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Advan. Enzyme Regul. 47 (2007) 169–183

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Regulation of calcium signalling by the smallGTP-binding proteins Ras and Rac1

Karl Malya,�, Georg Hechenbergera, Kukka Stresea,Ingeborg Tinhoferb, Irene Wedea,

Wolfgang Dopplera, Hans H. Grunickea

aInnsbruck Biocenter, Division of Medical Biochemistry, Medical University of Innsbruck, AustriabLaboratory of Immunology and Molecular Cancer Research, 3rd Medical Department of Haematology,

Oncology, University Hospital Salzburg, Austria

Introduction

Increases in the concentration of intracellular free Ca2+-ions act as ubiquitous signals inalmost any type of living cells ranging from bacteria to mammalian systems (Berridge,1997; Berridge et al., 2000). Ca2+-signals have been shown to regulate a wide variety ofbiological processes including proliferation, fertilization, differentiation, contraction,secretion, learning and memory (Berridge et al., 2000; Bootman et al., 2001). In spite of itsbiological significance, many aspects of the molecular mechanisms regulating signalling byCa2+ have remained unclear. This applies in particular to Ca2+-signalling in non-excitablecells stimulated by growth factors acting through receptor tyrosine kinases. Stimulation ofthese receptors results in an activation of the Ras4Raf4ERK pathway accompanied byan increase in cytosolic free Ca2+ (Schlessinger and Ullrich, 1992; Grunicke, 1995).Although data suggesting a regulation of Ras by Ca2+ have been published (Aspenstrom,2004; Cullen and Lockyer, 2002), evidence for a regulatory function of Ras upstream ofCa2+ has also been presented (Wakelam et al., 1986; Tinhofer et al., 1996; Obermeieret al., 1996). Elevated levels of cytosolic free Ca2+ are seen after expression oftransforming Ras (Hancock et al.,1988; Chen et al., 1988; Lang et al., 1991; Maly et al.,1995). Furthermore, it had been demonstrated that the Ca2+ signal exerted by activationof the epidermal growth factor receptor (EGFR) is inhibited by expression of dominant

see front matter r 2007 Elsevier Ltd. All rights reserved.

.advenzreg.2006.12.017

nding author. Tel.: +43512 900370160; fax: +43 512 900373130.

dress: Karl.Maly@i-med.ac.at (K. Maly).

ARTICLE IN PRESSK. Maly et al. / Advan. Enzyme Regul. 47 (2007) 169–183170

negative Ras or microinjection of neutralizing anti-Ras antibody, findings which point to aregulation of Ca2+-signalling by Ras (Tinhofer et al., 1996). As shown by other authors,the EGF-induced Ca2+ signal is also completely abolished by expression of a dominantnegative mutant of the Ras-related small GTP-binding protein Rac1 (Peppelenbosch et al.,1996). As Ras may initiate a pathway leading to an activation of Rac1 (Nobes and Hall,1995; Prendergast et al., 1995; Qiu et al., 1995), both small G-Proteins may be elements ofthe same signalling cascade. The detailed mechanism, however, underlying the effects ofRas and Rac on Ca2+ signalling remained unclear. The studies presented here have beeninitiated to gain further insight into the interaction of Ras and Rac with mechanismsresponsible for the increase in intracellular free Ca2+ following activation of either theEGFR or the receptor for nerve growth factor (TRK).

Materials and methods

Materials

Fura-2/AM was obtained from Molecular Probes; culture media and sera were fromVWR International; EGF, FITC-Dextran and hygromycin B was purchased fromSigma, Transfast from Promega, antibodies and peptides from Santa Cruz; myo-[2-3H]inositol was from NEN, UK; pEXV-Asn17-Ha-Ras and pEXV-Asn17-Rac1 werekindly provided by A. Hall, MRC Laboratory for Molecular Cell Biology, CancerResearch UK Oncogene and Signal Transduction Group, University College London andpEF-neo GFP-S65T by S. Geley, Department of Pathophysiology, Medical Universityof Innsbruck, pSV2M2.6-Nl7Ras (metallothionein promoter controlled) was kindlyprovided by W. Birchmeier, Max Delbrueck Centre for Molecular Medicine, Berlin(Hartmann et al., 1994).

Cell culture and generation of inducible cell lines

NIH3T3 fibroblasts expressing EGFR, a chimeric EGFR/TRK or the mutant receptorsEGF-R.X or EGFR/TRK.X (Obermeier et al., 1996) were grown in Dulbecco’s modifiedEagle’s medium, supplemented with 10% fetal calf serum and 2mM L-glutamine at 37 1Cin a humidified atmosphere (95% air, 5% CO2). pEXV-Asn17-Ha-Ras and pEXV-Asn17-Rac1 constructs were subcloned into BIG2-N17Ras or BIG2-N17Rac. The stabletransfections of pSV2M2.6-N17Ras, BIG2-N17Ras and BIG2-N17Rac together with thepY-3 hygromycin-resistence-gene were performed with Transfasts and the cells wereselected with 200 mg/ml hygromycin B.

Transient transfection and microinjection of peptides

EGFR or EGFR/TRK fibroblasts were transfected by microinjection of single cells(Eppendorf micromanipulator 5171) as described (Tinhofer et al., 1996). Microinjectionexperiments were performed by injection of approximately 50 fl of 1 mg/ml RasGaP(sc-4018) and PLCg-peptide (sc-4019) together with 20 mg/ml FITC-dextran.

