Abstract
The fission yeast Schizosaccharomyces pombe has no detectable endogenous receptor tyrosine kinases or associated signalling apparatus, and we have used this cell system to reconstitute mammalian platelet-derived growth factor beta (PDGF beta) receptor-linked activation of phospholipase C gamma 2 (PLC gamma 2). The PDGF beta receptor migrates as a glycosylated protein of 165 kDa associated exclusively with membrane fractions. No tyrosine autophosphorylation was detected when PDGF beta was expressed alone. PLC gamma 2 appears as a 140-kDa protein distributed between particulate and soluble fractions which exhibits characteristic selectivity for phosphatidylinositol 4,5-bisphosphate and is sensitive to powerful activation by Ca2+. When coexpressed, both PDGF beta and PLC gamma 2 undergo tyrosine phosphorylation, and this is accompanied by a > 26-fold increase in [3H]inositol 4,5-biphosphate ([3H]IP2) and [3H]inositol 1,4,5-triphosphate [3H]IP3 production. Treatment with the tyrosine phosphatase inhibitor pervanadate further increased PLC gamma 2 tyrosine phosphorylation as well as [3H]IP2 and [3H]IP3 generation. Phosphorylated PLC gamma 2 was found predominantly in membrane fractions. To test a nonreceptor tyrosine kinase, we then expressed the human proto-oncogene c-src together with its negative regulator Csk. These were immunodetectable as bands at 60 kDa (c-Src) and 50 kDa (Csk) and distributed between membrane and cytosolic fractions. When yeast coexpressing c-Src, Csk, and PLC gamma 2 was incubated with pervanadate, PLC gamma 2 was tyrosine phosphorylated and [3H]IP2 and [3H]IP3 production increased 11.0- and 7.0-fold, respectively. Csk expressed alone with PLC gamma 2 was ineffective. Similar PLC gamma 2 activation was observed upon in vitro mixing with the extracts expressing either c-Src or the PDGF beta receptor. In summary, this is the first report of a reconstitution of mammalian tyrosine kinase-linked effector activation in yeast cells and also the first demonstration of direct PLC gamma 2 activation by the proto-oncogene c-src. These observations indicate that S. pombe provides a powerful cell system in which to study critical molecular interactions and activities underlying receptor and nonreceptor tyrosine kinase-dependent cell signaling.
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- Atherton-Fessler S., Hannig G., Piwnica-Worms H. Reversible tyrosine phosphorylation and cell cycle control. Semin Cell Biol. 1993 Dec;4(6):433–442. doi: 10.1006/scel.1993.1051. [DOI] [PubMed] [Google Scholar]
- Berridge M. J. Inositol trisphosphate and diacylglycerol: two interacting second messengers. Annu Rev Biochem. 1987;56:159–193. doi: 10.1146/annurev.bi.56.070187.001111. [DOI] [PubMed] [Google Scholar]
- Boulton T. G., Stahl N., Yancopoulos G. D. Ciliary neurotrophic factor/leukemia inhibitory factor/interleukin 6/oncostatin M family of cytokines induces tyrosine phosphorylation of a common set of proteins overlapping those induced by other cytokines and growth factors. J Biol Chem. 1994 Apr 15;269(15):11648–11655. [PubMed] [Google Scholar]
