Characterization of the tyrosine phosphorylation of calpactin I (annexin II) induced by platelet-derived growth factor.
Stimulation in vivo of Swiss 3T3 fibroblasts with platelet-derived growth factor (PDGF) in the presence of orthovanadate induces the tyrosine phosphorylation of a 39 kDa protein, identified as the phosphorylated slow-migrating form of calpactin I (annexin II) heavy chain, p36. In fact, in PDGF-stimulated cells, anti-(calpactin I) antibodies recognize a doublet of bands, p36 and p39, and the latter disappears upon treatment with phosphatase. In many regards phosphorylation of p39 differs from the rapid and transient phosphorylation of the PDGF receptor and of other substrates: (a) it has slower kinetics but is then stable for longer periods of time; (b) it occurs at 37 degrees C but not at 4 degrees C; and (c) whereas most of the tyrosine-phosphorylated proteins are associated with membrane-enriched preparations, membrane association of p39 only occurs in the presence of Ca2+. Moreover, calpactin I leaks out of permeabilized cells at 0.1 microM free Ca2+, whereas it remains associated with the cells at concentrations of Ca2+ greater than or equal to 1 microM. PDGF does not stimulate phosphoinositide turnover (and thus Ca2+ mobilization) at 4 degrees C; thus it can be suggested that the Ca(2+)-dependent translocation of the protein to membrane/cytoskeletal structures is a necessary condition for its phosphorylation. In addition, calpactin I may not be a direct substrate for the PDGF receptor kinase, but rather the substrate of another tyrosine kinase activated by the receptor.
Brambilla
R
R
Department of General Physiology and Biochemistry, University of Milan, Italy.
Zippel
R
R
Sturani
E
E
Morello
L
L
Peres
A
A
Alberghina
L
L
D000818:Animals; D017302:Annexins; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002460:Cell Line; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D010716:Phosphatidylinositols / Q000378:metabolism; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D017302:Annexins; D002135:Calcium-Binding Proteins; D008565:Membrane Proteins; D010716:Phosphatidylinositols; D010982:Platelet-Derived Growth Factor; D014638:Vanadates; D014443:Tyrosine