[Ca2+] measurements

The cells (104/ml) were plated on coverslips (diameter 22mm) in 35-mm dishes (6-wellplates) and cultured for 1 day. Loading with fura-2 was performed by incubation with1 mM fura-2/AM for 15min. Then the cells were washed with HEPES buffer (HBS:

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140mM NaCl, 5mM KCl, 1mM CaCl2, 0.5mM MgCl2, 5.5mM glucose, 20mM HEPES/NaOH, pH 7.4) and kept in HEPES buffer at room temperature. For determination of thecytoplasmic Ca2+ concentration ([Ca2+]i) a single cell imaging system (Magical, AppliedImaging, Sunderland SR53HD, UK, Nikon Diaphot microscope and Metafluors—Visitron Systems) was used. The cytoplasmic Ca2+ concentration was calculated asdescribed (Tinhofer et al., 1996). Fura-2 Fluorescence Quench by Mn2+ for thedetermination of the calcium influx was measured as described previously (Tinhoferet al., 1996).

Cell lysis, immunoprecipitation, and western blotting

Prior to experiments, cells were cultured for 48 h in the presence or the absence ofdoxycycline or Zn2+ for induction of N17Ras or N17Rac expression and treated withEGF as indicated. Precleared lysates were immunoprecipitated, subjected to gelelectrophoresis and immunoblotted as described (Seedorf et al., 1994). Where indicated0.5 mg/ml PLCg-peptide (sc-4019) was added to the immunoprecipitation buffer before thepulldown by EGFR-antibodies.

Analysis of inositol phosphates and phosphoinositols

Cells were labelled with 10 mCi/ml myo-[2-3H]inositol and inositolphosphates (InsP3)were analyzed as described (Woll et al., 1992). For analysis of PIP2 the cells were scrapedfrom the ground and the solution transferred to a test tube. 1.2ml of a further solutionconsisting of chloroform and methanol in a ratio of 5:1 and 50 ml of 0.5M NaOH wereadded for deacylation. The sample was then vortexed for 20min and centrifuged for 10minat 3000 rpm. 1ml of the upper phase was used for HPLC analysis as described (Woll et al.,1992).

Results and discussion

Expression of dominant negative Asn17-Ras abrogates EGF receptor-mediated Ca2+ release

from internal stores

Fig. 1A demonstrates a representative single cell recording of an EGF-induced Ca2+

signal in cells overexpressing the EGF receptor (EGFR). For these experiments, fura-2loaded cells plated on cover slips were kept in a Ca2+-free medium. Addition of EGFresults in an increase in cytosolic free Ca2+ due to a release from internal stores. Thiscalcium transient is blocked by the dominant negative Ras mutant—a finding confirmingpreviously published data from this laboratory (Tinhofer et al., 1996). Addition of 1mMCa2+ to the medium results in a second Ca2+ peak representing Ca2+ influx probablythrough store-operated calcium channels and release of refilled stores. This second peak isalso suppressed by Asn17 Ras (data not shown). As described elsewhere (Tinhofer et al.,1996), the same results could be obtained by microinjection of a neutralizing anti-Rasantibody (Y13-238) which interacts with the effector binding domain of Ras. Neitherdominant negative Ras nor anti-Ras antibody were found to interfere with thapsigargin-induced calcium influx excluding an effect of Ras on store-operated calcium channels ofthe plasma membrane. (Tinhofer et al., 1996).

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Fig. 1. Effect of Asn17Ras on Ca2+ release from intracellular stores induced by the activated EGF receptor

(A) and the EGF/TRK receptor (B).

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Ca2+-release induced by the activated nerve growth factor receptor (NGFR/TRK) is not

affected by dominant negative Ras

In order to investigate the effect of a different Ras-activating receptor tyrosine kinase onCa2+ signalling, we employed an EGFR/TRK chimeric receptor consisting of anextracellular EGFR domain and the cytosolic domain of TRK expressed in the sameNIH3T3 cell line used for the experiments described in Fig. 1A. This system described inprevious publications (Tinhofer et al., 1996; Obermeier et al., 1996) permits the study ofboth receptors in the same cellular environment employing the same agonist, i.e. EGF. Inagreement with previously published data (Tinhofer et al., 1996), the Ca2+ transientinduced by activation of TRK is not affected by dominant negative Ras (Fig. 1B).

Ras dependence of receptor-mediated Ca2+ signalling is determined by the phospholipase C g1

(PLCg1) binding domain

It is generally accepted that the Ca2+-release induced by receptor tyrosine kinases ismediated by PLC g (EC 3.1.4.10). Binding and activation of PLCg results in a release ofinositol 1,4,5-trisphosphate (InsP3) which in turn mediates rapid Ca2+ store releasethrough the activation of InsP3 receptors in the endoplasmic reticulum membrane(Berridge et al., 2000). EGFR and NGFR/TRK differ markedly with regard to their PLCgbinding sites. Compared to the EGFR, the affinity of NGFR/TRK to PLCg is �100-foldhigher (Obermeier et al., 1993). The high affinity of NGFR/TRK was shown to bedetermined by 75 amino acid residues flanking phosphorylated tyrosine 785 (Obermeieret al., 1996). In contrast to NGFR/TRK, PLCg1 binds to the EGFR with significantlylower affinity to several phosphorylated tyrosines including Y992, Y1148, Y1173, Y1086and Y 1068. None of them is selective for PLCg1 but bind other SH2 proteins—especiallyRas-GAP with similar affinities (Rotin et al., 1992; Soler et al., 1994; Milarski et al., 1993).In order to investigate whether the Ras dependence of the calcium signal is determined

by the structure of the PLCg-binding domain of the receptor, exchange mutantswere employed, in which the entire PLCg-binding site of he EGFR was replaced by the

