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Platelet-derived Growth Factor-dependent Cellular Transformation Requires Either Phospholipase Cγ or Phosphatidylinositol 3 Kinase*

  • Kris A. DeMali
    Affiliations
    Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts 02114

    University of Colorado Health Sciences Center, Department of Pharmacology, Denver, Colorado 80262, and
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  • Craig C. Whiteford
    Affiliations
    Section of Virology and Oncology, Division of Biology, Kansas State University, Manhattan, Kansas 66506
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  • Emin T. Ulug
    Affiliations
    Section of Virology and Oncology, Division of Biology, Kansas State University, Manhattan, Kansas 66506
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  • Andrius Kazlauskas
    Correspondence
    To whom correspondence should be addressed
    Affiliations
    Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts 02114

    University of Colorado Health Sciences Center, Department of Pharmacology, Denver, Colorado 80262, and
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  • Author Footnotes
    * This research was supported in part by National Institutes of Health Grants CA58291 (to E. T. U.) and GM-48339 (to A. K.) and by a grant-in aid from the American Heart Association, Kansas Affiliate (to E. T. U.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:April 04, 1997DOI:https://doi.org/10.1074/jbc.272.14.9011
      Although it has been well established that constitutive activation of receptor tyrosine kinases leads to cellular transformation, the signal relay pathways involved have not been systematically investigated. In this study we used a panel of platelet-derived growth factor (PDGF) β receptor mutants (β-PDGFR), which selectively activate various signal relay enzymes to define which signaling pathways are required for PDGF-dependent growth of cells in soft agar. The host cell line for these studies was Ph cells, a 3T3-like cell that expresses normal levels of the β-PDGFR but no PDGF-α receptor (α-PDGFR). Hence, this cell system can be used to study signaling of mutant αPDGFRs or α/β chimeras. We constructed chimeric receptors containing the αPDGFR extracellular domain and the βPDGFR cytoplasmic domain harboring various phosphorylation site mutations. The mutants were expressed in Ph cells, and their ability to drive PDGF-dependent cellular transformation (growth in soft agar) was assayed. Cells infected with an empty expression vector failed to grow in soft agar, whereas introduction of the chimera with a wild-type β-PDGFR cytoplasmic domain gave rise to a large number of colonies. In contrast, the N2F5 chimera, in which the binding sites for phospholipase Cγ (PLC-γ), RasGTPase-activating protein, phosphatidylinositol 3 kinase (PI3K), and SHP-2 were eliminated, failed to trigger proliferation. Restoring the binding sites for RasGTPase-activating protein or SHP-2 did not rescue the PDGF-dependent response. In contrast, receptors capable of associating with either PLC-γ or PI3K relayed a growth signal that was comparable to wild-type receptors in the soft agar growth assay. These findings indicate that the PDGF receptor activates multiple signaling pathways that lead to cellular transformation, and that either PI3K or PLC-γ are key initiators of such signal relay cascades.

