Activation of phospholipase C gamma in Schizosaccharomyces pombe by coexpression of receptor or nonreceptor tyrosine kinases.
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.
Arkinstall
S
S
Glaxo Institute for Molecular Biology, Plan-les-Ouates, Geneva, Switzerland.
Payton
M
M
Maundrell
K
K
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D003001:Cloning, Molecular; D004789:Enzyme Activation; D015870:Gene Expression; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D010716:Phosphatidylinositols / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D000090063:Proto-Oncogene Mas; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D020797:Receptor, Platelet-Derived Growth Factor beta; D017479:Receptors, Platelet-Derived Growth Factor / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012568:Schizosaccharomyces / Q000378:metabolism*; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D007527:Isoenzymes; C000715470:MAS1 protein, human; D010716:Phosphatidylinositols; D000090063:Proto-Oncogene Mas; D011994:Recombinant Proteins; C076715:pervanadate; D014638:Vanadates; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases; D020797:Receptor, Platelet-Derived Growth Factor beta; D017479:Receptors, Platelet-Derived Growth Factor; D010738:Type C Phospholipases; D002118:Calcium