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PLCg-binding domain of TRK and vice versa. Thus, the exchange mutant EGF-R.Xcarried the TRK residues 780–790 in place of the EGFR amino acids 987–997; in thechimeric EGFR/TRK receptor, TRK residues 780–790 were replaced by the EGFRsequence 987–997, to generate the EGFR/TRK-R.X. Generation of these receptors andthe biological properties of the corresponding cell lines over-expressing these constructshad been described previously (Tinhofer et al., 1996; Obermeier et al., 1996). A schematicrepresentation of the receptor constructs is shown in Fig. 2.

Fig. 3 demonstrates that the sensitivity of the Ca2+ signal to dominant negative Ras isstrictly dependent on the structure of the PLCg-binding site. Expression of Asn17Ras nolonger affects the Calcium transient induced by the activated EGFR carrying the TRKPLCg-binding site, whereas the Ca2+ signal mediated by the EGFR/TRK receptor withthe EGFR PLCg-binding site is now sensitive to dominant negative Ras. The data clearlydemonstrate that the function of Ras in regulating EGFR-induced Ca2+ release dependsentirely on the structure of the PLCg-binding domain in agreement with previous findings(Tinhofer et al., 1996).

If possible interactions of Ras and PLC are discussed, a potential role of the morerecently discovered PLC isoform PLCe has to be considered. In contrast to PLCg isozymeswhich are regulated by tyrosine kinases and membrane translocation (Berridge et al.,2000), PLCe contains two Ras-binding domains (Kelley et al., 2001; Song et al., 2001).Association of Ras and Rho proteins with these regions activates the phospholipase

EGF-R EGF-R.XEGF/TRK-R EGF/ TRK-R.X

Fig. 2. Schematic representation of receptor constructs. EGF-R: Epidermal growth factor receptor. EGF/TRK-

R: Chimeric receptor containing the extracellular portion of the EGF-R and the cytoplamic part of the nerve

growth factor receptor (TRK). The horizontal bar indicates the plasma membrane. Below the horizontal bar the

cytoplasmic portions of the receptors are drawn with the tyrosine kinase domains indicated by boxes. Open boxes

represent the tyrosine kinase of the EGFR, closed boxes symbolize the tyrosine kinase of the TRK receptor. The

PLCg-binding sites are symbolized by open (J) or closed (K) circles. Open circle: PLCg-binding site of the

EGFR; closed circle: PLCg-binding site of the TRK receptor. In the exchange mutant EGF-R.X the PLCg-binding site of EGF-R is replaced by the PLCg-binding region of TRK. EGF/TRK-R.X contains the PLCg-binding site of EGF-R instead of the PLCg-binding site of TRK.

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Fig. 3. Effect of dominant negative Ras on Ca2+ signals induced by activated exchange mutant receptors EGF/

TRK-R.X or EGF-R.X. Asn17Ras construct or the empty control vector were cotransfected with the green

fluorescence marker construct by microinjection. One day after transfection, the cells were loaded with fura-2, and

the Ca2+ influx was estimated fromMn2+-dependent fura-2 quench detected 60 s after addition of 100mMMnCl2(final concentration). The fura-2 quench was calculated as the percentage of fluorescence decrease of the initial

fura-2 fluorescence. Error bars indicate SEM, n4 ¼ 5.

K. Maly et al. / Advan. Enzyme Regul. 47 (2007) 169–183174

activity of the enzyme (Kelley et al., 2001; Song et al., 2001). In addition to the Ras-binding regions RA1 and RA2, PLCe contains a CDC25 domain, capable of activatingRas and/or Rap1 (Kelley et al., 2001; Song et al., 2001). Thus, PLCe can act as a Raseffector, but may also function as a regulator of Ras.However, the data shown in Fig. 3 clearly demonstrate that the role of Ras in EGFR- or

TRK-induced Ca2+ signalling is exclusively determined by the structure of thecorresponding PLCg-binding site. As PLCe is not activated by receptor association, itcan be concluded that PLCe is not involved in the depression of EGFR-induced Ca2+

release by dominant negative Ras.

