PubMed Health⌕ Search

Biomedical subjects

T Leung

Publications and source records attributed to T Leung.

At least 73 records · Page 4Linked to original sources
PubMed1996

The p160 RhoA-binding kinase ROK alpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton.

The GTPase RhoA has been implicated in various cellular activities, including the formation of stress fibers, motility, and cytokinesis. We recently reported on a p150 serine/threonine kinase (termed ROK alpha) binding RhoA only in its active GTP-bound state and on its cDNA; introduction of RhoA into HeLa cells resulted in translocation of the cytoplasmic kinase to plasma membranes, consistent with ROK alpha being a target for RhoA (T. Leung, E. Manser, L. Tan, and L. Lim, J. Biol. Chem. 256:29051-29054, 1995). Reanalysis of the cDNA revealed that ROK alpha contains an additional N-terminal region. We also isolated another cDNA which encoded a protein (ROK beta) with 90% identity to ROK alpha in the kinase domain. Both ROK alpha and ROK beta, which had a molecular mass of 160 kDa, contained a highly conserved cysteine/histidine-rich domain located within a putative pleckstrin homology domain. The kinases bound RhoA, RhoB, and RhoC but not Rac1 and Cdc42. The Rho-binding domain comprises about 30 amino acids. Mutations within this domain caused partial or complete loss of Rho binding. The morphological effects of ROK alpha were investigated by microinjecting HeLa cells with DNA constructs encoding various forms of ROK alpha. Full-length ROK alpha promoted formation of stress fibers and focal adhesion complexes, consistent with its being an effector of RhoA. ROK alpha truncated at the C terminus promoted this formation and also extensive condensation of actin microfilaments and nuclear disruption. The proteins exhibited protein kinase activity which was required for stress fiber formation; the kinase-dead ROK alpha K112A and N-terminally truncated mutants showed no such promotion. The latter mutant instead induced disassembly of stress fibers and focal adhesion complexes, accompanied by cell spreading. These effects were mediated by the C-terminal region containing Rho-binding, cysteine/histidine-rich, and pleckstrin homology domains. Thus, the multidomained ROK alpha appears to be involved in reorganization of the cytoskeleton, with the N and C termini acting as positive and negative regulators, respectively, of the kinase domain whose activity is crucial for formation of stress fibers and focal adhesion complexes.

Amino Acid Sequence↗
PubMed1996

A Drosophila homolog of the Rac- and Cdc42-activated serine/threonine kinase PAK is a potential focal adhesion and focal complex protein that colocalizes with dynamic actin structures.

Changes in cell morphology are essential in the development of a multicellular organism. The regulation of the cytoskeleton by the Rho subfamily of small GTP-binding proteins is an important determinant of cell shape. The Rho subfamily has been shown to participate in a variety of morphogenetic processes during Drosophila melanogaster development. We describe here a Drosophila homolog, DPAK, of the serine/threonine kinase PAK, a protein which is a target of the Rho subfamily proteins Rac and Cdc42. Rac, Cdc42, and PAK have previously been implicated in signaling by c-Jun amino-terminal kinases. DPAK bound to activated (GTP-bound) Drosophila Rac (DRacA) and Drosophila Cdc42. Similarities in the distributions of DPAK, integrin, and phosphotyrosine suggested an association of DPAK with focal adhesions and Cdc42- and Rac-induced focal adhesion-like focal complexes. DPAK was elevated in the leading edge of epidermal cells, whose morphological changes drive dorsal closure of the embryo. We have previously shown that the accumulation of cytoskeletal elements initiating cell shape changes in these cells could be inhibited by expression of a dominant-negative DRacA transgene. We show that leading-edge epidermal cells flanking segment borders, which express particularly large amounts of DPAK, undergo transient losses of cytoskeletal structures during dorsal closure. We propose that DPAK may be regulating the cytoskeleton through its association with focal adhesions and focal complexes and may be participating with DRacA in a c-Jun amino-terminal kinase signaling pathway recently demonstrated to be required for dorsal closure.

Actins↗
PubMed1995

A novel serine/threonine kinase binding the Ras-related RhoA GTPase which translocates the kinase to peripheral membranes.