- Coggeshall K. M., McHugh J. C., Altman A. Predominant expression and activation-induced tyrosine phosphorylation of phospholipase C-gamma 2 in B lymphocytes. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5660–5664. doi: 10.1073/pnas.89.12.5660. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Emori Y., Homma Y., Sorimachi H., Kawasaki H., Nakanishi O., Suzuki K., Takenawa T. A second type of rat phosphoinositide-specific phospholipase C containing a src-related sequence not essential for phosphoinositide-hydrolyzing activity. J Biol Chem. 1989 Dec 25;264(36):21885–21890. [PubMed] [Google Scholar]
- Goldschmidt-Clermont P. J., Kim J. W., Machesky L. M., Rhee S. G., Pollard T. D. Regulation of phospholipase C-gamma 1 by profilin and tyrosine phosphorylation. Science. 1991 Mar 8;251(4998):1231–1233. doi: 10.1126/science.1848725. [DOI] [PubMed] [Google Scholar]
- Goode N. T., Parker P. J. A phorbol ester-responsive PKC-zeta generated by fusion with the regulatory domain of PKC-delta. FEBS Lett. 1994 Feb 28;340(1-2):145–150. doi: 10.1016/0014-5793(94)80190-8. [DOI] [PubMed] [Google Scholar]
- Gout I., Dhand R., Hiles I. D., Fry M. J., Panayotou G., Das P., Truong O., Totty N. F., Hsuan J., Booker G. W. The GTPase dynamin binds to and is activated by a subset of SH3 domains. Cell. 1993 Oct 8;75(1):25–36. [PubMed] [Google Scholar]
- Guinebault C., Payrastre B., Sultan C., Mauco G., Breton M., Levy-Toledano S., Plantavid M., Chap H. Tyrosine kinases and phosphoinositide metabolism in thrombin-stimulated human platelets. Biochem J. 1993 Jun 15;292(Pt 3):851–856. doi: 10.1042/bj2920851. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Heffetz D., Bushkin I., Dror R., Zick Y. The insulinomimetic agents H2O2 and vanadate stimulate protein tyrosine phosphorylation in intact cells. J Biol Chem. 1990 Feb 15;265(5):2896–2902. [PubMed] [Google Scholar]
- Homma Y., Sakamoto H., Tsunoda M., Aoki M., Takenawa T., Ooyama T. Evidence for involvement of phospholipase C-gamma 2 in signal transduction of platelet-derived growth factor in vascular smooth-muscle cells. Biochem J. 1993 Mar 15;290(Pt 3):649–653. doi: 10.1042/bj2900649. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hughes D. A., Ashworth A., Marshall C. J. Complementation of byr1 in fission yeast by mammalian MAP kinase kinase requires coexpression of Raf kinase. Nature. 1993 Jul 22;364(6435):349–352. doi: 10.1038/364349a0. [DOI] [PubMed] [Google Scholar]
- Jensen R. A., Beeler J. F., Heidaran M. A., LaRochelle W. J. Characterization of baculovirus-expressed human alpha and beta platelet-derived growth factor receptors. Biochemistry. 1992 Nov 10;31(44):10887–10892. doi: 10.1021/bi00159a032. [DOI] [PubMed] [Google Scholar]
- Kodaki T., Woscholski R., Hallberg B., Rodriguez-Viciana P., Downward J., Parker P. J. The activation of phosphatidylinositol 3-kinase by Ras. Curr Biol. 1994 Sep 1;4(9):798–806. doi: 10.1016/s0960-9822(00)00177-9. [DOI] [PubMed] [Google Scholar]
- Kuhné M. R., Zhao Z., Rowles J., Lavan B. E., Shen S. H., Fischer E. H., Lienhard G. E. Dephosphorylation of insulin receptor substrate 1 by the tyrosine phosphatase PTP2C. J Biol Chem. 1994 Jun 3;269(22):15833–15837. [PubMed] [Google Scholar]
- Kumjian D. A., Barnstein A., Rhee S. G., Daniel T. O. Phospholipase C gamma complexes with ligand-activated platelet-derived growth factor receptors. An intermediate implicated in phospholipase activation. J Biol Chem. 1991 Feb 25;266(6):3973–3980. [PubMed] [Google Scholar]
- Kundra V., Escobedo J. A., Kazlauskas A., Kim H. K., Rhee S. G., Williams L. T., Zetter B. R. Regulation of chemotaxis by the platelet-derived growth factor receptor-beta. Nature. 1994 Feb 3;367(6462):474–476. doi: 10.1038/367474a0. [DOI] [PubMed] [Google Scholar]