Expression of dominant negative Ras reduces the binding of PLCg to EGFR not, however, to

the TRK receptor. The interference with PLCg-binding is accompanied by a reduction of

InsP3 formation

The data presented so far indicate that the inhibition of the Ca2+ signal by dominantnegative Ras is inversely related to the affinity of PLCg to its binding sites on thecorresponding receptors. The low affinity of the PLCg-binding domain of the EGFRrenders the receptor-induced Ca2+ release sensitive to the expression of the dominantnegative Ras mutant. These findings suggest that dominant negative Ras may interferewith the binding of PLCg to the receptor. An interference with PLCg-binding to EGFRshould result in reduced formation of InsP3 and a consecutive depression of the Ca2+

release. In order to check this hypothesis, cells were stably transfected with an Asn17 Rasconstruct under control of a metallothionein promoter. Induction of dominant negative

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Ras by Zn2+ results in a significant reduction of EGF-induced PLCg-binding to theEGFR (Fig. 4A) whereas the association of PLCg with TRK was not affected (Fig. 4B). Asmight be expected, binding of PLCg to the TRK receptor is markedly enhanced afteraddition of the ligand EGF. In case of the EGFR, however, addition of the growth factorleads surprisingly to a small but significant decrease of PLCg-binding. This observationmay indicate that activation of EGFR results in an increased competition of proteins withSH2-domains with PLCg for the same low- affinity binding sites of the receptor. Evidencefor the existence of such competition will be presented in the following section.Nevertheless, the data of Fig. 4 clearly demonstrate that induction of dominant negativeRas markedly supresses the binding of PLCg to the receptor.

Whether the reduced binding of PLCg to the EGFR seen after induction of dominantnegative Ras leads to a reduced formation of EGF-induced InsP3 levels was studiedin cells stably transfected with an Asn 17 -Ras construct under transcriptional controlof a doxycycline-on promoter. As shown in Fig. 5A, induction of dominant negativeRas by Dox results in a significant depression of EGF-induced InsP3 formation incells expressing EGFR, whereas the increase in InsP3 levels after activation of theTRK receptor was not found to be affected by expression of the dominant negativeRas construct (Fig. 5B). The inhibition of InsP3 formation after induction ofdominant negative Ras by Dox is accompanied by a depression of the EGF-inducedCa2+ signal in cells over-expressing the EGF receptor (Fig. 6A), not, however, afteractivation of TRK (Fig. 6B).

In summary, the data presented so far are in agreement with a model in which dominantnegative Ras depresses the EGFR-induced Ca2+ release by interfering with PLCg bindingto EGFR and a consecutive reduction in EGF-mediated InsP3 formation.

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Fig. 4. EGF-induced binding of PLCg to the EGF receptor (A) or the chimeric EGF/TRK receptor (B) after

expression of Asn17Ras. The bars represent densitometric determinations of the coprecipitated PLCg peptides

from the immunoblots shown underneath. Error bars indicate SEM, n ¼ 4.

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Fig. 5. Effect of dominant negative Ras on InsP3 formation after activation of the EGFR (A) or the TRK

receptor (B) respectively. Cells stably transfected with Asn17 Ras under control of a tetracycline-on promoter were

stimulated by EGF. Where indicated, Asn17 Ras was induced by doxycycline. Doxycycline-mediated induction of

Asn17Ras is demonstrated by the immunoblot underneath. Inositol-1,3,5-trisphosphate (InsP3) was determined as

described under Methods. Error bars indicate SEM, n4 ¼ 5.

K. Maly et al. / Advan. Enzyme Regul. 47 (2007) 169–183176

PLCg competes with other SH2-domain-carrying proteins for binding to the EGF receptor

By what mechanism could dominant negative Ras interfere with the binding of PLCg tothe EGFR? As outlined above, binding of PLCg to EGFR occurs through several low-affinity binding sites. This is in contrast to TRK which binds PLCg with high affinity to aregion75 amino acids around tyrosine 785 (Obermeier et al., 1996, 1993). The low-affinitybinding sites of the EGFR bind other SH2-domain carrying proteins with similar affinities.This applies in particular to Ras-GAP and the p85 subunit of phosphatidylinositol-30-kinase (PI3K) (EC 2.7.1.137). Ras-GAP and PLCg have been shown to be equallyeffective in binding to tyrosine 992 of EGFR (Milarski et al., 1993). Mutation of tyrosine1173 significantly reduces the binding of both PLCg and PI3K (Soler et al., 1994).Exchange of tyrosine 1148 (one of the PLCg binding sites) by phenylalanine inhibits thebinding of Ras-GAP to the EGFR (Soler et al., 1993). Mutation of both tyrosines 1173and 1148 resulted in a marked reduction of PLCg to the EGFR (Milarski et al., 1993).Both Ras-GAP and PI3K bind to activated, GTP-charged Ras, not, however, to inactiveGDP-Ras (Marshall, 1996; Serth et al., 1992). Dominant negative Ras interferes with Rasactivation and could thereby lead to increased levels of free p85 PI3K and Ras-GAP whichcould occupy PLCg-binding sites of EGFR and thereby reduce the amount of bound andactivated PLCg.In order to test this model it was investigated whether microinjection of Ras-GAP into

cells over-expressing either the EGFR or the EGF/TRK chimera would interfere with the

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Fig. 6. Effects of dominant negative mutant of Ras on Ca2+ signals induced by activation of either EGFR (A) or

TRK receptor (B). Cells stably transfected with Asn17 Ras or under control of a tetracycline-on promoter were

stimulated with EGF and the resulting increase in cytosolic free Ca2+ was determined. Where indicated, the

expression of the dominant negative mutants was induced by doxycycline (Dox). The bars represent peak Ca2+

levels 7 SEM, n4 ¼ 10.