We previously reported the cloning of a serine/threonine kinase, PAK (for p21 (Cdc42/Rac)-activated kinase), which binds to the Ras-related GTPases Cdc42Hs and Rac1 (Manser, E., Leung, T., Salihuddin, H., Zhao, Z-s., and Lim, L. (1994) Nature 367, 40-46). These p21 proteins together with RhoA comprise the Rho subfamily of proteins that are involved in morphological events. We now report the isolation of a rat cDNA encoding a 150-kDa protein, which specifically binds RhoA in its GTP form and contains an N-terminal serine/threonine kinase domain highly related to the human myotonic dystrophy kinase and a cysteine-rich domain toward the C terminus. The RhoA binding domain is unrelated to other p21 binding domains. Antibody raised against the kinase domain of the predicted protein, termed ROK alpha (for ROK alpha, RhoA-binding kinase), recognized a ubiquitous 150-kDa protein. The brain p150 purified by affinity chromatography with RhoA exhibited serine/threonine kinase activity. In cultured cells, immunoreactive p150 was recruited to membranes upon transfection with dominant positive RhoAV14 mutant and was localized with actin microfilaments at the cell periphery. These results are consistent with a role for the kinase ROK alpha as an effector for RhoA.

3T3 Cells↗
PubMed1995

Molecular cloning of a new member of the p21-Cdc42/Rac-activated kinase (PAK) family.

A number of "target" proteins for the Rho family of small GTP-binding proteins have now been identified, including the protein kinases ACK and p65PAK (Manser, E., Leung, T., Salihuddin, H., Zhao, Z.-S., and Lim, L. (1994) Nature 367, 40-46). The purified serine/threonine kinase p65PAK has been shown to be directly activated by GTP-Rac1 or GTP-Cdc42. Here we report the cDNA sequence encoding a new brain-enriched PAK isoform beta-PAK, which shares 79% amino acid identity with the previously described alpha-isoform. Their mRNAs are differentially expressed in the brain, with alpha-PAK mRNA being particularly abundant in motor-associated regions. In vitro translation products of the alpha- and beta-PAK cDNAs exhibited relative molecular masses of 68,000 and 65,000, respectively, by SDS-polyacrylamide analysis. A specific beta-PAK peptide sequence was obtained from rat brain-purified p65PAK. Recombinant alpha- and beta-PAKs exhibited an increase in kinase activity mediated by GTP-p21 induced autophosphorylation. Cdc42 was a more potent activator in vitro of alpha-PAK kinase, and the fully activated enzyme is 300 times more active than the unphosphorylated form. Interestingly the down-regulation in the binding of p21s to recombinant beta-PAK and brain p65PAK, which is observed upon kinase activation does not occur with recombinant alpha-PAK.

Amino Acid Sequence↗
PubMed1995

Germ cell tumors express a specific alpha-fetoprotein variant detectable by isoelectric focusing.

BACKGROUND: Many nonseminomatous germ cell tumors (NSGCTs) express elevated levels of serum alpha-fetoprotein (AFP), which is widely used for diagnosis and monitoring therapy. Alpha-fetoprotein is also expressed in patients with hepatocellular carcinoma (HCC). In these sera, several variants, some highly specific, recently have been detected by isoelectric focusing. METHODS: Sera were collected from nine patients with NSGCTs, five pregnant women, and five patients with HCC. After isoelectric focusing, the proteins were transferred to nitrocellulose membrane by blotting and incubated with polyclonal rabbit antihuman AFP conjugated with horseradish peroxidase. The enhanced chemiluminescence detection system and Hyperfilm-ECL were used to render the protein bands visible and each was quantified as a percentage of the total AFP using a densitometer. RESULTS: Isoelectric focusing of serum alpha-fetoprotein from the patients with NSGCTs (four testicular, five mediastinal) revealed a consistent pattern characterized by a specific band (band +III). A second band (band +II) that is also seen in sera from patients with hepatocellular carcinoma was present in all patients. The overall pattern was distinct from that of AFP from HCC and that produced during late pregnancy. The two characteristic bands (AFP +II and +III) were responsible for the great majority of total AFP in these patients; estimation of serial sera in patients undergoing treatment showed that total and "malignant" AFP changed in parallel. Banding patterns, apart from one patient, were identical in patients with testicular and mediastinal NSGCTs. CONCLUSION: NSGCTs tumors express a highly consistent and specific pattern of AFP variants. More detailed analysis of AFP patterns in larger numbers of patients may provide further insight into the cellular heterogeneity of germ cell tumors.

Adolescent↗
PubMed1995

Cross-infection compliance of UK dental staff and students.

