12&154) and 157 uninjected (range 134-198) cells per coverslip were counted. Results are expressed as the percentage of stained cells exhibiting nuclear fluorescence. Each valueis presented as the means 2 S.E. of three experiments. T-24 ras

Insulin

Basal Uniniected

Iniected

mg / ml

0 0.1 24.6 0.4 0.8 1.6

Uniniected

Iniected

82.6 f 1.1 83.624.1 77.5 f 8.6 84.3 f 0.8 80.4f3.9

79.42 2.3 80.1 k 3 . 4 81.42 3.1 80.3k 4.5 83.5k 1.2

%

17.5 -c 1.6 19.1 -c 1.1 2 2.6 18.72 3.8 21.6 2 5.4

22.6 k 3.6 31.2k4.5 81.9 k 2.1 79.8 k 4.2 79.3 k 2.6

"I

I

Basal

Basal

Basal

+

+

+

N17

T-24

N17 N17

Insulin Insulin

+

Insulin

+

+

T-24 T-24

FIG.6. Oncogenic p21" (T-24) rescues the HIRc-B cells from the inhibitory effect of N17 rm. Serum-deprived HIRc-Bcells were injected with either T-24 rus protein (0.8 mg/ml), N17 rus (1.5 mg/ml), or T-24 ras (0.8mg/ml) alongwith N17 rus (1.5 mg/ml). All microinjection protein mixtures included control rabbit IgG (3 mg/ml).After stabilization for 2 h, cells were incubated in the absence or presence of insulin (100 ng/ml). DNA synthesis was determined as described in the legend of Fig. 2. An average of 168 injected (range 143-198) cells per coverslip were counted. Theresults represent the mean (&.E) of three experiments.

rate of formation, via GTP for GDP exchange on p2lrUSGDP, versus the rate of conversion of p2lrUSGTPto p21raSGDP(9). The latter occurs by stimulation of the intrinsicGTPase activity of the rasprotein by GTPase-activating protein (GAP). The former occurs through interactionof guanine nucleotide-releasing factors (GNRF), which promote the dissociation of GDP from p2lrUs,which is then replaced by GTP. Although evidence exists thatsome growth factors, such asplatlet-derived growth factor, influence p21""GTP by modulating GAP activity, this may not be the case for insulin. Benito et al. (26) have shown that insulin increases p21ra"GTP content in 3T3-Ll cells, but has no effect on GAP phosphorylation or activity. Additionally, several reports haveshown that epidermalgrowth factor (35), nerve growth factor (361, platelet-derived growth factor (37), and insulin (38) can increase GNRF activity. Our own studies have shown that, inHIRc cells, insulin does not influence GAP phosphorylation or activity, but has a striking effect to stimulate GNRF activity (39).Consequently, GNRF may be a signaling component linking activated insulin receptors to p21""GTP accumulation. Insulin has a number of metabolic effects in addition to mitogenesis, such as increased hexose and amino acid transport, increased lipid accumulation, and increased glycogen synthesis. It will be of greatinteresttodetermine whether p21raSGTPis also a component of insulin-stimulated metabolic signaling. We also conducted microinjection studies with a constitu-

tively active oncogenic ~21"" protein (T-24 ras). Microinjection of T-24 ras activated the mitogenic pathway in a dose-responsivefashion and at submaximal concentrations ofT-24 ras these effects were additive to the stimulatoryeffects of insulin. When N17 and T-24 ras proteins were co-injected, DNA synthesis wasfully activated in the presence or absence of insulin. This indicates that thesite of action of T-24 ras is distal to the locus at which N17 ras exerts itsinhibitory effects. These findings areconsistent with the formulation that N17 ras inhibits GNRF activity, whereas T-24 ras is oncogenic because it lacks GTPase activity. Since the latter follows the former in the ras activation pathway, this would explain why T-24 ras rescues cells from N17 ras blockade (or conversely, why N17 ras does not block the effects of oncogenic T-24 ras). In the current studies, we have found that overexpression of competent insulin receptors in Rat I fibroblasts markedly enhances insulin's ability to stimulatep21""GTP formation. N17 ras is a dominant-interfering mutant ~21"" protein which strongly interacts with and inhibits GNRF. When microinjected into HIRc-B cells, anti-p2lraSmonoclonal antibodies or N17 ras inhibited insulin and IGF-I-induced DNA synthesis and c-fos protein expression in HIRc-B cells, demonstrating that p21"" is required for these acute and long term effects mediated by insulin. Study of the molecular mechanisms, which link activated insulin receptors to stimulation of p21""GTP and which couple activated ~21"" to downstream targets, holds great promise for the eventualelucidation of insulin's cellularmechanisms of action. Acknowledgments-We thank Dr. James Feramisco for the kind gift of N17 rus protein used here, and Dr. Lynn Seely for critically reading the manuscript. We are grateful to Elizabeth Martinezfor help in preparing this manuscript. REFERENCES 1. Rosen, 0.M. (1987) Science 237, 1452-1458 2. Olefsky, J. M. (1990) Diabetes 39, 1009-1016 3. Ullrich, A., Bell, J. R., Chen, E. Y., Herrera, R., Petruzzelli, L. M., Dull, T.J., Gray, A., Coussens, L., Liao, Y.-O., Tsubokawa, M., Mason, A., Seeburg, P. M., Grunfeld, C., Rosen, 0. M., and Ramachandran, J. (1985)Nature 313, 756761 4. McClain, D. A,, Maegawa, H., Lee, J., Dull, T. J., Ullrich, A,, and Olefsky, J. M. (1987)J. Biol. Chem. 262, 14663-14671 5. Chou, C. K., Dull. T. J., Russell, D. S., Gherzi, R., Lebwohl, D., Ullrich, A,, and Rosen, 0.M. (1987)J . Bid. Chem. 262, 1842-1847 6. Ebina, Y., Araki, E., Taira, M., Shimada, F., Mori, M., Craik, C. S., Siddle, K., Pierce, S. B., Roth, R. A., and Rutter, W. J. (1987) Proc. Natl. Acad. Sei. U. S. A. 84, 704-708 7. Ellis, L., Clauser, E., Morgan, D. O., Edery, M., Roth, R. A,, and Rutter, W. J. (1986) Cell 45, 721-732 8. Barbacid, M. (1987)Annu. Reu. Bioehem. 56, 779-827 9. Hall, A. (1990) Science 249, 635440 10. Mulcahy, L. S., Smith, M. R., and Stacey, D. W. (1985)Nature 313, 241-243 11. Hagag, N., Halegoua, S., and Viola, M. (1986) Nature 319, 680-682 12. Satoh, T.,Endo, M., Nakafuku, M., Nakamura,S., and Kaziro, Y. (1990)Proc. Natl. Acad. Sci. U. S. A. 87, 5993-5997 13. Satoh, T.,Endo, M., Nakafuku,M., Akiyama, T., Yamamoto, T., and Kaziro, Y. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 79267929 14. Osterop, A. P. R., Medema, R. H., Bos, J. L., Zon, G. C. M., Moller, D. E., Flier, J. S., Moller, W., and Maassen,J. A. (199215. Biol. Chem. 267, 14647-14653 15. Porras, A., Nebreda, A. R., Benito, M., and Santos, E. (1992)J . Bid. Chem. 267, 2112621131 16. Maassen, J. A., Burgering, B. M. T.,Medema, R. H., Osterop, A. P. R. M., van der Zon, G . C. M., Moller, W., and Bos, J. L (1992) Horm. Metab. Res. 24, 214-218 17. Gibbs, J. B., Marshall, M. S., Scolnick, E. M., Dixon, R. A. F., and Vogel, U. S. (1990) J. Biol. Chem. 266, 20437-20442 18. Downward, J., Graves, J. D., Warne, P. M., Rayter, S., and Cantrell, D. A. (1990) Nature 346, 719-723 19. Feramisco, J. R. (1979)Proc. Natl. Acad. Sci. U. S. A. 76, 3967-3971 20. Burgering, B. M. T., Medema, R. H., Maassen, J. A., van de Wetering, M. L.. van der Eb, A. J., McCormick, E , and Bos, J. L (1991)EMBO J . 10, 11031109 21. Szeberenyli, J., Cai, H., and Cooper, G. M. (1990)Mol. Cell B i d . 10,5324-5332 22. Feig, L. A., and Cooper, G. M. (1988)Mol. Cell. Biol. 8, 3255-3243 23. Feramisco, J. R., Gross, M., Kamata, T., Rosenberg, M., ans Sweet, R. W. (1984) Cell 38, 109-117 24. Stacey, D. W., Watson, T., Kung, H.-F., andCurran, T.(1987)Mol. Cell. Biol. 7, 523-527

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