K. Maly et al. / Advan. Enzyme Regul. 47 (2007) 169–183 177

ligand-induced Ca2+ signal. The results shown in Fig. 7 demonstrate that microinjectedRas-GAP significantly reduces the Ca2+ signal after activation of the EGFR, whereasthe Ca2+ transient induced by activation of the TRK receptor remained unaffectedby the increased intracellular concentration of Ras-GAP (Fig. 7A and D) Similarresults were obtained after microinjection of a lipase-free PLCg fragment containingthe SH2-binding domain of the protein (Fig. 7B,E). Again, only in cells expressing thewild-type EGFR could microinjection of the SH2-protein interfere with the EGF-inducedincrease in cytosolic free Ca2+. The relevance of the SH2 domain for competitionwith PLCg binding is demonstrated in Fig. 7C. For these experiments cells weremicroinjected with a Janus kinase binding protein (JAB) which normally binds to tyrosinephosphorylated Janus kinase (JAK) through its SH2-domain (Yasukawa et al., 1999).This protein also acts as an efficient competitor of PLCg and significantly reducesthe calcium signal after activation of the EGFR by its ligand. Deletion of the SH2regions renders the protein inefficient with regard to an interference with the EGFR-induced Ca2+ signal (Fig. 7C). Cbl may represent another example for a SH2-domaincontaining protein competing with PLCg-1 for common binding sites of the EGFR (Choiet al., 2003).

The data shown in Fig. 7 support the hypothesis that the inhibition of the EGFR-induced Ca2+ release by dominant negative Ras is caused by a suppression of PLCgbinding to the receptor. The reduced association with the receptor is the result of elevatedlevels of free SH2-domain carrying proteins—especially Ras-GAP and probably also thep85 subunit of PI3K—which compete with PLCg for the same binding sites of the EGFRand thereby reduce the amount of receptor-bound and activated PLCg.

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Fig. 7. Microinjection of SH2-containing proteins interferes with the Ca2+ signal following activation of the EGF

receptor (panels A–C) not, however, after activation of the TRK receptor (panels D and E). Error bars indicate

SEM, n4 ¼ 8.

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The high affinity binding site for PLCg of the TRK receptor which is highly selective forPLCg and does not associate with other SH2-containing proteins is not affected by thepresence of dominant negative Ras.

The Ras-related small GTP-binding protein Rac1 is required for EGFR and NGFR/TRK-

induced Ca2+ signalling

Other authors had shown that the dominant negative Asn17 mutant of the Ras-relatedsmall G-Protein Rac1 inhibits the EGF induced Ca2+ signal (Peppelenbosch et al., 1996).Ras has been shown to be able of activating Rac1 (Nobes and Hall, 1995; Prendergastet al., 1995; Qiu et al., 1995). An activation of Rac1 had also been demonstrated in cellsexpressing transforming V12Ras (Coso et al., 1995; Minden et al., 1995). It appearedpossible, therefore, that Ras and Rac1 act as elements of the same pathway leading to theEGFR-mediated increase in cytosolic free Ca2+. At first glance, this supposition issupported by the data shown in Fig. 8A, employing cells stably transfected with adominant negative Rac construct under transcriptional control of a doxycyclin-onpromoter. Induction of Asn17Rac caused a marked inhibition of EGF-induced Ca2+ signalin cells over-expressing EGFR. However, dominant negative Rac1 also acts as a potentinhibitor of the TRK-mediated Ca2+ signal (Fig. 8B), in marked contrast to the behavior

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Fig. 8. Effects of dominant negative mutant of Rac1 on Ca2+ signals induced by activation of either EGFR (A)

or TRK receptor (B). Cells stably transfected with Asn17Rac under control of a tetracycline-on promoter were

stimulated with EGF and the resulting increase in cytosolic free Ca2+ was determined. Where indicated, the

expression of the dominant negative mutants was induced by doxycycline (Dox). The bars represent peak Ca2+

levels 7 SEM, n4 ¼ 10.

K. Maly et al. / Advan. Enzyme Regul. 47 (2007) 169–183 179

of dominant negative Ras which did not affect the TRK-induced calcium transient(Fig. 6B). The differential effects of Ras and Rac1 on EGFR- and TRK-receptor-mediatedCa2+ signalling, respectively, strongly suggest that Rac1 affects receptor-mediated Ca2+

release by a mechanism quite different from the action of Ras. The data depicted in Fig. 9may provide an explanation for the mechanism underlying the inhibition of calciumsignalling by dominant negative Rac1. After induction of Asn17Rac in cells stablytransfected with a dominant negative Rac under control of a tet-on promoter, the EGF-induced formation of phosphatidylinositol-4,5-bisphosphate (PIP2) was significantlyreduced. The inhibition was seen in cells expressing the EGF-receptor wild -type as wellas in those over-expressing the EGFR/TRK chimera. The reduction of PIP2 wasaccompanied by a depression of the Ca2+ signal in both systems, i.e. after activation ofEGFR as well as after stimulation of TRK. In accordance with data shown above,dominant negative Ras did not affect the increase in cytosolic free Ca2+ after activation ofthe TRK receptor. The inhibition of PIP2 formation by dominant negative Rac is inagreement with data supporting regulation by Rac1 of phosphatidylinositol-40 kinase (EC2.7.1.67) and phosphatidylinositol4-phosphate-50 kinase (EC 2.7.1.68) (Hartwig et al.,1995; Chong et al., 1994; Tolias et al., 1995; Weernink et al., 2004). Both enzymes arenecessary for the generation of PIP2, the substrate of PLC. A depression of PIP2 formationshould, therefore, result in a decreased release of InsP3 and in turn to a suppressed calciumsignal.