- Lammers R., Bossenmaier B., Cool D. E., Tonks N. K., Schlessinger J., Fischer E. H., Ullrich A. Differential activities of protein tyrosine phosphatases in intact cells. J Biol Chem. 1993 Oct 25;268(30):22456–22462. [PubMed] [Google Scholar]
- Li W., Deanin G. G., Margolis B., Schlessinger J., Oliver J. M. Fc epsilon R1-mediated tyrosine phosphorylation of multiple proteins, including phospholipase C gamma 1 and the receptor beta gamma 2 complex, in RBL-2H3 rat basophilic leukemia cells. Mol Cell Biol. 1992 Jul;12(7):3176–3182. doi: 10.1128/mcb.12.7.3176. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Liao F., Shin H. S., Rhee S. G. In vitro tyrosine phosphorylation of PLC-gamma 1 and PLC-gamma 2 by src-family protein tyrosine kinases. Biochem Biophys Res Commun. 1993 Mar 31;191(3):1028–1033. doi: 10.1006/bbrc.1993.1320. [DOI] [PubMed] [Google Scholar]
- Liao F., Shin H. S., Rhee S. G. Tyrosine phosphorylation of phospholipase C-gamma 1 induced by cross-linking of the high-affinity or low-affinity Fc receptor for IgG in U937 cells. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3659–3663. doi: 10.1073/pnas.89.8.3659. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Maa M. C., Leu T. H., Trandel B. J., Chang J. H., Parsons S. J. A protein that is highly related to GTPase-activating protein-associated p62 complexes with phospholipase C gamma. Mol Cell Biol. 1994 Aug;14(8):5466–5473. doi: 10.1128/mcb.14.8.5466. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Margolis B., Rhee S. G., Felder S., Mervic M., Lyall R., Levitzki A., Ullrich A., Zilberstein A., Schlessinger J. EGF induces tyrosine phosphorylation of phospholipase C-II: a potential mechanism for EGF receptor signaling. Cell. 1989 Jun 30;57(7):1101–1107. doi: 10.1016/0092-8674(89)90047-0. [DOI] [PubMed] [Google Scholar]
- Margolis B., Zilberstein A., Franks C., Felder S., Kremer S., Ullrich A., Rhee S. G., Skorecki K., Schlessinger J. Effect of phospholipase C-gamma overexpression on PDGF-induced second messengers and mitogenesis. Science. 1990 May 4;248(4955):607–610. doi: 10.1126/science.2333512. [DOI] [PubMed] [Google Scholar]
- Maundrell K. Thiamine-repressible expression vectors pREP and pRIP for fission yeast. Gene. 1993 Jan 15;123(1):127–130. doi: 10.1016/0378-1119(93)90551-d. [DOI] [PubMed] [Google Scholar]
- Meisenhelder J., Suh P. G., Rhee S. G., Hunter T. Phospholipase C-gamma is a substrate for the PDGF and EGF receptor protein-tyrosine kinases in vivo and in vitro. Cell. 1989 Jun 30;57(7):1109–1122. doi: 10.1016/0092-8674(89)90048-2. [DOI] [PubMed] [Google Scholar]
- Mohammadi M., Honegger A. M., Rotin D., Fischer R., Bellot F., Li W., Dionne C. A., Jaye M., Rubinstein M., Schlessinger J. A tyrosine-phosphorylated carboxy-terminal peptide of the fibroblast growth factor receptor (Flg) is a binding site for the SH2 domain of phospholipase C-gamma 1. Mol Cell Biol. 1991 Oct;11(10):5068–5078. doi: 10.1128/mcb.11.10.5068. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mohammadi M., Honegger A., Sorokin A., Ullrich A., Schlessinger J., Hurwitz D. R. Aggregation-induced activation of the epidermal growth factor receptor protein tyrosine kinase. Biochemistry. 1993 Aug 31;32(34):8742–8748. doi: 10.1021/bi00085a004. [DOI] [PubMed] [Google Scholar]
