Src-family tyrosine kinases (SFKs), which are non-receptor-type tyrosine kinases, consist of proto-oncogene products and structurally related proteins and include at least eight highly homologous proteins: Src, Lyn, Fyn, Yes, Fgr, Hck, Lck and Blk (Brown and Cooper, 1996;Thomas and Brugge, 1997). SFKs are activated by various stimuli, including growth factors and adhesion proteins, and are involved in a wide range of signaling events at the plasma membrane, resulting in cell proliferation, differentiation, migration, and cell-shape changes. Src, Yes, Lyn and Fyn are widely expressed in a variety of cell types, whereas Blk, Fgr, Hck and Lck are found primarily in hematopoietic cells (Bolen and Brugge, 1997;Thomas and Brugge, 1997).SFKs are composed of (1) an N-terminal Src homology (SH) 4 domain that contains lipid modification sites; (2) a poorly conserved 'unique' domain; (3) an SH3 domain that can bind to specific proline-rich sequences; (4) an SH2 domain that can bind to specific sites of tyrosine phosphorylation; (5) an SH1 tyrosine kinase catalytic domain; and (6) a negative regulatory tail for autoinhibition of kinase activity (Brown and Cooper, 1996;Thomas and Brugge, 1997). All members of the Src family are cotranslationally myristoylated at Gly2 and, with the exception of Src and Blk, are also post-translationally palmitoylated at Cys3, Cys5 or Cys6 (Paige et al., 1993;Alland et al., 1994;Koegl et al., 1994;Resh, 1994; Shenoy-Scaria et al., 1994; Kasahara et al., 2007a). Fatty acylation of SFKs has been shown to influence their interactions with cell membranes (McCabe and Berthiaume, 1999;Resh, 1999) and as a consequence their intracellular distribution.It is generally thought that SFKs are predominantly located at the cytoplasmic face of the plasma membrane through posttranslational myristoylation, usually with subsequent palmitoylation, but in fact, appreciable fractions are found at in variety of intracellular locations, such as endosomes, secretory granules or phagosomes and the Golgi complex (Kaplan et al., 1992;Mohn et al., 1995;Brown and Cooper, 1996;Thomas and Brugge, 1997;Kasahara et al., 2004). Although distinctive localizations of SFK members have been implicated in their specific functions, the mechanism that underlies the targeting of SFKs to their specific locations remains to be elucidated.We recently showed that Lyn, a palmitoylated SFK, is exocytosed to the plasma membrane via the Golgi region along the secretory pathway (Kasahara et al., 2004). More recently, we demonstrated that Src, a non-palmitoylated SFK, rapidly moves between the plasma membrane and late endosomes or lysosomes, and that mutation of Cys3 in Lyn allows Lyn to traffic in a similar manner to Src (Kasahara et al., 2007a), indicating the importance of palmitoylation for distinct trafficking between Lyn and Src.In this study, we investigate the localization and trafficking of other ubiquitously expressed SFKs, such as Yes and Fyn. We demonstrate that Lyn and Yes, which are monopalmitoylated SFKs, Src-family tyrosine kin...

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Src-family kinases (SFKs), which participate in various signaling events, are found at not only the plasma membrane but also several subcellular compartments, including the nucleus. Nuclear structural changes are frequently observed during transcription, cell differentiation, senescence, tumorigenesis, and cell cycle. However, little is known about signal transduction in the alteration of chromatin texture. Here, we develop a pixel imaging method for quantitatively evaluating chromatin structural changes. Growth factor stimulation increases euchromatic hypocondensation and concomitant heterochromatic hypercondensation in G(1) phase, and the levels reach a plateau by 30 min, sustain for at least 5 h and return to the basal levels after 24 h. Serum-activated SFKs in the nucleus were more frequently detected in the euchromatin areas than the heterochromatin areas. Nuclear expression of kinase-active SFKs, but not unrelated Syk kinase, drastically increases both euchromatinization and heterochromatinization in a manner dependent on the levels of nuclear tyrosine phosphorylation. However, growth factor stimulation does not induce chromatin structural changes in SYF cells lacking SFKs, and reintroduction of one SFK member into SYF cells can, albeit insufficiently, induce chromatin structural changes. These results suggest that nuclear tyrosine phosphorylation by SFKs plays an important role in chromatin structural changes upon growth factor stimulation.

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Background:We showed that nuclear tyrosine phosphorylation is involved in chromatin structural changes. Results: Several tyrosine kinases phosphorylate KAP1 at Tyr-449, Tyr-458, and Tyr-517 in the nucleus, resulting in a decrease of KAP1 association with heterochromatin. Conclusion: Tyrosine phosphorylation of KAP1 by nucleus-localized tyrosine kinases, including Src, involves heterochromatin structural changes. Significance: These findings provide a new insight into nuclear tyrosine phosphorylation signals.

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Introduction of genetic material into cells is an essential prerequisite for current research in molecular cell biology. Although transfection with commercially available reagents results in excellent gene expression, their high costs are obstacles to experimentation with a large number or large scales of transfection. The cationic polymer linear-polyethylenimine (MW 25,000) (PEI), one of the most cost-effective vehicles, facilitates DNA compaction by polyplex formation, which leads to efficient delivery of DNA into cells by endocytosis. However, the use of PEI is still limited because of substantial cytotoxicity and intolerable deterioration in transfection efficiency by its low stability. Here, we show that acidification of PEI is important for its transfection activity. Dissolving PEI powder in 0.2N HCl confers a long shelf-life for PEI storage at 4 and -80 degrees C, and the polyplex formation of plasmid DNA with PEI is optimized in lactate-buffered saline at pH 4.0. Furthermore, changing the culture medium at 8-12 h posttransfection can minimize the cytotoxicity of PEI without sacrificing the high transfection efficiency comparable to that of commercial reagents. The cost per test using acidified PEI is drastically reduced to approximately 1:10,000, compared with commercial reagents. Thus, we conclude that acidification of PEI satisfactorily accomplishes cost-effective, high-efficiency transfection.