Identification of platelet-derived growth factor (PDGF) receptor-specific-Ca²⁺-dependent tyrosine phosphorylated proteins

PDGF elicits mitogenesis and chemotaxis in target cells in a Ca²⁺-dependent fashion. We have begun investigating which proteins are tyrosine phosphorylated in a Ca²⁺-dependent fashion by Ca²⁺ and /or PDGF. Porcine aortic endothelial (PAE) cells transfected with wildtype (wt) or mutant (mt) PDGF α or β receptors were exposed to low (chemotactic) doses of PDGF or Thapsigargin (TG) in the presence or absence of extracellular Ca²⁺. Numerous proteins were found to be tyrosine phosphorylated in a dose, time and Ca²⁺-dependent fashion following TG treatment. The two major tyrosine phosphorylated bands were identified as focal adhesion kinase (FAK) and paxillin. lonomycin also caused tyrosine phosphorylation of these proteins, which was inhibited by the Ca²⁺ chelator BAPTA. PDGF resulted in phosphorylation of FAK and paxillin; however, only α receptor activation (with PDGF AA), resulted in a Ca²⁺-dependent increase in paxillin tyrosine phosphorylation, although lysophosphatidic acid (LPA) induced tyrosine phosphorylation of both FAK and paxillin in PAE cells expressing either wt or mt PDGF α receptors. Mutation of α tyrosine 768 to phenylalanine was found to prevent PDGF-induced phosphorylation of FAK. These findings suggest that PDGF-induced Ca²⁺-dependent tyrosine phosphorylation of FAK and paxillin may play a role in PDGF-induced chemotaxis.