Src-family tyrosine kinases (SFKs), which participate in a variety of signal transduction events, are known to localize to the cytoplasmic face of the plasma membrane through lipid modification. Recently, we showed that Lyn, an SFK member, is exocytosed to the plasma membrane via the Golgi region along the secretory pathway. We show here that SFK trafficking is specified by the palmitoylation state. Yes is also a monopalmitoylated SFK and is biosynthetically transported from the Golgi pool of caveolin to the plasma membrane. This pathway can be inhibited in the trans-Golgi network (TGN)-to-cell surface delivery by temperature block at 19°C or dominant-negative Rab11 GTPase. A large fraction of Fyn, a dually palmitoylated SFK, is directly targeted to the plasma membrane irrespective of temperature block of TGN exit. Fyn(C6S), which lacks the second palmitoylation site, is able to traffic in the same way as Lyn and Yes. Moreover, construction of Yes(S6C) and chimeric Lyn or Yes with the Fyn N-terminus further substantiates the importance of the dual palmitoylation site for plasma membrane targeting. Taken together with our recent finding that Src, a nonpalmitoylated SFK, is rapidly exchanged between the plasma membrane and late endosomes/lysosomes, these results suggest that SFK trafficking is specified by the palmitoylation state in the SH4 domain.

Src family non-receptor-type tyrosine kinases regulate a wide variety of cellular events including cell cycle progression in G(2)/M phase. Here, we show that Src signaling regulates the terminal step in cytokinesis called abscission in HeLa cells. Abscission failure with an unusually elongated intercellular bridge containing the midbody is induced by treatment with the chemical Src inhibitors PP2 and SU6656 or expression of membrane-anchored Csk chimeras. By anti-phosphotyrosine immunofluorescence and live cell imaging, completion of abscission requires Src-mediated tyrosine phosphorylation during early stages of mitosis (before cleavage furrow formation), which is subsequently delivered to the midbody through Rab11-driven vesicle transport. Treatment with U0126, a MEK inhibitor, decreases tyrosine phosphorylation levels at the midbody, leading to abscission failure. Activated ERK by MEK-catalyzed dual phosphorylation on threonine and tyrosine residues in the TEY sequence, which is strongly detected by anti-phosphotyrosine antibody, is transported to the midbody in a Rab11-dependent manner. Src kinase activity during the early mitosis mediates ERK activation in late cytokinesis, indicating that Src-mediated signaling for abscission is spatially and temporally transmitted. Thus, these results suggest that recruitment of activated ERK, which is phosphorylated by MEK downstream of Src kinases, to the midbody plays an important role in completion of abscission.