      INTRODUCTION

      Several lines of evidence implicate PDGF
      The abbreviations used are: PDGF
      platelet-derived growth factor
      PDGFR
      PDGF receptor
      PI3K
      phosphatidylinositol 3 kinase
      PLC-γ
      phospholipase Cγ
      DMEM
      Dulbecco's modified Eagle's medium
      HPLC
      high performance liquid chromatography
      PIPES
      1,4-piperazinediethanesulfonic acid
      WT
      wild-type
      GAP
      GTPase-activating protein
      and its receptor (PDGFR) as key members in the genesis of certain forms of cancer. First, the B chain of the PDGF ligand is identical to the transforming protein of the v-sis oncogene (
      • Waterfield M.D.
      • Scrace G.T.
      • Whittle N.
      • Stroobant P.
      • Johnsson A.
      • Wasteson A.
      • Westermark B.
      • Heldin C.-H.
      • Huang J.S.
      • Deuel T.F.
      ). Second, several studies have shown that co-expression of PDGF and its receptor results in cellular transformation (
      • Gazit A.
      • Igarashi H.
      • Chiu I.-M.
      • Srinivasan A.
      • Yaniv A.
      • Tronick S.R.
      • Robbins K.C.
      • Aaronson S.A.
      ), whereas expression of dominant negative constructs of the PDGF reagents can reverse the transformed phenotype of naturally occurring tumor cell lines (
      • Shamah S.M.
      • Stiles C.D.
      • Guhu A.
      ,
      • Vassbotn F.S.
      • Andersson M.
      • Westermark B.
      • Heldin C.-H.
      • Österman A.
      ). Third, a fusion between the β-PDGFR and tel (an ets-like transcription factor) is implicated in the progression of chronic myelogenous leukemia patients to an acute chromic myelomonocytic leukemic state (
      • Golub T.R.
      • Barker G.F.
      • Lovett M.
      • Gilliland D.G.
      ). Collectively these findings suggest that deregulation of PDGF-dependent pathways leads to cellular transformation.
      There are three isoforms of the PDGF ligand, PDGF AA, PDGF BB, and PDGF AB, which differ in their transforming efficiencies (
      • Beckmann M.P.
      • Betsholtz C.
      • Heldin C.-H.
      • Westermark B.
      • DiMarco E.
      • Di Fiore P.P.
      • Robbins K.C.
      • Aaronson S.A.
      ,
      • Kim H.R.C.
      • Upadhyay S.
      • Korsmeyer S.
      • Deuel T.F.
      ). PDGF AA activates only the α-PDGFR isoform, whereas the PDGF BB ligand activates both α-PDGFRs and β-PDGFRs (
      • Heldin C.H.
      ). The PDGF BB ligand is functionally identical to v-sis (
      • Waterfield M.D.
      • Scrace G.T.
      • Whittle N.
      • Stroobant P.
      • Johnsson A.
      • Wasteson A.
      • Westermark B.
      • Heldin C.-H.
      • Huang J.S.
      • Deuel T.F.
      ) and can drive cellular transformation in NIH3T3 cells. In contrast, PDGF AA is less potent at mediating this response. The ability of the PDGF BB ligand to drive cellular transformation of NIH3T3 cells more efficiently than the PDGF AA ligand is believed to be due to either activation of the β-PDGFR or due to the simultaneous activation of both types of receptors.
      The majority of efforts to elucidate β-PDGFR signal relay has focused on the role of the receptor-associated proteins in mediating regulated growth. In contrast, relatively little is known regarding the signal transduction pathways important for driving abnormal, unregulated growth akin to that which would be present in a cancerous or transformed state. Careful studies comparing the α- and β-PDGFRs suggest that the β-PDGFR more efficiently transforms cells and that a region in the tail of the β-PDGFR is critical for this effect (
      • Beckmann M.P.
      • Betsholtz C.
      • Heldin C.-H.
      • Westermark B.
      • DiMarco E.
      • Di Fiore P.P.
      • Robbins K.C.
      • Aaronson S.A.
      ,
      • Uren A.
      • Yu J.C.
      • Li W.
      • Chung I.Y.
      • Mahadevan D.
      • Pierce J.H.
      • Heidaran M.A.
      ). It has not yet been determined what signaling pathways are being modulated by this region of the β-PDGFR to enhance the transformation response.
      To better understand the role of PI3K, RasGAP, SHP-2, and PLC-γ in PDGF-dependent cellular transformation, we constructed a panel of chimeric PDGFR mutants. Each construct contained the extracellular α-PDGFR domain and the intracellular β-PDGFR domain with a tyrosine to phenylalanine substitution at the tyrosine(s) required for binding the receptor-associated proteins. The chimeric constructs were stably expressed in fibroblast cells lacking α-PDGFRs and evaluated for their ability to promote PDGF-dependent transformation by assaying growth in soft agar. The chimeric receptor that binds all of the PDGFR-associated proteins, N2WT, was capable of driving PDGF-dependent foci formation, whereas the chimera in which the binding sites for PI3K, RasGAP, SHP-2, and PLC-γ were mutated failed to promote growth in soft agar. These observations suggested that activation of the kinase activity of the receptor was not enough to drive cellular transformation, and that one or more of the signaling enzymes recruited to the receptor are required. Using the panel of β-PDGFR mutants, we found that activation of either the PI3K or PLC-γ signaling cascades restored PDGF-dependent growth in soft agar. We conclude that similar signaling cascades are used to drive normal as well as deregulated growth of cells.

