Kikuko Ikeda

Src signaling regulates completion of abscission in cytokinesis through ERK/MAPK activation at the midbody

Src family non-receptor-type tyrosine kinases regulate a wide variety of cellular events including cell cycle progression in G2/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.

Role of src-family kinases in formation and trafficking of macropinosomes

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. J. Cell. Physiol. 211: 220–232, 2007.

Trafficking of Lyn through the Golgi caveolin involves the charged residues on αE and αI helices in the kinase domain

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.

Nuclear localization of Lyn tyrosine kinase mediated by inhibition of its kinase activity

Src-family kinases, cytoplasmic enzymes that participate in various signaling events, are found at not only the plasma membrane but also subcellular compartments, such as the nucleus, the Golgi apparatus and late endosomes/lysosomes. Lyn, a member of the Src-family kinases, is known to play a role in DNA damage response and cell cycle control in the nucleus. However, it is still unclear how the localization of Lyn to the nucleus is regulated. Here, we investigated the mechanism of the distribution of Lyn between the cytoplasm and the nucleus in epitheloid HeLa cells and hematopoietic THP-1 cells. Lyn was definitely detected in purified nuclei by immunofluorescence and immunoblotting analyses. Nuclear accumulation of Lyn was enhanced upon treatment of cells with leptomycin B (LMB), an inhibitor of Crm1-mediated nuclear export. Moreover, Lyn mutants lacking the sites for lipid modification were highly accumulated in the nucleus upon LMB treatment. Intriguingly, inhibition of the kinase activity of Lyn by SU6656, Csk overexpression, or point mutation in the ATP-binding site induced an increase in nuclear Lyn levels. These results suggest that Lyn being imported into and rapidly exported from the nucleus preferentially accumulates in the nucleus by inhibition of the kinase activity and lipid modification.

Role of Src-family kinases in formation of the cortical actin cap at the dorsal cell surface.

Protein-tyrosine phosphorylation is regulated by protein-tyrosine kinases and protein-tyrosine phosphatases (PTPs). Src-family tyrosine kinases (SFKs) participate in the regulation of the actin cytoskeleton. Actin filaments can be accumulated in a cap at the dorsal cell surface, which is called the cortical actin cap. Here, we show that SFKs play an important role in formation of the cortical actin cap. HeLa cells normally exhibit the cortical actin cap, one of the major sites of tyrosine phosphorylation. The cortical actin cap is disrupted by SFK inhibitors or overexpression of the Lyn SH3 domain. Csk-knockout cells form the cortical actin cap when the level of tyrosine phosphorylation is increased by Na(3)VO(4), a PTP inhibitor, and the formation of the cortical actin cap is inhibited by SFK inactivation with re-introduction of Csk. SYF cells lacking SFKs minimally exhibit the cortical actin cap even in the presence of Na(3)VO(4), and transfection with Lyn restores the cortical actin cap in the presence of Na(3)VO(4). Disruption of the cortical actin cap by dominant-negative Cdc42 causes loss of tyrosine phosphorylation at the cell top. These results suggest that SFK(s) is involved in formation of the cortical actin cap, which may serve as a platform of tyrosine phosphorylation signaling.

Hypoxia induces expression of a GPI-anchorless splice variant of the prion protein

The human prion protein (PrP) is a glycoprotein with a glycosylphosphatidylinositol (GPI) anchor at its C‐terminus. Here we report alternative splicing within exon 2 of the PrP gene (PRNP) in the human glioblastoma cell line T98G. The open reading frame of the alternatively spliced mRNA lacked the GPI anchor signal sequence and encoded a 230 amino acid polypeptide. Its product, GPI‐anchorless PrP (GPI− PrPSV), was unglycosylated and soluble in non‐ionic detergent, and was found in the cytosolic fraction. We also detected low levels of alternatively spliced mRNA in human brain and non‐neuronal tissues. When long‐term passaged T98G cells were placed in a low‐oxygen environment, alternatively spliced mRNA expression increased and expression of normally spliced PrP mRNA decreased. These findings imply that oxygen tension regulates GPI− PrPSV expression in T98G cells.

Requirement of the SH4 and tyrosine-kinase domains but not the kinase activity of Lyn for its biosynthetic targeting to caveolin-positive Golgi membranes.

BACKGROUND The Src-family non-receptor-type tyrosine kinase Lyn, which is often associated with chemotherapeutic resistance in cancer, localizes not only to the plasma membrane but also Golgi membranes. Recently, we showed that Lyn, which is synthesized in the cytosol, is transported from the Golgi to the plasma membrane along the secretory pathway. However, it is still unclear how Golgi targeting of newly synthesized Lyn is regulated. METHODS Subcellular localization of Lyn and its mutants was determined by confocal microscopy. RESULTS We show that the kinase domain, but not the SH3 and SH2 domains, of Lyn is required for the targeting of Lyn to the Golgi, whereas the N-terminal lipids of the Lyn SH4 domain are not sufficient for its Golgi targeting. Although intact Lyn, which colocalizes with caveolin-positive Golgi membranes, can traffic toward the plasma membrane, kinase domain-deleted Lyn is immobilized on caveolin-negative Golgi membranes. GENERAL SIGNIFICANCE Besides the SH4 domain, the Lyn kinase domain is important for targeting of newly synthesized Lyn to the Golgi, especially caveolin-positive transport membranes. Our results provide a novel role of the Lyn catalytic domain in the Golgi targeting of newly synthesized Lyn in a manner independent of its kinase activity.

1-Oxidopyridin-2-yldiazomethane: a water-soluble alkylating agent for nucleosides and nucleotides

The water-soluble alkylating agent: 1-oxido-pyridin-2-yldiazomethane (I) introduces the 1-oxido-pyridin-2-ylmethyl protecting group into acidic substances (pKa < 9·8) including nucleotides; it can be removed by treatment with acetic anhydride followed by methanolic ammonia.

Expression of a splice variant of prion protein during hypoxia in human glioblastoma cell line T98G

Human prion diseases occur in sporadic, genetic, and transmitted forms. It is associated with the conversion of normal cellular prion protein (PrPC) into a protease-resistant isoform (PrPres). We have shown previously that the human glioblastoma cell line T98G had no coding-region mutation of the prion protein gene (PRNP) which was of the 129 M/V genotype and produced a form of proteinase K-resistant prion protein (PrP) fragment following long-term culture and high passage number (Kikuchi et. al., 2004, J. Gen. Virol. 85, 3449–3457). In this study, we identified an unusual alternative splicing occurring within the exon 2, which resulted in the generation of mRNA laking a C-terminal glycosylphosphatidylinositol (GPI) anchor signal sequence. Only a low level of an alternative spliced form of PRNP was identified in T98G cells under normoxia. Under hypoxia, however, expression levels of the alternatively spliced mRNA were increased. Treatment with cobalt chloride, which mimics anoxia, also increased its expression levels in T98G cells. These results indicate that decrease in oxygen pressure may modulate the PRNP gene expression. The alternatively spliced mRNA encoded the 231 amino acids of polypeptide, which consisted of the amino-terminal portion of PrP and additional residues at its carboxy terminus.