Tr-kit-induced resumption of the cell cycle in mouse eggs requires activation of a Src-like kinase.
Microinjection in mouse eggs of tr-kit, a truncated form of the c-kit tyrosine kinase present in mouse spermatozoa, causes resumption of meiosis through activation of phospholipase Cgamma1 (PLCgamma1) and Ca(2+) mobilization from intracellular stores. We show that the Src-like kinase Fyn phosphorylates Tyr161 in tr-kit and that this residue is essential for tr-kit function. Fyn is localized in the cortex region underneath the plasma membrane in mouse oocytes. Using several approaches, we demonstrate that Fyn associates with tr-kit and that the interaction requires Tyr161. The interaction between tr-kit and Fyn triggers activation of the kinase as monitored by both autophosphorylation and phosphorylation of PLCgamma1. Co-injection of tr-kit with the SH2 domain of Fyn, or pre-treatment with a Fyn inhibitor, impairs oocyte activation, suggesting that activation of Fyn by tr-kit also occurs in vivo. Finally, microinjection of constitutively active Fyn triggers oocyte activation downstream of tr-kit but still requires PLC activity. We suggest that the mechanism by which tr-kit triggers resumption of meiosis of mouse eggs requires a functional interaction with Fyn and phosphorylation of PLCgamma1.
Sette
Claudio
C
Department of Public Health and Cell Biology, Section of Anatomy, University of Rome Tor Vergata, Rome, Italy. claudio.sette@uniroma2.it
Paronetto
Maria Paola
MP
Barchi
Marco
M
Bevilacqua
Arturo
A
Geremia
Raffaele
R
Rossi
Pellegrino
P
D000818:Animals; D001665:Binding Sites; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D005306:Fertilization / Q000502:physiology; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism; D008297:Male; D008540:Meiosis / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008845:Microinjections; D008954:Models, Biological; D009865:Oocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D010446:Peptide Fragments / Q000008:administration & dosage / Q000737:chemistry / Q000378:metabolism; D051966:Phospholipase C gamma; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn; D019009:Proto-Oncogene Proteins c-kit / Q000008:administration & dosage / Q000737:chemistry / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry; D019061:src-Family Kinases / Q000737:chemistry / Q000378:metabolism*
D007527:Isoenzymes; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D014443:Tyrosine; D019009:Proto-Oncogene Proteins c-kit; C494960:Fyn protein, mouse; D051076:Proto-Oncogene Proteins c-fyn; D019061:src-Family Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma