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...

The production of cells capable of expressing gene(s) of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and animal transgenesis. The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis. Recent work on integrases from ΦC31, R4, TP901-1 and Bxb1 phages demonstrated that these recombinases catalyze site-specific recombination in mammalian cells. In the present study, we examined the activities of integrases on site-specific recombination and gene expression in mammalian cells. We designed a human artificial chromosome (HAC) vector containing five recombination sites (ΦC31 attP, R4 attP, TP901-1 attP, Bxb1 attP and FRT; multi-integrase HAC vector) and de novo mammalian codon-optimized integrases. The multi-integrase HAC vector has several functions, including gene integration in a precise locus and avoiding genomic position effects; therefore, it was used as a platform to investigate integrase activities. Integrases carried out site-specific recombination at frequencies ranging from 39.3–96.8%. Additionally, we observed homogenous gene expression in 77.3–87.5% of colonies obtained using the multi-integrase HAC vector. This vector is also transferable to another cell line, and is capable of accepting genes of interest in this environment. These data suggest that integrases have high DNA recombination efficiencies in mammalian cells. The multi-integrase HAC vector enables us to produce transgene-expressing cells efficiently and create platform cell lines for gene expression.

Src-family kinases, known to participate in signaling pathways of a variety of surface receptors, are localized to the cytoplasmic side of the plasma membrane through lipid modification. We show here that Lyn, a member of the Src-family kinases, is biosynthetically transported to the plasma membrane via the Golgi pool of caveolin along the secretory pathway. The trafficking of Lyn from the Golgi apparatus to the plasma membrane is inhibited by deletion of the kinase domain or Csk-induced “closed conformation” but not by kinase inactivation. Four residues (Asp346 and Glu353 on αE helix, and Asp498 and Asp499 on αI helix) present in the C-lobe of the kinase domain, which can be exposed to the molecular surface through an “open conformation,” are identified as being involved in export of Lyn from the Golgi apparatus toward the plasma membrane but not targeting to the Golgi apparatus. Thus, the kinase domain of Lyn plays a role in Lyn trafficking besides catalysis of substrate phosphorylation.

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 membraneanchored 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.

The respiratory chain of Gram-negative marine and halophilic bacteria has a Na(+)-dependent NADH-quinone reductase that functions as a primary Na(+) pump. The Na(+)-translocating NADH-quinone reductase (NQR) from the marine Vibrio alginolyticus is composed of six structural genes (nqrA to nqrF). The NqrF subunit has non-covalently bound FAD. There are conflicting results on the existence of other flavin cofactors. Recent studies revealed that the NqrB and NqrC subunits have a covalently bound flavin, possibly FMN, which is attached to a specified threonine residue. A novel antibiotic, korormicin, was found to specifically inhibit the NQR complex. From the homology search of the nqr operon, it was found that the Na(+)-pumping NQR complex is widely distributed among Gram-negative pathogenic bacteria.

The Na + -translocating NADH-quinone reductase (NQR) from Vibrio alginolyticus is composed of six subunits (NqrA to NqrF). We previously demonstrated that both NqrB and NqrC subunits contain a flavin cofactor covalently attached to a threonine residue. Fluorescent peptide fragments derived from the NqrB and NqrC subunits were applied to a matrixassisted laser desorption ionization time-of-flight mass spectrometer, and covalently attached flavin was identified as FMN in both subunits. From post-source decay fragmentation analysis, it was concluded that FMN is attached by a phosphate group to Thr-235 in the NqrB subunit and to Thr-223 in the NqrC subunit. The phosphoester binding of FMN to a threonine residue reported here is a new type of flavin attachment to a polypeptide. ß

Src-family kinases that localize to the cytoplasmic side of cellular membranes through lipid modification play a role in signaling events including membrane trafficking. Macropinocytosis is an endocytic process for solute uptake by large vesicles called macropinosomes. Although macropinosomes can be visualized following uptake of fluorescent macromolecules, little is known about the dynamics of macropinosomes in living cells. Here, we show that constitutive c-Src expression generates macropinosomes in a kinase-dependent manner. Live-cell imaging of GFP-tagged c-Src (Src-GFP) reveals that c-Src associates with macropinosomes via its N-terminus continuously from their generation at membrane ruffles, through their centripetal trafficking, to fusion with late endosomes and lysosomes. Fluorescence recovery after photobleaching (FRAP) of Src-GFP shows that Src-GFP is rapidly recruited to macropinosomal membranes from the plasma membrane and intracellular organelles through vesicle transport even in the presence of a protein synthesis inhibitor. Furthermore, using a HeLa cell line overexpressing inducible c-Src, we show that following stimulation with epidermal growth factor (EGF), high levels of c-Src kinase activity promote formation of macropinosomes associated with the lysosomal compartment. Unlike c-Src, Lyn and Fyn, which are palmitoylated Src kinases, only minimally induce macropinosomes, although a Lyn mutant in which the palmitoylation site is mutated efficiently induces macropinocytosis. We conclude that kinase activity of nonpalmitoylated Src kinases including c-Src may play an important role in the biogenesis and trafficking of macropinosomes.

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