Biomedical subjects

H S Chou

Publications and source records attributed to H S Chou.

11 recordsLinked to original sources

PubMed1998

Human and yeast cdk-activating kinases (CAKs) display distinct substrate specificities.

Cell cycle progression is controlled by the sequential functions of cyclin-dependent kinases (cdks). Cdk activation requires phosphorylation of a key residue (on sites equivalent to Thr-160 in human cdk2) carried out by the cdk-activating kinase (CAK). Human CAK has been identified as a p40(MO15)/cyclin H/MAT1 complex that also functions as part of transcription factor IIH (TFIIH) where it phosphorylates multiple transcriptional components including the C-terminal domain (CTD) of the large subunit of RNA polymerase II. In contrast, CAK from budding yeast consists of a single polypeptide (Cak1p), is not a component of TFIIH, and lacks CTD kinase activity. Here we report that Cak1p and p40(MO15) have strikingly different substrate specificities. Cak1p preferentially phosphorylated monomeric cdks, whereas p40(MO15) preferentially phosphorylated cdk/cyclin complexes. Furthermore, p40(MO15) only phosphorylated cdk6 bound to cyclin D3, whereas Cak1p recognized monomeric cdk6 and cdk6 bound to cyclin D1, D2, or D3. We also found that cdk inhibitors, including p21(CIP1), p27(KIP1), p57(KIP2), p16(INK4a), and p18(INK4c), could block phosphorylation by p40(MO15) but not phosphorylation by Cak1p. Our results demonstrate that although both Cak1p and p40(MO15) activate cdks by phosphorylating the same residue, the structural mechanisms underlying the enzyme-substrate recognition differ greatly. Structural and physiological implications of these findings will be discussed.

CDC2-CDC28 Kinases↗

PubMed1998

Requirement of cyclin E-Cdk2 inhibition in p16(INK4a)-mediated growth suppression.

Loss-of-function mutations of p16(INK4a) have been identified in a large number of human tumors. An established biochemical function of p16 is its ability to specifically inhibit cyclin D-dependent kinases in vitro, and this inhibition is believed to be the cause of the p16-mediated G1 cell cycle arrest after reintroduction of p16 into p16-deficient tumor cells. However, a mutant of Cdk4, Cdk4(N158), designed to specifically inhibit cyclin D-dependent kinases through dominant negative interference, was unable to arrest the cell cycle of the same cells (S. van den Heuvel and E. Harlow, Science 262:2050-2054, 1993). In this study, we determined functional differences between p16 and Cdk4(N158). We show that p16 and Cdk4(N158) inhibit the kinase activity of cellular cyclin D1 complexes through different mechanisms. p16 dissociated cyclin D1-Cdk4 complexes with the release of bound p27(KIP1), while Cdk4(N158) formed complexes with cyclin D1 and p27. In cells induced to overexpress p16, a higher portion of cellular p27 formed complexes with cyclin E-Cdk2, and Cdk2-associated kinase activities were correspondingly inhibited. Cells engineered to express moderately elevated levels of cyclin E became resistant to p16-mediated growth suppression. These results demonstrate that inhibition of cyclin D-dependent kinase activity may not be sufficient to cause G1 arrest in actively proliferating tumor cells. Inhibition of cyclin E-dependent kinases is required in p16-mediated growth suppression.

Animals↗

PubMed1997

New functional activities for the p21 family of CDK inhibitors.

The association of cdk4 with D-type cyclins to form functional kinase complexes is comparatively inefficient. This has led to the suggestion that assembly might be a regulated step. In this report we demonstrate that the CDK inhibitors p21(CIP), p27(KIP), and p57(KIP2) all promote the association of cdk4 with the D-type cyclins. This effect is specific and does not occur with other cdk inhibitors or cdk-binding proteins. Both in vivo and in vitro, the abundance of assembled cdk4/cyclin D complex increases directly with increasing inhibitor levels. The promotion of assembly is not attributable to a simple cell cycle block and requires the function of both the cdk and cyclin-binding domains. Kinetic studies demonstrate that p21 and p27 lead to a 35- and 80-fold increase in K(a), respectively, mostly because of a decrease in K(off). At low concentrations, p21 promotes the assembly of active kinase complexes, whereas at higher concentrations, it inhibits activity. Moreover, immunodepletion experiments demonstrate that most of the active cdk4-associated kinase activity also associates with p21. To confirm these results in a natural setting, we examine the assembly of endogenous complexes in mammary epithelial cells after release from a G(0) arrest. In agreement with our other data, cyclin D1 and p21 bind concomitantly to cdk4 during the in vivo assembly of cdk4/cyclin D1 complexes. This complex assembly occurs in parallel to an increase in cyclin D1-associated kinase activity. Immunodepletion experiments demonstrate that most of the cellular cyclin D1-associated kinase activity is also p21 associated. Finally, we find that all three CIP/KIP inhibitors target cdk4 and cyclin D1 to the nucleus. We suggest that in addition to their roles as inhibitors, the p21 family of proteins, originally identified as inhibitors, may also have roles as adaptor proteins that assemble and program kinase complexes for specific functions.