OBJECTIVES: The objectives of the study were to determine the compliance of a group of dental health care workers and students in aspects of cross-infection control. DESIGN: The dentists and dental students working in a dental clinic were observed by a 'hidden' ceiling-mounted video camera. Procedures were recorded onto videotape and the actions of the observed clinicians and students assessed by two suitably qualified assessors. SETTING: An emergency Oral Medicine clinic in a dental hospital. SUBJECTS: Seventy nine dentists and 35 dental undergraduates. METHODS: The action of staff and students were recorded on videotape and assessed retrospectively. MAIN OUTCOME MEASURES: Degree of compliance with recognised infection control policies. RESULTS: Compliance with recommended guidelines for control of cross-infection was poor. In only 56% of health care worker-patient contacts were protective gloves changed between patients. Facemasks and protective eyewear were only worn in 38% and 29% of such contacts respectively. CONCLUSIONS: Compliance with cross-infection control measures are poor in dentistry--even when clinicians are provided with appropriate facilities.

Attitude of Health Personnel↗
PubMed1995

Pheromone signalling in Saccharomyces cerevisiae requires the small GTP-binding protein Cdc42p and its activator CDC24.

Pheromone signalling in Saccharomyces cerevisiae is mediated by the STE4-STE18 G-protein beta gamma subunits. A possible target for the subunits is Ste20p, whose structural homolog, the serine/threonine kinase PAK, is activated by GTP-binding p21s Cdc42 and Rac1. The putative Cdc42p-binding domain of Ste20p, expressed as a fusion protein, binds human and yeast GTP-binding Cdc42p. Cdc42p is required for alpha-factor-induced activation of FUS1.cdc24ts strains defective for Cdc42p GDP/GTP exchange show no pheromone induction at restrictive temperatures but are partially rescued by overexpression of Cdc42p, which is potentiated by Cdc42p12V mutants. Epistatic analysis indicates that CDC24 and CDC42 lie between STE4 and STE20 in the pathway. The two-hybrid system revealed that Ste4p interacts with Cdc24p. We propose that Cdc42p plays a pivotal role both in polarization of the cytoskeleton and in pheromone signalling.

Amino Acid Sequence↗
PubMed1995

An audit study of defaulters of regular psychiatric outpatient appointments in Hong Kong.

This paper describes an audit study of general psychiatric outpatient defaulters in Hong Kong. Defaulters were increased among those who were married, unemployed, housewives, seen within one year, receiving medications and previously admitted to hospital. Clinical diagnoses (ICD-10 Axis-1) of the F1, F2, F3 and F7 groups were also associated with increased defaulting behaviour. However, fewer defaulters were found among those who were aged below 19, single, employed, students, and had diagnostic groups of F4 and F9. These results added new information on the non-attendance behavioral pattern of Chinese patients.

Adolescent↗
PubMed1994

Cerebellar beta 2-chimaerin, a GTPase-activating protein for p21 ras-related rac is specifically expressed in granule cells and has a unique N-terminal SH2 domain.

beta-Chimaerin, a 30-kDa GTPase-activating protein (GAP) for the p21 Ras-related Rac, is expressed specifically in late stage spermatocytes (Leung, T., How, B.-E., Manser, E., and Lim, L. (1993) J. Biol. Chem. 268, 3813-3816). Using antibodies raised against beta-chimaerin, we detected a major 46-kDa RacGAP in the rat cerebellum. With beta-chimaerin cDNA as a probe and using polymerase chain reaction, cDNAs from both human and rat cerebellum were isolated. The human and rat cDNAs encoded sequences containing cysteine-rich and GAP domains identical to those of testis beta-chimaerin. The cDNAs also encoded an additional N-terminal SH2 (Src homology 2) domain, probably derived from the beta-chimaerin gene by alternate splicing. This SH2 domain of the predicted 54-kDa protein was strikingly similar to that of alpha 2-chimaerin, including replacement by glutamic acid of the invariant tryptophan present at the start of other SH2 domains. The SH2 domains of alpha- and beta-chimaerin thus represent a subset of SH2 domains. The cerebellar beta-chimaerin (beta 2-) is expressed mainly in granule cells and exhibits postnatal developmental increases. beta 2-Chimaerin was enriched in particulate/synaptosomal fractions. In the mouse weaver mutant lacking mature granule cells, there is a corresponding decrease in beta 2-chimaerin, which could well serve as a marker of granule cell differentiation.

Amino Acid Sequence↗
PubMed1994

A brain serine/threonine protein kinase activated by Cdc42 and Rac1.

A new brain serine/threonine protein kinase may be a target for the p21ras-related proteins Cdc42 and Rac1. The kinase sequence is related to that of the yeast protein STE20, implicated in pheromone-response pathways. The kinase complexes specifically with activated (GTP-bound) p21, inhibiting p21 GTPase activity and leading to kinase autophosphorylation and activation. Autophosphorylated kinase has a decreased affinity for Cdc42/Rac, freeing the p21 for further stimulatory activities or downregulation by GTPase-activating proteins. This bimolecular interaction provides a model for studying p21 regulation of mammalian phosphorylation signalling pathways.