In summary, both dominant negative Ras as well as dominant negative Rac inhibitthe EGFR and NGFR/TRK-induced Ca2+ signal by interfering with phospholipidmetabolism, although the two small G-proteins act at different steps in the pathwayleading to receptor-mediated release of Ca2+ from intracellular stores.

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Fig. 9. Inhibition of phosphatidylinositol-4,5-bisphosphate (PIP2) formation by dominant negative Rac after

activation of either the EGF (A)—or the TRK-receptor (B). Cells stably transfected with either Asn17Rac or

Asn17Ras both under control of a tetracycline-on promoter were stimulated with EGF and the resulting PIP2

formation was determined as described under Methods. Where indicated, the expression of the dominant negative

mutants was induced by doxycycline (Dox). Closed bars: Cells transfected with N17 Rac; open bars: cells

transfected with N17 Ras. Error bars indicate SEM, n4 ¼ 3.

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Biological significance of the observed interference of Ras and Rac mutants with calcium

signalling mechanisms

The role of Ras on Ca2+ signalling presented here was obtained with cells over-expressing a dominant negative Ras mutant. It may be argued, therefore, that theeffects presented here represent experimentally induced situations which cannotnecessarily be extrapolated to biological conditions. It should be emphasized, however,that the results obtained with the dominant negative Ras mutants could also be observedafter microinjection of neutralizing anti-Ras antibody, i.e. without over-expressionof the Ras construct (Tinhofer et al., 1996). These findings point to a biological roleof Ras in regulating the EGFR-mediated Ca2+ signal by providing a cross-talkbetween the Ras4Raf4Erk pathway and Ca2+ regulated mechanisms. The inhibitionby Grb2 of PLCg represents an additional mechanism for such a cross-talk (Choiet al., 2005). The adaptor protein Grb2 is a key element involved in receptor-mediatedRas activation (Grunicke and Maly, 1993). Conditions favoring an increase in freeGrb2 would be the same as those leading to an elevation of unbound Ras-GAP.Other mechanisms providing a cross-talk between Ras- and Calcium signallinginclude the direct activation of PLCe by Ras (Kelley et al., 2001; Song et al., 2001) andthe inhibition of plasma membrane Ca2+ channels by PLCg (Patterson et al., 2002),although these last mentioned two mechanisms do not appear to be of relevance for theeffects described here as outlined above. In summary, evidence is accumulating supporting

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a signalling relationship between Ras and Ras-like GTP binding proteins and Ca2+

signalling.The observation that the NGFR/TRK-mediated calcium transient is not affected by

dominant negative Ras although this receptor like EGFR also signals through Ras isprobably due to the different biological functions of these two receptor types. In PC12 cellsunder physiological conditions, EGFR and NGFR induce opposite effects, activation ofEGFR elicits proliferation, whereas stimulation of NGFR leads to differentiation(Obermeier et al., 1994; Ginty et al., 1994; van der Geer et al., 1994; Marshall, 1995;Chao, 1992). When expressed in fibroblasts, however, NGFR leads to a proliferativeresponse (Cordon-Cardo et al., 1991). In addition to the mitogenic response, the receptorconstructs employed in these studies had also been shown to exhibit an oncogenic potentialwhich was found to be inversely correlated to the PLCg affinity (Obermeier et al., 1996).Considering the different biological function of the two receptor types it appearsconceivable that they are subject to different regulatory mechanisms.

Summary

In order to investigate a possible interaction of the small GTP-binding proteins Ras andRac1 with Ca2+-mediated signalling cascades the effects of dominant negative mutantsof Ras and Rac1 on Ca2+ signalling have been studied after stimulation of either theEGFR or the nerve growth factor receptor (TRK). Expression of dominant negative Rasblocks the release of Ca2+ from internal stores after activation of EGFR whereas thecalcium signal elicited by the activated TRK receptor is unaffected. The sensitivity todominant negative Ras is determined by the structure of the PLCg-binding sites ofthe corresponding receptors. Exchange of the PLCg-binding domain of the EGFR by thePLCg-binding site of TRK renders the EGFR-induced calcium signal insensitive tothe expression of dominant negative Ras. Substitution of the PLCg-binding site ofTRK by the PLCg-binding region of EGFR renders TRK sensitive to dominant negativeRas. The inhibition of Ca2+ release by dominant negative Ras is accompanied by areduction in PLCg binding to the EGFR and a concomitant decrease of EGF-inducedinositol-1,3,5-trisphosphate (InsP3) formation. The depression of PLCg binding to EGFRis explained by a competition of PLCg with other SH2-domain containing proteins for thesame low affinity binding regions of the EGFR. This conclusion is supported by theobservation that microinjection of several SH2-domain containing proteins including Ras-GP, lipase-free fragment of PLCg or Janus kinase binding protein (JAB), reduces theassociation of PLCg to the EGFR, not, however, to TRK. In contrast to dominantnegative Ras which does not affect the Ca2+ transient induced by the activation of theTRK receptor, a dominant negative mutant of Rac significantly depresses the Ca2+ signalsinduced by EGFR as well as by TRK. The different behavior of Rac and Ras supports thenotion that the two small GTP-binding proteins act through separate pathways. It isdemonstrated that dominant negative Rac significantly reduces the formation ofphosphatidylinositol-4,5-bisphosphate (PIP2), the substrate of PLCg. This effect is notobserved after expression of dominant negative Ras. In summary, the data provide furtherevidence for a cross-talk between small GTP-binding proteins and Ca2+ signalling inwhich both G-proteins interfere with the formation of InsP3 although by differentmechanisms.

