In vivo and in vitro phosphorylation and subcellular localization of trypanosomatid cytoskeletal giant proteins

Promastigote forms of Phytomonas serpens, Leptomonas samueli, and Leishmania tarentolae express cytoskeletal giant proteins with apparent molecular masses of 3,500 kDa (Ps 3500), 2,500 kDa (Ls 2500), and 1,200 kDa (Lt 1200). respectively. Polyclonal antibodies to it 1200 and to Ps 3500 specifically recognize similar polypeptides of the same genera of parasite. in addition to reacting with giant polypeptides of the Leptomonas species, anti-is 2500 also cross reacts with Ps 3500, and with a 500-kDa polypeptide of Leishmania. Confocal immunofluorescence and immunogold electron microscopy showed major differences in topological distribution of these three proteins, though they partially share a common localization at the anterior end of the cell body skeleton. Furthermore, Ps 3500. Ls 2500, and it 1200 are in vivo phosphorylated at serine and threonine residues, whereas, in vitro phosphorylation of cytoskeletal fractions reveal that only Ps 3500 and Ls 2500 are phosphorylated. Heat treatment (100 degrees C) of high salt cytoskeletal extracts demonstrates that Ps 3500 and Ls 2500 remain stable in solution, whereas it 1200 is denatured. Kinase assays with immunocomplexes of heat-treated giant proteins show that only Ps 3500 and Ls 2500 are phosphorylated. These results demonstrate the existence of a novel class of megadalton phosphoproteins in promastigote forms of trypanosomatids that appear to be genera specific with distinct cytoskeletal functions. in addition, there is also evidence that Ps 3500 and is 2500, in contrast to it 1200, seem to be autophosphorylating serine and threonine protein kinases, suggesting that they might play regulatory roles in the cytoskeletal organization. (C) 2000 Wiley-Liss, Inc. Univ São Paulo, Dept Parasitol, Inst Ciencias Biomed, BR-05508900 São Paulo, Brazil Universidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, Escola Paulista Med, São Paulo, Brazil Universidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, Escola Paulista Med, São Paulo, Brazil Web of Science