Showing papers by "Erich A. Nigg published in 1995"

Cyclin-dependent protein kinases: key regulators of the eukaryotic cell cycle.

Abstract: Passage through the cell cycle requires the successive activation of different cyclin-dependent protein kinases (CDKs). These enzymes are controlled by transient associations with cyclin regulatory subunits, binding of inhibitory polypeptides and reversible phosphorylation reactions. To promote progression towards DNA replication, CDK/cyclin complexes phosphorylate proteins required for the activation of genes involved in DNA synthesis, as well as components of the DNA replication machinery. Subsequently, a different set of CDK/cyclin complexes triggers the phosphorylation of numerous proteins to promote the profound structural reorganizations that accompany the entry of cells into mitosis. At present, much research is focused on elucidating the links between CDK/cyclin complexes and signal transduction pathways controlling cell growth, differentiation and death. In future, a better understanding of the cell cycle machinery and its deregulation during oncogenesis may provide novel opportunities for the diagnostic and therapeutic management of cancer and other proliferation-related diseases.

Cell cycle regulation of the activity and subcellular localization of Plk1, a human protein kinase implicated in mitotic spindle function.

Abstract: Correct assembly and function of the mitotic spindle during cell division is essential for the accurate partitioning of the duplicated genome to daughter cells. Protein phosphorylation has long been implicated in controlling spindle function and chromosome segregation, and genetic studies have identified several protein kinases and phosphatases that are likely to regulate these processes. In particular, mutations in the serine/threonine-specific Drosophila kinase polo, and the structurally related kinase Cdc5p of Saccharomyces cerevisae, result in abnormal mitotic and meiotic divisions. Here, we describe a detailed analysis of the cell cycle-dependent activity and subcellular localization of Plk1, a recently identified human protein kinase with extensive sequence similarity to both Drosophila polo and S. cerevisiae Cdc5p. With the aid of recombinant baculoviruses, we have established a reliable in vitro assay for Plk1 kinase activity. We show that the activity of human Plk1 is cell cycle regulated, Plk1 activity being low during interphase but high during mitosis. We further show, by immunofluorescent confocal laser scanning microscopy, that human Plk1 binds to components of the mitotic spindle at all stages of mitosis, but undergoes a striking redistribution as cells progress from metaphase to anaphase. Specifically, Plk1 associates with spindle poles up to metaphase, but relocalizes to the equatorial plane, where spindle microtubules overlap (the midzone), as cells go through anaphase. These results indicate that the association of Plk1 with the spindle is highly dynamic and that Plk1 may function at multiple stages of mitotic progression. Taken together, our data strengthen the notion that human Plk1 may represent a functional homolog of polo and Cdc5p, and they suggest that this kinase plays an important role in the dynamic function of the mitotic spindle during chromosome segregation.

MAT1 ('menage à trois') a new RING finger protein subunit stabilizing cyclin H-cdk7 complexes in starfish and Xenopus CAK.

Abstract: The kinase responsible for Thr161-Thr160 phosphorylation and activation of cdc2/cdk2 (CAK:cdk-activating kinase) has been shown previously to comprise at least two subunits, cdk7 and cyclin H. An additional protein co-purified with CAK in starfish oocytes, but its sequencing did not reveal any similarity with any known protein. In the present work, a cDNA encoding this protein is cloned and sequenced in both starfish and Xenopus oocytes. It is shown to encode a new member of the RING finger family of proteins with a characteristic C3HC4 motif located in the N-terminal domain. We demonstrate that the RING finger protein (MAT1: 'menage a trois') is a new subunit of CAK in both vertebrate and invertebrates. However, CAK may also exist in oocytes as heterodimeric complexes between cyclin H and cdk7 only. Stable heterotrimeric CAK complexes were generated in reticulocyte lysates programmed with mRNAs encoding Xenopus cdk7, cyclin H and MAT1. In contrast, no heterodimeric cyclin H-cdk7 complex could be immunoprecipitated from reticulocyte lysates programmed with cdk7 and cyclin H mRNAs only. Stabilization of CAK complexes by MAT1 does not involve phosphorylation of Thr176, as the Thr176-->Ala mutant of Xenopus cdk7 could engage as efficiently as wild-type cdk7 in ternary complexes. Even though starfish MAT1 is almost identical to Xenopus MAT1 in the RING finger domain, the starfish subunit could not replace the Xenopus subunit and stabilize cyclin H-cdk7 in reticulocyte lysate, suggesting that the MAT1 subunit does not (or not only) interact with cyclin H-cdk7 through the RING finger domain.

In vitro assembly of a functional human cdk7-cyclin h complex requires mat1, a novel 36 kda ring finger protein

Abstract: It is proposed that the CDK7-cyclin H complex functions in cell cycle progression, basal transcription and DNA repair. Here we report that in vitro reconstitution of an active CDK7-cyclin H complex requires stoichiometric amounts of a novel 36 kDa assembly factor termed MAT1 (menage a trois 1). Sequencing of MAT1 reveals a putative zinc binding motif (a C3HC4 RING finger) in the N-terminus; however, this domain is not required for ternary complex formation with CDK7-cyclin H. MAT1 is associated with nuclear CDK7-cyclin H at all stages of the cell cycle in vivo. Ternary complexes of CDK7, cyclin H and MAT1 display kinase activity towards substrates mimicking both the T-loop in CDKs and the C-terminal domain of RNA polymerase II, regardless of whether they are immunoprecipitated from HeLa cells or reconstituted in a reticulocyte lysate. MAT1 constitutes the first example of an assembly factor that appears to be essential for the formation of an active CDK-cyclin complex.

