Gastric ezrin is a 80kDa protein, which is highly enriched in apical microvilli of gastric parietal cells and with sequence homology to talin and erythrocyte band 4.1. Ezrin has beensuggested to play a key role in mediating the apical surface rearrangements involved in acid secretion by parietal cells. The mediation of stimulation of acid secretion by the cAMP-dependent protein kinase A pathway has been correlated with the phosphorylation of ezrin. These studies suggest that phosphorylation is at serine/threonine residues and not at tyrosine residues. On the other hand, ezrin in A431 cells which overexpresses EGF receptors was characterized as a substrate for an EGF-stimulated tyrosine kinase and was phosphorylated at bothserine and tyrosine residues (Brestcher et al., 1989). EGF inhibited acid secretion in parietal cells and it has been suggested that EGF-stimulated tyrosine phosphorylation of ezrin might itself inhibit stimulation. In this project we propose to examine the site-directed phosphorylation of ezrin by histamine and EGF which stimulate and inhibit acid secretion respectively and identify the in vivo phosphorylation sites in each case. Our experimental strategy would involve proteolysis of ezrin in gel slices, followed by purification of phosphopeptides on iron-loaded (FeIII) nitriloacetic (NTA) sepaharose columns, the separation of the phosphopeptides by HPLC and the identification of the specific Ser, Thr or Tyr phosphorylated, by MALDI mass spectrometry and post-source decay analysis.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Biotechnology Resource Grants (P41)
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