Amino Acid Sequence↗
PubMed1994

Behaviour of fibroblasts during initial attachment to a glass-ceramic implant material in vitro: a time-lapse video-micrographic study.

The in vitro behaviour of 3T3 fibroblasts plated on a glass-ceramic implant material, Apoceram, and on tissue culture polystyrene (TCP) was monitored using phase-contrast, time-lapse video microscopy and scanning electron microscopy. Significant differences were observed in the short-term cellular response to TCP and Apoceram in the timing of the fibroblasts' morphological changes, with earlier spreading and stabilization of the cells occurring on TCP. Cell surface ruffling activity was greater on the glass-ceramic, reflecting weaker adhesions to the substratum. The experimental method used facilitates direct analysis of the dynamic activity of cells attaching to artificial substrata.

Animals↗
PubMed1994

A cancer attitude survey among medical undergraduates in Hong Kong.

A cancer attitude survey was administered to a cohort of 152 final-year medical undergraduates in Hong Kong from mid-1988 to mid-1990, on the first and last days of a four-week oncology module. Significant differences in pre- and post-module scores suggest that the course had significant impact on attitudes toward early diagnosis, treatment aggressiveness, and acceptance of death. The changes are generally regarded by the teachers as desirable. Comparison with a similar survey performed ten years earlier on a group of American second-year medical undergraduates shows significant differences in most of the pre-course scores. Factor analysis shows that the items in the survey could not be readily categorized by a limited number of factors, thus casting doubt on the validity of the instrument in reflecting attitudinal attributes as described in the original survey.

Attitude to Death↗
PubMed1993

The human active breakpoint cluster region-related gene encodes a brain protein with homology to guanine nucleotide exchange proteins and GTPase-activating proteins.

GTPase-activating proteins (GAPs) modulate the activity of the ras superfamily of proteins by converting active GTP-bound to inactive GDP-bound p21s. Employing a novel GAP overlay assay (Manser, E., Leung, T., Monfries, C., Teo, M., Hall, C., and Lim, L. (1992) J. Biol. Chem. 267, 16025-16028), we demonstrated a diversity of proteins with GAP activities in different tissues. Using a polymerase chain reaction strategy exploiting conserved residues in the GAP domains of n-chimaerin and the product of the breakpoint cluster region gene (BCR), we isolated a human brain 5.3-kilobase cDNA containing a 486-base pair region with complete identity to a previously reported active BCR-related (ABR) gene sequence on human chromosome 17. The brain cDNA encoded a 98-kDa protein (ABR) resembling BCR (68% identity), containing both the oncogene dbl-related domain at the N terminus and the GAP domain at the C terminus; however, it lacks the N-terminal BCR protein kinase domain. The ABR GAP domain expressed as an Escherichia coli fusion protein was active against Rac1 and Cdc42 of the rho subfamily. The ABR mRNA is highly enriched in the brain. ABR probably corresponds to the brain-enriched 100-kDa GAP for Rac and Cdc42Hs previously detected. The relationship of ABR to Miller-Dieker syndrome, a neurological disorder co-mapping to 17p13.3, is discussed.

Amino Acid Sequence↗
PubMed1993

A non-receptor tyrosine kinase that inhibits the GTPase activity of p21cdc42.

The Ras-related Rho subfamily of GTP-binding proteins (p21s), which includes Rho, Rac and Cdc42Hs, is implicated in different aspects of cytoskeletal organization. These proteins behave like Ras (p21ras) in that their active GTP-bound form is inactivated by intrinsic hydrolysis of the nucleotide gamma-phosphate, which can be stimulated by GTPase-activating proteins (GAPs). We have previously shown that there is a diversity of GAPs that recognize this subfamily, including n-chimaerin, which is enriched in the hippocampus; we also detected proteins that bind these p21 proteins and seem to inhibit GTP hydrolysis. We now report the characterization of a hippocampal complementary DNA encoding a tyrosine kinase that specifically binds Cdc42Hs in its GTP-bound form. This binding is mediated by a unique sequence of 47 amino acids C-terminal to an SH3 domain and inhibits both the intrinsic and GAP-stimulated GTPase activity of Cdc42Hs. Our findings indicate that there may be a regulatory mechanism that sustains the GTP-bound active form of Cdc42Hs and which is directly linked to a tyrosine phosphorylation pathway.

Amino Acid Sequence↗