Inhibition of phosphotyrosine phosphatases reveals candidate substrates of the PDGF receptor kinase.
In normal fibroblasts stimulated by platelet derived growth factor (PDGF), PDGF receptors are transiently phosphorylated on tyrosine and represent the major phosphotyrosine containing protein. The phosphate of the phosphotyrosine groups turns over rapidly, and extensive evidence indicates a dynamic balance between phosphorylation and dephosphorylation reactions. Thus, the effect of an inhibitor of phosphatases, orthovanadate, on the pattern of the tyrosine phosphorylations induced by PDGF in Swiss 3T3 fibroblasts was investigated. Western blot analysis with antibodies against phosphotyrosine indicated that whereas in unstimulated cells no phosphotyrosine containing proteins were detected, treatment of cells with orthovanadate alone elicited the slow phosphorylation of several proteins including a 170 kDa component that was recognized to be the phosphorylated PDGF receptor. Addition of PDGF to cells shortly pretreated with vanadate highly increased the intensity of the 170 kDa band corresponding to the phosphorylated receptor and caused its stabilization during time. In addition, the phosphorylation on tyrosine of other proteins (molecular mass 116, 80, 73, 60, 50 and 39 kDa) was also induced. Both the receptor and the other tyrosine phosphorylated proteins appeared to be associated with the detergent insoluble matrix.
Zippel
R
R
Department of General Physiology and Biochemistry, University of Milan, Italy.
Morello
L
L
Brambilla
R
R
Comoglio
P M
PM
Alberghina
L
L
Sturani
E
E
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D005347:Fibroblasts; D051379:Mice; D010449:Peptide Mapping; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000378:metabolism / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases; D011506:Proteins / Q000032:analysis / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism; D017479:Receptors, Platelet-Derived Growth Factor; D014443:Tyrosine / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
D010982:Platelet-Derived Growth Factor; D011506:Proteins; D011956:Receptors, Cell Surface; D014638:Vanadates; D014443:Tyrosine; D011494:Protein Kinases; D017479:Receptors, Platelet-Derived Growth Factor; D010749:Phosphoprotein Phosphatases; D017027:Protein Tyrosine Phosphatases