The applicant recently demonstrated the rapid reversible phosphorylation of two histamine residues in the cytoplasmic domain of PECAM during activation of platelets. In this proposal, the applicant postulates the existence of a new cell activation signaling pathway involving transient phosphohistidine formation in mammalian cells. The application describes experiments to determine whether additional platelet proteins undergo transient phosphorylation on histamine residues and to identify and characterize the protein kinases and phosphatases involved. To determine the generality of cell activation-dependent phosphorylation/dephosphorylation on protein histidine residues, known platelet proteins for which there is a change in phosphorylation state upon platelet activation will be examined for phosphohistidine content. Anti-phosphohistidine antibodies will be developed and used to screen more broadly for the activation dependent phosphorylation/dephosphorylation of platelet proteins. A few known platelet kinases and phosphatases, for which there are literature data to suggest a potential role in histidine phosphorylation, will be examined for histidine kinase or phosphohistidine phosphatase activity. The two hybrid screening method will be used to identify novel platelet histidine kinases and phosphatases. An alternative to finding such novel kinases and phosphatases may be direct purification from platelet lysates. In either case, the newly identified histidine kinases and phosphohistidine phosphatases will be thoroughly characterized with regard to enzymatic activity and cell localization. Successful isolation of histidine activities are regulated by cell activation will confirm the postulate stated above. As proteins and enzymes that regulate serine, threonine and tyrosine phosphorylation have been associated with regulation of cell growth and attachment and in malignant transformation, identification of novel signal transduction pathways is important for understanding these processes. Regulation of phosphorylation of platele proteins is initimately involved with the biochemical and morphologic changes that occur in these cells upon activation and thus the role of platelets in hemostatic and thrombotic processes.
| Furie, B; Furie, B C; Flaumenhaft, R (2001) A journey with platelet P-selectin: the molecular basis of granule secretion, signalling and cell adhesion. Thromb Haemost 86:214-21 |
| Flaumenhaft, R; Furie, B; Furie, B C (1999) Alpha-granule secretion from alpha-toxin permeabilized, MgATP-exposed platelets is induced independently by H+ and Ca2+. J Cell Physiol 179:1-10 |
| Flaumenhaft, R; Croce, K; Chen, E et al. (1999) Proteins of the exocytotic core complex mediate platelet alpha-granule secretion. Roles of vesicle-associated membrane protein, SNAP-23, and syntaxin 4. J Biol Chem 274:2492-501 |
