Fyn membrane localization is necessary to induce the constitutive tyrosine phosphorylation of Sam68 in the nucleus of T lymphocytes.
A close relationship between Sam68, a tyrosine and proline-rich RNA-binding protein, and Src protein tyrosine kinases (PTK) has already been established, also in T lymphocytes. A constitutive phosphorylation of the molecule has also been documented in various transformed T cells, which probably reflects an increased expression of PTK of the Src family. Using the hybridoma T cell line, T8.1, or Jurkat T cells, we investigated the respective contribution of the two Src kinases Fyn and Lck, expressed in T cells, in this phenomenon. By overexpressing the two proteins, we show that the constitutive phosphorylation of Sam68 in vivo directly correlates with cellular Fyn levels, but not with Lck expression, despite the capacity of the PTK to strongly phosphorylate the molecule in vitro. Overexpressed Fyn is mainly localized at the cell membrane. We find that Sam68 phosphorylation, including in the nuclear fraction in which the molecule is predominantly expressed, is lost with a delocalized Fyn mutant deleted of its N-terminal membrane-anchoring domain. Finally, we demonstrate, using a construct encoding a Sam68 molecule without its nuclear localization signal, that nuclear expression of Sam68 is not required for phosphorylation. We conclude that the constitutive phosphorylation of Sam68 in T cells is a Fyn-dependent process occurring in a cell-membrane compartment from which phospho-Sam68 molecules can thereafter accumulate into the nucleus.
Lang
V
V
Laboratoire d'Immunologie Cellulaire, Centre National de la Recherche Scientifique UMR 7627, Centre Hospitalier Pitié-Salpêtrière, CERVI, Paris, France.
Semichon
M
M
Michel
F
F
Brossard
C
C
Gary-Gouy
H
H
Bismuth
G
G
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002467:Cell Nucleus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D006801:Humans; D006825:Hybridomas; D007424:Intracellular Fluid / Q000201:enzymology; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism; D051379:Mice; D019913:Nuclear Localization Signals / Q000276:immunology; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D013601:T-Lymphocytes / Q000201:enzymology* / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; C489559:KHDRBS1 protein, human; C513253:Khdrbs1 protein, mouse; D019913:Nuclear Localization Signals; D011518:Proto-Oncogene Proteins; D016601:RNA-Binding Proteins; D014443:Tyrosine; C494957:FYN protein, human; C494960:Fyn protein, mouse; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051076:Proto-Oncogene Proteins c-fyn; D019061:src-Family Kinases