Biological Transport↗

PubMed1988

T-cell receptor VT beta genes in natural populations of mice.

The composition of 15 VT beta gene subfamilies has been examined by Southern hybridization among a broad spectrum of colony bred rat and mouse species extending phylogenetically from Rattus to Mus musculus domesticus. Most mouse species contain a similar content of VT beta genes as determined by the number of hybridizing restriction fragment (RF) bands. Furthermore, the extent of restriction fragment length polymorphism (RFLP) appears to be limited. Some VT beta gene families, however, are missing from Rattus (VT beta 7, VT beta 12) and M. shortridgei (VT beta 9, VT beta 16). Extension of the VT beta survey to a panel of 38 wild caught mice reveals that nearly a third lack specific hybridization to the VT beta 5 probe. Previous reports have established that the mouse inbred strains SJL, C57BR, C57L, and SWR lack 50% of their VT beta repertoire, including VT beta 5 (Behlke et al. 1985). This study demonstrates that natural populations of mice also carry a significantly reduced VT beta gene repertoire.

Animals↗

PubMed1988

A conserved sequence in the T-cell receptor beta-chain promoter region.

The antigen-specific receptors of T and B lymphocytes are distinct, though structurally related, molecules. During development, lymphoid cells assemble functional variable (V) region genes for each receptor chain from separate multimember gene families by somatic DNA rearrangements of individual germ-line segments. Transcription may play a role in regulating the tissue and stage specificity of these rearrangements by controlling the accessibility of germ-line loci to the recombinational machinery. Immunoglobulin V-region genes are transcribed from tissue-specific promoters that have been well characterized. We report here the characterization of 14 T-cell receptor beta-chain V-region gene promoters. Sequence analysis indicates that these promoters do not contain the conserved octamer that is located upstream of all immunoglobulin genes. However, a unique decanucleotide sequence, not present in immunoglobulin genes, is conserved in the promoter region of murine and human V beta genes. We identify this sequence as a potential regulatory element, based on its position, conservation, and sequence homology to sites known to bind transcription-activating factors. The possibility that the distinct structures of immunoglobulin and T-cell receptor gene promoters may contribute to the tissue-specific rearrangement and expression of receptor gene families is discussed.

Animals↗

PubMed1987

Germline organization of the murine T cell receptor beta-chain genes.

The complete germline organization of the beta-chain genes of the murine T cell receptor was elucidated in order to obtain the structural basis for understanding the mechanisms of somatic DNA rearrangements. Twenty of the 22 known variable (V beta) genes are clustered within 250 kilobases of DNA 5' to the constant region (C beta) genes. These V beta genes share the same transcriptional orientation as the diversity (D beta), joining (J beta), and C beta genes, which implies that chromosomal deletion is the mechanism for most V beta to D beta-J beta rearrangements. Within this V beta cluster, the distance between the most proximal V beta gene and the D beta-J beta-C beta cluster is 320 kilobases, as determined by field-inversion gel electrophoresis. The large distance between V beta and D beta, relative to that between D beta and J beta, may have significant implications for the ordered rearrangement of the T cell receptor beta-chain genes.

Animals↗

PubMed1987

Receptor diversity of insulin-specific T cell lines from C57BL (H-2b) mice.