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Acknowledgements

The expert technical assistance of Elisabeth Kindler-Maly and Anto Nogalo is gratefullyacknowledged. We thank Dr. W. Birchmeier for providing the metallothionein inducibleAsn17 Ras construct and Dr. A. Hall for Asn17 Ras and Asn17 Rac1 plasmids. This studyhas been supported by Austrian Science Fund (FWF), Grant no. P12547-MOB.

References

Aspenstrom P. Integration of signalling pathways regulated by small GTPases and calcium. Biochim Biophys

Acta 2004;1742:51–8.

Berridge MJ. Elementary and global aspects of calcium signalling. J Physiol 1997;499:291–306.

Berridge MJ, Lipp P, Bootman MD. The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol

2000;1:11–21.

Bootman MD, Lipp P, Berridge MJ. The organisation and functions of local Ca(2+) signals. J Cell Sci

2001;114:2213–22.

Chao MV. Growth factor signaling: where is the specificity? Cell 1992;68:995–7.

Chen CF, Corbley MJ, Roberts TM, Hess P. Voltage-sensitive calcium channels in normal and transformed 3T3

fibroblasts. Science 1988;239:1024–6.

Choi JH, Hong WP, Yun S, Kim HS, Lee JR, Park JB, et al. 2005 Cbl competitively inhibits epidermal growth

factor-induced activation of phospholipase C-gamma1. Mol Cells 2003;15:245–55.

Choi JH, Bae SS, Park JB, Ha SH, Song H, Kim JH, et al. Grb2 negatively regulates epidermal growth factor-

induced phospholipase C-gamma1 activity through the direct interaction with tyrosine-phosphorylated

phospholipase C-gamma1. Cell Signal 2005;17:1289–99.

Chong LD, Traynor-Kaplan A, Bokoch GM, Schwartz MA. The small GTP-binding protein Rho regulates a

phosphatidylinositol 4-phosphate 5-kinase in mammalian cells. Cell 1994;79:507–13.

Cordon-Cardo C, Tapley P, Jing SQ, Nanduri V, O’Rourke E, Lamballe F, et al. The trk tyrosine protein kinase

mediates the mitogenic properties of nerve growth factor and neurotrophin-3. Cell 1991;66:173–83.

Coso OA, Chiariello M, Yu JC, Teramoto H, Crespo P, Xu N, et al. The small GTP-binding proteins Rac1 and

Cdc42 regulate the activity of the JNK/SAPK signaling pathway. Cell 1995;81:1137–46.

Cullen PJ, Lockyer PJ. Integration of calcium and Ras signalling. Nat Rev Mol Cell Biol 2002;3:339–48.

Ginty DD, Bonni A, Greenberg ME. Nerve growth factor activates a Ras-dependent protein kinase that

stimulates c-fos transcription via phosphorylation of CREB. Cell 1994;77:713–25.

Grunicke HH. Signal transduction mechanisms in cancer. Heidelberg, Germany: Springer; 1995.

Grunicke HH, Maly K. Role of GTPases and GTPase regulatory proteins in oncogenesis. Crit Rev Oncog

1993;4:389–402.

Hancock JF, Marshall CJ, McKay IA, Gardner S, Houslay MD, Hall A, et al. Mutant but not normal p21 ras

elevates inositol phospholipid breakdown in two different cell systems. Oncogene 1988;3:187–93.

Hartmann G, Weidner KM, Schwarz H, Birchmeier W. The motility signal of scatter factor/hepatocyte growth

factor mediated through the receptor tyrosine kinase met requires intracellular action of Ras. J Biol Chem

1994;269:21936–9.

Hartwig JH, Bokoch GM, Carpenter CL, Janmey PA, Taylor LA, Toker A, et al. Thrombin receptor ligation and

activated Rac uncap actin filament barbed ends through phosphoinositide synthesis in permeabilized human

platelets. Cell 1995;82:643–53.

Kelley GG, Reks SE, Ondrako JM, Smrcka AV. Phospholipase C(epsilon): a novel Ras effector. EMBO J

2001;20:743–54.

Lang F, Friedrich F, Kahn E, Woll E, Hammerer M, Waldegger S, et al. Bradykinin-induced oscillations of cell

membrane potential in cells expressing the Ha-ras oncogene. J Biol Chem 1991;266:4938–42.

Maly K, Kindler E, Tinhofer I, Grunicke HH. Activation of Ca2+ influx by transforming Ha-ras. Cell Calcium

1995;18:120–34.

Marshall CJ. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-

regulated kinase activation. Cell 1995;80:179–85.

Marshall CJ. Ras effectors. Curr Opin Cell Biol 1996;8:197–204.

ARTICLE IN PRESSK. Maly et al. / Advan. Enzyme Regul. 47 (2007) 169–183 183

Milarski KL, Zhu G, Pearl CG, McNamara DJ, Dobrusin EM, MacLean D, et al. Sequence specificity in

recognition of the epidermal growth factor receptor by protein tyrosine phosphatase 1B. J Biol Chem

1993;268:23634–9.