      DISCUSSION

      In this study we have evaluated the importance of RasGAP, SHP-2, PLC-γ, and PI3K in promoting PDGF-dependent growth of mouse Ph fibroblasts in soft agar. We observed that receptors capable of associating with PI3K and PLC-γ, but not RasGAP or SHP-2, initiate pathways that result in foci formation.
      There is considerable controversy concerning the role of PI3K and PLC-γ in PDGF-dependent cell cycle progression. One important variable is that, depending on the type of PDGF used, one or more isoform of the PDGF receptor can be activated. Different signaling pathways are used by the α- and β-PDGFRs; hence, the downstream effects are not identical. Studies by Yu et al. (
      • Yu J.-C.
      • Gutkind J.S.
      • Mahadevan D.
      • Li W.
      • Meyers K.A.
      • Pierce J.H.
      • Heidaran M.A.
      ,
      • Yu J.C.
      • Li W.
      • Wang L.-M.
      • Uren A.
      • Pierce J.H.
      • Heidaran M.A.
      ) suggest that stimulation of chimeric receptor constructs that contained colony-stimulating factor-1 receptor extracellularly and α-PDGFR receptor intracellularly promoted growth in soft agar in response to colony-stimulating factor stimulation. Mutation of one or both of the sites required for binding of PI3K and PLC-γ to the α-PDGFR did not compromise the ability of these chimeric constructs to promote growth in soft agar (
      • Yu J.-C.
      • Gutkind J.S.
      • Mahadevan D.
      • Li W.
      • Meyers K.A.
      • Pierce J.H.
      • Heidaran M.A.
      ,
      • Yu J.C.
      • Li W.
      • Wang L.-M.
      • Uren A.
      • Pierce J.H.
      • Heidaran M.A.
      ). These findings suggest that for the α-PDGFR, PI3K and PLC-γ are dispensable for PDGF-dependent growth in soft agar. In contrast, using the β-PDGFR chimera system described here, we find that the β-PDGFR initiates multiple pathways that lead to growth in soft agar, and that PI3K and PLC-γ are required to engage these events. The extent to which these pathways converge downstream of PI3K and PLC-γ remains to be investigated.
      In comparison with the α-PDGFR, the β-PDGFR more efficiently stimulates growth of cells in soft agar, and recent studies (
      • Uren A.
      • Yu J.C.
      • Li W.
      • Chung I.Y.
      • Mahadevan D.
      • Pierce J.H.
      • Heidaran M.A.
      ) identified a region in the β-PDGFR tail that is important for conferring the enhanced transforming activity of the β-PDGFR. These studies were carried out by swapping a piece of the β-PDGFR into α-PDGFR, which included the PLC-γ binding site for both of the receptors. As a result the swapped receptor is an α-PDGFR with a β-PDGFR PLC-γ binding site. The α-PDGFRs and β-PDGFRs have been compared with respect to PLC-γ binding and activation (
      • Eriksson A.
      • Nanberg E.
      • Ronnstrand L.
      • Engstrom U.
      • Hellman U.
      • Rupp E.
      • Carpenter G.
      • Heldin C.H.
      • Claesson-Welsh L.
      ), and based on these findings, one would predict that replacing the PLC-γ binding site of the α-PDGFR with the PLC-γ binding site of the β-PDGFR would decrease binding of PLC-γ and increase tyrosine phosphorylation and activation of PLC-γ. Although the ability of the swapped receptor to activate PLC-γ has not been reported, the hybrid receptor should activate PLC-γ better than wild-type α-PDGFR. This is consistent with our observation that PLC-γ is required for soft agar growth by the β-PDGFR. However, it is not consistent with findings by Obermeier et al. (
      • Obermeier A.
      • Tinhofer I.
      • Grunicke H.H.
      • Ullrich A.
      ), which suggest that PLC-γ activation inversely correlates with transformation. Although all of these studies indicate that PLC-γ is important in cellular transformation, the exact role of this signaling molecule, as well as whether it has a positive or negative effect, has not yet emerged.
      An unexpected observation that arose from our studies was the ability of the chimeric receptors to bind SHP-2 independent of phosphorylation of tyrosine 1009 (
      • Valius M.
      • Kazlauskas A.
      ,
      • Lechleider R.