To characterize the T cell receptor repertoire in an immune response in which the Ia and nominal antigenic determinants are defined and limited, we have cloned and sequenced the expressed receptors from four independent, beef insulin-specific T cell lines from C57BL mice. Each of these lines responded to beef but not to the pork insulin, thus defining the nominal antigenic determinant recognized. Furthermore, each of these lines could only be presented antigen by B6 but not mutant B6.C-H-2bm12 antigen-presenting cells, thus defining the requisite Ia recognition or antigen-association site. In spite of this functional similarity in ligand specificity, each of these T cell lines was found to use different V alpha and V beta gene segments. Moreover, structural comparisons of implied protein sequences of each of these receptors showed no stretches of conserved amino acid residues that could be implicated in ligand interaction. However, the V alpha genes used by these four clones appeared considerably more homologous to each other than were their V beta genes.

Animals↗

PubMed1987

Tandem linkage and unusual RNA splicing of the T-cell receptor beta-chain variable-region genes.

The variable-region (V) genes of the murine T-cell receptor beta chain exist largely as single-element subfamilies. The V beta 5 and V beta 8 genes belong to the only two known three-member V beta subfamilies. We present studies on the linkage of these six genes and show that the genomic organization is that of alternating V beta 5 and V beta 8 genes. Our analysis suggests that these genes were tandemly duplicated, the unit of duplication being a pair of V beta 5 and V beta 8 genes. This tandem organization permits transcripts to initiate from the promoter of an unrearranged V beta located upstream of the rearranged V beta gene. These transcripts can generate functional beta-chain gene messages by novel RNA splicing of the upstream leader exon to the V beta coding exon of the downstream rearranged gene. We extend the analysis of the T-cell receptor genomic organization to include 12 V beta genes and suggest that all V beta genes are closely linked on chromosome 6. In addition, we discuss the possible implications of the close linkage of the V beta genes on the development of the T-cell receptor beta-chain gene repertoire.

Amino Acid Sequence↗

PubMed1986

T cell receptor genes in an alloreactive CTL clone: implications for rearrangement and germline diversity of variable gene segments.

Both cDNA and genomic clones of the T cell receptor (TCR) alpha- and beta-chain genes of the alloreactive cytotoxic T lymphocyte (CTL) clone F3 were examined. Two distinct rearrangement events, one functional and one non-functional, were found for both the alpha and beta loci. Thus only a single functional TCR alpha beta heterodimer could be defined, consistent with allelic exclusion in the TCR genes. The V alpha gene employed by F3 is part of a six-member V alpha subfamily. Genomic clones containing each member of this subfamily were isolated and the V alpha nucleotide sequences determined. Five of these six genes are functional; these genes differ from each other by 7-14% at the amino acid level. A single dominant hypervariable region was defined within this subfamily, in contrast to the pattern of variability seen between V alpha genes in general.

Alleles↗

PubMed1986

Murine T-cell receptor mutants with deletions of beta-chain variable region genes.

Genomic Southern blots of DNA from eight strains of mice were examined for restriction fragment length polymorphisms in their loci encoding the variable region of the T-cell receptor beta chain (V beta), using 16 different V beta-specific probes. Mouse strains BALB/c, C57BL/6, C3H, and PL were identical, while strains SJL, C57BR, C57L, and SWR shared several polymorphisms with respect to the other four strains. In addition, SJL, C57L, C57BR, and SWR DNAs were missing 50% of the hybridizing bands visualized in BALB/c DNA. A cDNA library from concanavalin A-stimulated SJL spleen blasts was constructed and examined for V beta gene usage. Ten genes were found to account for all V beta-containing clones isolated, including three newly identified V beta genes. All 10 of these genes were found to be present in BALB/c mice. We conclude that SJL, C57L, C57BR, and SWR mice represent V beta deletion mutants of the BALB/c genotype.

Amino Acid Sequence↗

PubMed1985

T-cell receptor beta-chain expression: dependence on relatively few variable region genes.

Fifteen independently isolated complementary DNA clones that contain T-cell receptor (TCR) V beta genes were sequenced and found to represent 11 different V beta genes. When compared with known sequences, 14 different V beta genes could be defined from a total of 25 complementary DNA's; 11 clones therefore involved repeated usage of previously identified V beta's. Based on these data, we calculate a maximum likelihood estimate of the number of expressed germline V beta genes to be 18 with an upper 95 percent confidence bound of 30 genes. Southern blot analysis has shown that most of these genes belong to single element subfamilies which show very limited interstrain polymorphism. The TCR beta-chain diversity appears to be generated from a limited V beta gene pool primarily by extensive variability at the variable-diversity-joining (V-D-J) junctional site, with no evidence for the involvement of somatic hypermutation.

Alleles↗