Minden A, Lin A, Claret FX, Abo A, Karin M. Selective activation of the JNK signaling cascade and c-Jun

transcriptional activity by the small GTPases Rac and Cdc42Hs. Cell 1995;81:1147–57.

Nobes CD, Hall A. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes

associated with actin stress fibers, lamellipodia, and filopodia. Cell 1995;81:53–62.

Obermeier A, Halfter H, Wiesmuller KH, Jung G, Schlessinger J, Ullrich A. Tyrosine 785 is a major determinant

of Trk—substrate interaction. EMBO J 1993;12:933–41.

Obermeier A, Bradshaw RA, Seedorf K, Choidas A, Schlessinger J, Ullrich A. Neuronal differentiation signals

are controlled by nerve growth factor receptor/Trk binding sites for SHC and PLC gamma. EMBO J 1994;

13:1585–90.

Obermeier A, Tinhofer I, Grunicke HH, Ullrich A. Transforming potentials of epidermal growth factor and nerve

growth factor receptors inversely correlate with their phospholipase C gamma affinity and signal activation.

EMBO J 1996;15:73–82.

Patterson RL, van Rossum DB, Ford DL, Hurt KJ, Bae SS, Suh PG, et al. Phospholipase C-gamma is required

for agonist-induced Ca2+ entry. Cell 2002;111:529–41.

Peppelenbosch MP, Tertoolen LG, Vries-Smits AM, Qiu RG, M’Rabet L, Symons MH, et al. Rac-dependent and

-independent pathways mediate growth factor-induced Ca2+ influx. J Biol Chem 1996;271:7883–6.

Prendergast GC, Khosravi-Far R, Solski PA, Kurzawa H, Lebowitz PF, Der CJ. Critical role of Rho in cell

transformation by oncogenic Ras. Oncogene 1995;10:2289–96.

Qiu RG, Chen J, Kirn D, McCormick F, Symons M. An essential role for Rac in Ras transformation. Nature

1995;374:457–9.

Rotin D, Margolis B, Mohammadi M, Daly RJ, Daum G, Li N, et al. SH2 domains prevent tyrosine

dephosphorylation of the EGF receptor: identification of Tyr992 as the high-affinity binding site for SH2

domains of phospholipase C gamma. EMBO J 1992;11:559–67.

Schlessinger J, Ullrich A. Growth factor signaling by receptor tyrosine kinases. Neuron 1992;9:383–91.

Seedorf K, Kostka G, Lammers R, Bashkin P, Daly R, Burgess WH, et al. Dynamin binds to SH3 domains of

phospholipase C gamma and GRB-2. J Biol Chem 1994;269:16009–14.

Serth J, Weber W, Frech M, Wittinghofer A, Pingoud A. Binding of the H-ras p21 GTPase activating protein by

the activated epidermal growth factor receptor leads to inhibition of the p21 GTPase activity in vitro.

Biochemistry 1992;31:6361–5.

Soler C, Beguinot L, Sorkin A, Carpenter G. Tyrosine phosphorylation of ras GTPase-activating protein does not

require association with the epidermal growth factor receptor. J Biol Chem 1993;268:22010–9.

Soler C, Beguinot L, Carpenter G. Individual epidermal growth factor receptor autophosphorylation sites do not

stringently define association motifs for several SH2-containing proteins. J Biol Chem 1994;269:12320–4.

Song C, Hu CD, Masago M, Kariyai K, Yamawaki-Kataoka Y, Shibatohge M, et al. Regulation of a novel

human phospholipase C, PLCepsilon, through membrane targeting by Ras. J Biol Chem 2001;276:2752–7.

Tinhofer I, Maly K, Dietl P, Hochholdinger F, Mayr S, Obermeier A, et al. Differential Ca2+ signaling induced

by activation of the epidermal growth factor and nerve growth factor receptors. J Biol Chem 1996;271:

30505–9.

Tolias KF, Cantley LC, Carpenter CL. Rho family GTPases bind to phosphoinositide kinases. J Biol Chem

1995;270:17656–9.

van der Geer P, Hunter T, Lindberg RA. Receptor protein-tyrosine kinases and their signal transduction

pathways. Annu Rev Cell Biol 1994;10:251–337.

WakelamMJ, Davies SA, Houslay MD, McKay I, Marshall CJ, Hall A. Normal p21N-ras couples bombesin and

other growth factor receptors to inositol phosphate production. Nature 1986;323:173–6.

Weernink PA, Meletiadis K, Hommeltenberg S, Hinz M, Ishihara H, Schmidt M, et al. Activation of type I

phosphatidylinositol 4-phosphate 5-kinase isoforms by the Rho GTPases, RhoA, Rac1, and Cdc42. J Biol

Chem 2004;279:7840–9.

Woll E, Waldegger S, Lang F, Maly K, Grunicke H. Mechanism of intracellular calcium oscillations in fibroblasts

expressing the ras oncogene. Pflugers Arch 1992;420:208–12.

Yasukawa H, Misawa H, Sakamoto H, Masuhara M, Sasaki A, Wakioka T, et al. The JAK-binding protein JAB

inhibits Janus tyrosine kinase activity through binding in the activation loop. EMBO J 1999;18:1309–20.