      • Sugimoto S.
      • Bennett A.
      • Kashishian A.
      • Cooper J.
      • Shoelson S.
      • Walsh C.
      • Neel B.
      ). Previous studies have shown that mutating the tyrosine at position 1009 in the WT receptor largely eliminates SHP-2 binding, whereas restoring tyrosine at 1009 in the F5 receptor enables the phosphorylated β-PDGFR to bind SHP-2 stably (
      • Valius M.
      • Kazlauskas A.
      ). One difference between these studies is the host cell line in which these receptor constructs were expressed. The chimeric receptors were expressed in Ph cells, whereas the β-PDGFR mutants were expressed in HepG2 cells. Consequently, we tested whether the unusual binding of SHP-2 was cell line-dependent and found that this was not the case.
      Unpublished observations.
      Although SHP-2 binding is still dependent on tyrosine phosphorylation of the receptor, it appears that the chimera is able to bind SHP-2 in a Tyr1009-independent manner. It is possible that creation of the chimeric receptors alters receptor conformation such that some sequences that were previously unavailable for SHP-2 binding are now accessible. Studies are currently under way to understand better the binding of SHP-2 to the chimeric PDGFRs independent of an intact binding site.
      Although SHP-2 associated with all of the receptors used in these studies, we do not think that this changes the interpretation of the soft agar data. Previous studies from our laboratory have suggested that SHP-2 does not play a central role in relaying signals that lead to a proliferative response (
      • Valius M.
      • Secrist J.P.
      • Kazlauskas A.
      ,
      • Klinghoffer R.A.
      • Duckworth B.
      • Valius M.
      • Cantley L.
      • Kazlauskas A.
      ). Furthermore, restoring the binding site for SHP-2 to β-PDGFRs capable of associating with PLC-γ had no effect on PLC-γ binding, activation of PLC-γ, or the DNA synthesis response (
      • Valius M.
      • Secrist J.P.
      • Kazlauskas A.
      ). Similarly, restoration of the SHP-2 binding site to receptors capable of associating with PI3K did not inhibit PDGF-dependent activation of PI3K (
      • Klinghoffer R.A.
      • Kazlauskas A.
      ). Thus SHP-2 binding does not negatively affect signaling by either PLC-γ or PI3K, and binding of SHP-2 itself does not mediate DNA synthesis. These data are consistent with the idea that SHP-2 may not play a pivitol role in regulating the PDGF-dependent growth of cells in soft agar.
      Although we have focused on binding of PI3K, PLC-γ, SHP-2, and RasGAP to the chimeric receptor mutants described in these experiments, it is likely that several other proteins associate with these receptors as well. For instance, Src associates with the F5 receptor to near-WT levels when expressed in HepG2 or A431 cell lines.
      J. P. Secrist, J. A. Gelderloos, R. R. Vaillancourt, and A. Kazlauskas, manuscript in preparation.
      The chimeric N2F5 receptor was unable to mediate PDGF-dependent growth of cells in soft agar, indicating that Src and other proteins that may associate with the N2F5 receptor are not sufficient to trigger a biological response. However, it is possible that Src or these other receptor-associated proteins play a co-operative role in mediating this response.
      In summary, these studies suggest that PI3K or PLC-γ binding to chimeric α/β receptors is required for PDGF-dependent growth of cells in soft agar. Previous reports (
      • Valius M.
      • Kazlauskas A.
      ) have suggested that activation of PI3K or PLC-γ is sufficient to promote PDGF-dependent DNA synthesis. The data present herein indicate that PI3K or PLC-γ are required for PDGF-dependent transformation and suggest that the early steps in the pathways leading to normal or cancerous growth are surprisingly similar.

      Acknowledgments

      We thank Charlie Hart for the PDGF AA, Dan Bowen-Pope for the Ph cells, Chuck Stiles for the BALB/C 3T3/v-sis cells, and members of the Kazlauskas laboratory for critically reviewing the manuscript.

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