Publications (73) View all
-
Article: Regulation of the antioncogenic Chk2 kinase by the oncogenic Wip1 phosphatase.
[show abstract] [hide abstract]
ABSTRACT: The antioncogenic Chk2 kinase plays a crucial role in DNA damage-induced cell-cycle checkpoint regulation. Here we show that Chk2 associates with the oncogenic protein Wip1 (wild-type p53-inducible phosphatase 1) (PPM1D), a p53-inducible protein phosphatase. Phosphorylation of Chk2 at threonine68 (Thr68), a critical event for Chk2 activation, which is normally induced by DNA damage or overexpression of Chk2, is inhibited by expression of wild-type (WT), but not a phosphatase-deficient mutant (D314A) of Wip1 in cultured cells. Furthermore, an in vitro phosphatase assay revealed that Wip1 (WT), but not Wip1 (D314A), dephosphorylates Thr68 on phosphorylated Chk2 in vitro, resulting in the inhibition of Chk2 kinase activity toward glutathione S-transferase-Cdc25C. Moreover, inhibition of Wip1 expression by RNA interference results in abnormally sustained Thr68 phosphorylation of Chk2 and increased susceptibility of cells in response to DNA damage, indicating that Wip1 acts as a negative regulator of Chk2 in response to DNA damage.Cell Death and Differentiation 08/2006; 13(7):1170-80. · 8.85 Impact Factor -
Article: Involvement of SHP-1 tyrosine phosphatase in TCR-mediated signaling pathways in lipid rafts.
[show abstract] [hide abstract]
ABSTRACT: To elucidate the process of TCR-mediated signaling pathways in lipid rafts, we constructed a chimeric molecule that localizes activated SHP-1 to rafts. Raft targeting of activated SHP-1 in Jurkat-derived transfectants completely inhibited the expression of CD69 and transcriptional factors after TCR cross-linking. Whereas the inducible tyrosine phosphorylation of TCR zeta and ZAP-70 and the kinase activity of Lck were intact, phosphorylated LAT was rapidly dephosphorylated by raft targeting of activated SHP-1, leading to defects in LAT activation and subsequent downstream signaling events. Intriguingly, recruitment of endogenous SHP-1 to rafts and its association with LAT were dramatically increased after TCR engagement, suggesting that SHP-1 is involved in raft-mediated T cell activation.Immunity 07/2001; 14(6):669-80. · 21.64 Impact Factor -
Article: Down-regulation of alpha6 integrin, an anti-oncogene product, by functional cooperation of H-Ras and c-Myc.
[show abstract] [hide abstract]
ABSTRACT: The molecular basis of cooperation of H-Ras and c-Myc in regulating cellular behaviour, such as cell adhesiveness, is still poorly understood. To investigate the role of H-Ras and c-Myc in cell adhesiveness, a constitutively active H-RasV12 (H-RasV12) and c-Myc were stably expressed, singly or in combination in a haematopoietic cell line, and the expression and activity of cell adhesion molecules were monitored. We have shown that the ectopic expression of H-RasV12, but not c-Myc alone, in a haematopoietic cell line, induces the activation of very late antigen-6 (VLA-6, alpha6beta1) integrin. Co-expression of H-RasV12 and c-Myc in the same cells further resulted in the induction of expression of vascular cell adhesion molecule-1 (VCAM-1) and the inhibition of expression of alpha6 integrin, a candidate anti-oncogene product, leading to a loss of adhesiveness to laminin (Lm), a ligand for VLA-6. Cooperation of H-Ras and c-Myc reciprocally regulates expression of the adhesion molecules, alpha6 integrin and VCAM-1. Our results represent an unprecedented account of the cooperation of the oncogene products, H-Ras and c-Myc, to inhibit expression of an anti-oncogene product, alpha6 integrin.Genes to Cells 05/2001; 6(4):337-43. · 2.68 Impact Factor -
Article: A pivotal role of cysteine 3 of Lck tyrosine kinase for localization to glycolipid-enriched microdomains and T cell activation.
[show abstract] [hide abstract]
ABSTRACT: Lck, a Src family protein tyrosine kinase (PTKs), is post-translationally modified by palmitoylation, a process thought to regulate the biological function, membrane affinity and glycolipid-enriched microdomain (GEM) localization of this molecule. To examine the importance of palmitoylation sites Cys3 and Cys5 in Lck, one or both of these residues was mutated to serine to create mutants S3, S5, and S3,5, respectively. Immunofluorescence and confocal microscopy of COS-7 cells transfected with these constructs showed that while S5 and S3 localized to the plasma membrane, S3,5 was localized to the cytoplasm, suggesting that palmitoylation at at least one site is essential for membrane localization. Sucrose gradient based fractionation of these mutants expressed in COS-7 cells showed that while S5 localized to GEMs in similar fashion to the wild type, GEM localization of S3 was severely inhibited. Expression of these mutants in Lck-negative JCaM1 cells showed that although S5 reconstituted activation of nuclear factor NFAT as per the wild type, S3 expression failed to do so. These results suggest that Cys3 of Lck plays a more important role than Cys5 in GEM localization and T cell activation. Additionally, it was found that the degree of T cell function recovery is positively correlated with the degree of Lck expression in GEMs.Immunology Letters 04/2001; 76(2):133-8. · 2.53 Impact Factor -
Article: Serine 6 of Lck tyrosine kinase: a critical site for Lck myristoylation, membrane localization, and function in T lymphocytes.
[show abstract] [hide abstract]
ABSTRACT: Lck is a member of the Src family kinases expressed predominantly in T cells, and plays a pivotal role in TCR-mediated signal transduction. Myristoylation of glysine 2 in the N-terminal Src homology 4 (SH4) domain of Lck is essential for membrane localization and function. In this study, we examined a site within the SH4 domain of Lck regulating myristoylation, membrane localization, and function of Lck. A Lck mutant in which serine 6 (Ser6) was substituted by an alanine was almost completely cytosolic in COS-7 cells, and this change of localization was associated with a drastic inhibition of myristoylation in this mutant. To assess the role of Ser6 of Lck in T cell function, we established stable transfectants expressing various Lck mutants using Lck-negative JCaM1 cells. The Lck mutant of Ser6 to alanine, most of which did not target to the plasma membrane, was not able to reconstitute TCR-mediated signaling events in JCaM1 cells, as analyzed by tyrosine phosphorylation of intracellular proteins and CD69 expression. These results demonstrate that Ser6 is a critical factor for Lck myristoylation, membrane localization, and function in T cells, presumably because the residue is important for N-myristoyl transferase recognition.The Journal of Immunology 10/2000; 165(6):3226-31. · 5.79 Impact Factor
