Differential mitotic activation of endogenous c-Src, c-Yes, and Lyn in HeLa cells

The regulation of protein tyrosine phosphorylation is an important aspect during the cell cycle. From G2-M transition to mitotic anaphase, phosphorylation of Tyr421, Tyr466 and Tyr482 of cortactin, an actin-filament associated protein, is dramatically induced. The phosphorylated cortactin is almost exclusively associated with centrosomes or spindle poles during mitosis. At G2-M transition prior to the breakdown of the nuclear envelope, two duplicated centrosomes migrate towards opposite ends of the nucleus to form the spindle poles. This centrosome-separation process and also the start of mitosis are inhibited or delayed by the depolymerization of actin filaments. Also inhibited is the separation of centrosomes when a truncated form of cortactin is expressed, whose C-terminus contains the tyrosine phosphorylation region but lacks the actin-binding domains. We introduced mutations at the tyrosine phosphorylation sites in the truncated C-terminus of cortactin and found that the C-terminus could no longer interfere with centrosome separation process. Our study shows that, cortactin phosphorylated at Tyr421, Tyr466 and Tyr482 mediates the actin-filament-driven centrosome separation at G2-M transition by providing a bridge between the centrosome and actin-filaments.

Section: Resultssupporting

confidence: 63%

Centrosome separation driven by actin-microfilaments during mitosis is mediated by centrosome-associated tyrosine-phosphorylated cortactin

Wang

Chen

Ding

et al. 2008

“…Lyn and Yes were subcloned into the pcDNA4/TO vector (Invitrogen) as described (Kasahara et al, 2004;Kuga et al, 2007), and Fyn was also subcloned into pcDNA4/TO. Fyn24-Lyn was generated by fusion of the N-terminal sequence of Fyn (1-24; with 1 designating the initiator methionine) with Lyn lacking the N-terminal sequence (26-512), and the linker sequence Gly-Arg-Ser-Ser was inserted between Fyn(1-24) and Lyn(26-512).…”

Section: Methodsmentioning

confidence: 99%

“…Src, Lyn, Yes and Fyn are widely expressed in various cell types (Thomas and Brugge, 1997;Kuga et al, 2007). We recently showed that Lyn is biosynthetically transported to the plasma membrane via the Golgi pool of caveolin along the secretory pathway (Kasahara et al, 2004).…”

Section: Yes and Lyn Localize To The Golgi Regionmentioning

confidence: 99%

“…Immunodetection was performed by enhanced chemiluminescence (Amersham Biosciences) as described (Mera et al, 1999;Matsuda et al, 2006;Kuga et al, 2007;Kasahara et al, 2007c;Ikeda et al, 2008). Sequential reprobing of membranes with a variety of antibodies was performed after the complete removal of primary and secondary antibodies from membranes in stripping buffer or inactivation of HRP by 0.1% NaN 3 , according to the manufacturer's instructions.…”

Section: Western Blottingmentioning

confidence: 99%

See 1 more Smart Citation

Differential trafficking of Src, Lyn, Yes and Fyn is specified by the state of palmitoylation in the SH4 domain

Sato

Obata

Kasahara

et al. 2009

Self Cite

165

174

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...

“…Lyn, a member of Src-family tyrosine kinases, is widely expressed in a variety of organs, tissues and cell types, including epithelial, hematopoietic and neuronal cells, and has an important role in signal transduction at the cytoplasmic face of the plasma membrane (Tsukita et al, 1991;Hibbs et al, 1995;Nishizumi et al, 1995;Hirao et al, 1998;Hayashi et al, 1999;Sheets et al, 1999;Tada et al, 1999;Rivera and Olivera, 2007;Kuga et al, 2007;Kuga et al, 2008;Sohn et al, 2008). We recently showed that newly synthesized Lyn in the cytoplasm accumulates on Golgi membranes and is subsequently transported to the plasma membrane along the secretory pathway in a manner that is dependent on the Lyn kinase domain (Kasahara et al, 2004;Sato et al, 2009).…”

Section: Research Articlementioning

confidence: 99%

The Lyn kinase C-lobe mediates Golgi export of Lyn through conformation-dependent ACSL3 association

Obata

Fukumoto

Nakayama

et al. 2010

Self Cite

The Src-family tyrosine kinase Lyn has a role in signal transduction at the cytoplasmic face of the plasma membrane upon extracellular ligand stimulation. After synthesis in the cytoplasm, Lyn accumulates on the Golgi and is subsequently transported to the plasma membrane. However, the mechanism of Lyn trafficking remains elusive. We show here that the C-lobe of the Lyn kinase domain is associated with long-chain acyl-CoA synthetase 3 (ACSL3) on the Golgi in a manner that is dependent on Lyn conformation but is independent of its kinase activity. Formation of a closed conformation by CSK prevents Lyn from associating with ACSL3, resulting in blockade of Lyn export from the Golgi. Overexpression and knockdown of ACSL3 accelerates and blocks Golgi export of Lyn, respectively. The post-Golgi route of Lyn, triggered by ACSL3, is distinct from that of vesicular stomatitis virus glycoprotein (VSV-G) and of caveolin. Moreover, an ACSL3 mutant lacking the LR2 domain, which is required for the catalytic activity, retains the ability to associate with Lyn and accelerate Golgi export of Lyn. These results suggest that initiation of Golgi export of Lyn involves association of ACSL3 with the Lyn C-lobe, which is exposed to the molecular surface in an open conformation.