Glaxo Institute for Molecular Biology , Plan-les-Ouates , Geneva , Switzerland .
Mol Cell Biol 15 : 1431-8 ( 1995)
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 -kDa protein distributed between particulate and soluble fractions which exhibits characteristic selectivity for phosphatidylinositol , 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 , 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.(ABSTRACT TRUNCATED AT 250 WORDS )