- Mondesert O., Moreno S., Russell P. Low molecular weight protein-tyrosine phosphatases are highly conserved between fission yeast and man. J Biol Chem. 1994 Nov 11;269(45):27996–27999. [PubMed] [Google Scholar]
- Moreno S., Klar A., Nurse P. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 1991;194:795–823. doi: 10.1016/0076-6879(91)94059-l. [DOI] [PubMed] [Google Scholar]
- Mori S., Rönnstrand L., Yokote K., Engström A., Courtneidge S. A., Claesson-Welsh L., Heldin C. H. Identification of two juxtamembrane autophosphorylation sites in the PDGF beta-receptor; involvement in the interaction with Src family tyrosine kinases. EMBO J. 1993 Jun;12(6):2257–2264. doi: 10.1002/j.1460-2075.1993.tb05879.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Morrison D. K., Kaplan D. R., Rhee S. G., Williams L. T. Platelet-derived growth factor (PDGF)-dependent association of phospholipase C-gamma with the PDGF receptor signaling complex. Mol Cell Biol. 1990 May;10(5):2359–2366. doi: 10.1128/mcb.10.5.2359. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nishibe S., Wahl M. I., Hernández-Sotomayor S. M., Tonks N. K., Rhee S. G., Carpenter G. Increase of the catalytic activity of phospholipase C-gamma 1 by tyrosine phosphorylation. Science. 1990 Nov 30;250(4985):1253–1256. doi: 10.1126/science.1700866. [DOI] [PubMed] [Google Scholar]
- Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science. 1992 Oct 23;258(5082):607–614. doi: 10.1126/science.1411571. [DOI] [PubMed] [Google Scholar]
- Obermeier A., Bradshaw R. A., 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 Apr 1;13(7):1585–1590. doi: 10.1002/j.1460-2075.1994.tb06421.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Park D. J., Min H. K., Rhee S. G. IgE-induced tyrosine phosphorylation of phospholipase C-gamma 1 in rat basophilic leukemia cells. J Biol Chem. 1991 Dec 25;266(36):24237–24240. [PubMed] [Google Scholar]
- Park D. J., Rho H. W., Rhee S. G. CD3 stimulation causes phosphorylation of phospholipase C-gamma 1 on serine and tyrosine residues in a human T-cell line. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5453–5456. doi: 10.1073/pnas.88.12.5453. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Peles E., Levy R. B., Or E., Ullrich A., Yarden Y. Oncogenic forms of the neu/HER2 tyrosine kinase are permanently coupled to phospholipase C gamma. EMBO J. 1991 Aug;10(8):2077–2086. doi: 10.1002/j.1460-2075.1991.tb07739.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ren C. L., Morio T., Fu S. M., Geha R. S. Signal transduction via CD40 involves activation of lyn kinase and phosphatidylinositol-3-kinase, and phosphorylation of phospholipase C gamma 2. J Exp Med. 1994 Feb 1;179(2):673–680. doi: 10.1084/jem.179.2.673. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ren H. Y., Komatsu N., Shimizu R., Okada K., Miura Y. Erythropoietin induces tyrosine phosphorylation and activation of phospholipase C-gamma 1 in a human erythropoietin-dependent cell line. J Biol Chem. 1994 Jul 29;269(30):19633–19638. [PubMed] [Google Scholar]
- Rhee S. G., Choi K. D. Multiple forms of phospholipase C isozymes and their activation mechanisms. Adv Second Messenger Phosphoprotein Res. 1992;26:35–61. [PubMed] [Google Scholar]
- Schlessinger J., Ullrich A. Growth factor signaling by receptor tyrosine kinases. Neuron. 1992 Sep;9(3):383–391. doi: 10.1016/0896-6273(92)90177-f. [DOI] [PubMed] [Google Scholar]
- Sieh M., Batzer A., Schlessinger J., Weiss A. GRB2 and phospholipase C-gamma 1 associate with a 36- to 38-kilodalton phosphotyrosine protein after T-cell receptor stimulation. Mol Cell Biol. 1994 Jul;14(7):4435–4442. doi: 10.1128/mcb.14.7.4435. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sorokin A., Lemmon M. A., Ullrich A., Schlessinger J. Stabilization of an active dimeric form of the epidermal growth factor receptor by introduction of an inter-receptor disulfide bond. J Biol Chem. 1994 Apr 1;269(13):9752–9759. [PubMed] [Google Scholar]
- Stephens R. M., Loeb D. M., Copeland T. D., Pawson T., Greene L. A., Kaplan D. R. Trk receptors use redundant signal transduction pathways involving SHC and PLC-gamma 1 to mediate NGF responses. Neuron. 1994 Mar;12(3):691–705. doi: 10.1016/0896-6273(94)90223-2. [DOI] [PubMed] [Google Scholar]
- Sultzman L., Ellis C., Lin L. L., Pawson T., Knopf J. Platelet-derived growth factor increases the in vivo activity of phospholipase C-gamma 1 and phospholipase C-gamma 2. Mol Cell Biol. 1991 Apr;11(4):2018–2025. doi: 10.1128/mcb.11.4.2018. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Superti-Furga G., Fumagalli S., Koegl M., Courtneidge S. A., Draetta G. Csk inhibition of c-Src activity requires both the SH2 and SH3 domains of Src. EMBO J. 1993 Jul;12(7):2625–2634. doi: 10.1002/j.1460-2075.1993.tb05923.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Twamley-Stein G. M., Pepperkok R., Ansorge W., Courtneidge S. A. The Src family tyrosine kinases are required for platelet-derived growth factor-mediated signal transduction in NIH 3T3 cells. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7696–7700. doi: 10.1073/pnas.90.16.7696. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Valius M., Kazlauskas A. Phospholipase C-gamma 1 and phosphatidylinositol 3 kinase are the downstream mediators of the PDGF receptor's mitogenic signal. Cell. 1993 Apr 23;73(2):321–334. doi: 10.1016/0092-8674(93)90232-f. [DOI] [PubMed] [Google Scholar]
- Wahl M. I., Jones G. A., Nishibe S., Rhee S. G., Carpenter G. Growth factor stimulation of phospholipase C-gamma 1 activity. Comparative properties of control and activated enzymes. J Biol Chem. 1992 May 25;267(15):10447–10456. [PubMed] [Google Scholar]
- Weiss A., Koretzky G., Schatzman R. C., Kadlecek T. Functional activation of the T-cell antigen receptor induces tyrosine phosphorylation of phospholipase C-gamma 1. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5484–5488. doi: 10.1073/pnas.88.13.5484. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weiss A., Littman D. R. Signal transduction by lymphocyte antigen receptors. Cell. 1994 Jan 28;76(2):263–274. doi: 10.1016/0092-8674(94)90334-4. [DOI] [PubMed] [Google Scholar]
- Yang L. J., Rhee S. G., Williamson J. R. Epidermal growth factor-induced activation and translocation of phospholipase C-gamma 1 to the cytoskeleton in rat hepatocytes. J Biol Chem. 1994 Mar 11;269(10):7156–7162. [PubMed] [Google Scholar]
- Yarden Y., Escobedo J. A., Kuang W. J., Yang-Feng T. L., Daniel T. O., Tremble P. M., Chen E. Y., Ando M. E., Harkins R. N., Francke U. Structure of the receptor for platelet-derived growth factor helps define a family of closely related growth factor receptors. Nature. 1986 Sep 18;323(6085):226–232. doi: 10.1038/323226a0. [DOI] [PubMed] [Google Scholar]