Identification of human cyclin-dependent kinase 8, a putative protein kinase partner for cyclin C

Abstract: Metazoan cyclin C was originally isolated by virtue of its ability to rescue Saccharomyces cerevisiae cells deficient in G1 cyclin function. This suggested that cyclin C might play a role in cell cycle control, but progress toward understanding the function of this cyclin has been hampered by the lack of information on a potential kinase partner. Here we report the identification of a human protein kinase, K35 [cyclin-dependent kinase 8 (CDK8)], that is likely to be a physiological partner of cyclin C. A specific interaction between K35 and cyclin C could be demonstrated after translation of CDKs and cyclins in vitro. Furthermore, cyclin C could be detected in K35 immunoprecipitates prepared from HeLa cells, indicating that the two proteins form a complex also in vivo. The K35-cyclin C complex is structurally related to SRB10-SRB11, a CDK-cyclin pair recently shown to be part of the RNA polymerase II holoenzyme of S. cerevisiae. Hence, we propose that human K35(CDK8)-cyclin C might be functionally associated with the mammalian transcription apparatus, perhaps involved in relaying growth-regulatory signals.

Cyclin-dependent kinase

Abstract: This invention relates to a cyclin-dependent kinase (CDK), derivatives of said kinase, antibodies specific for said kinase, and to means and methods for the production thereof. The invention is also directed to nucleic acids coding for a kinase of the invention, to a method of obtaining such nucleic acid molecules, and to the expression thereof. Furthermore, the invention is directed to uses of the proteins and nucleic acids of the invention.

Protein kinases in the control of mitosis: focus on nucleocytoplasmic trafficking.

Abstract: The eukaryotic cell nucleus is a highly dynamic organelle. This is illustrated most dramatically during mitosis, when the nuclear envelope breaks down, the nuclear lamina disassembles, chromosomes condense, and a microtubule-based spindle apparatus distributes sister chromatids to the dividing daughter cells. Many of these dramatic changes in nuclear architecture and microtubule organization are controlled by phosphorylation and dephosphorylation events. Whereas the cardinal role of cyclin-dependent kinases (CDKs) in the regulation of mitosis is well established, there is now clear evidence for the requirement of additional mitotic protein kinases. Studies into the regulation of CDKs and other mitotic kinases have revealed that these enzymes undergo cell cycle dependent changes in subcellular distribution, suggesting that localization may contribute to regulating their activities. This article describes some recent findings relating to the nucleocytoplasmic translocation of CDK/cyclin complexes at the onset of mitosis. In addition, it summarizes recent information on two novel human protein kinases which have been implicated in the control of mitotic progression.

Ring finger protein

Abstract: This invention relates to a protein which is essential for the formation of an active cyclin-dependent kinase (CDK)/cyclin complex, derivatives of said protein, antibodies specific for said protein, and to means and methods for the production thereof. The invention is also directed to nucleic acids coding for a protein of the invention, to a method of obtaining such nucleic acid molecules, and to the expression thereof. Furthermore, the invention is directed to uses of the proteins and nucleic acids of the invention.

The Nuclear Lamina: Regulation of Assembly by Posttranslational Modifications

Abstract: The nuclear lamina is a proteinaceous meshwork underlying the inner nuclear membrane (Gerace and Burke 1988; Nigg 1989, 1992a, b; Moir and Goldman 1993). Its major constituents, the nuclear lamins, display a high degree of structural similarity to cytoplasmic intermediate filament (IF) proteins and hence are classified as type V IFs. The common feature of IF proteins is a central α-helical rod domain (of about 360 amino acids) which contains a heptad repeat of hydrophobic amino acids favoring the formation of two-stranded coiled-coil structures (Steinert et al. 1993; Stewart 1993; Heins and Aebi 1993; Fuchs and Weber 1994). When compared to vertebrate cytoplasmic IFs, the rod domain of lamins contains an extra 42 amino acids; however, an identically increased rod length is also seen in invertebrate cytoplasmic IFs (Weber et al. 1989), suggesting that lamins may be ancestral members of the IF family. This view is supported further by a striking conservation of the intron-exon structure among vertebrate lamins and invertebrate cytoplasmic IFs (Doring and Stick 1990; Dodemont et al. 1990; Lin and Worman 1993).

Control of Nuclear Lamina Assembly/Disassembly by Phosphorylation

Abstract: The nuclear envelope is a complex organelle that separates the nuclear and cytoplasmic compartments and plays a key role in communication between them. It consists of a double nuclear membrane, nuclear pore complexes, and the nuclear lamina. During mitosis, the nuclear envelope transiently disassembles, and recent results suggest that site specific phosphorylation of the nuclear lamins controls the polymerization state of the lamina during the cell cycle. The focus of this review is to summarize the different cell cycle dependent phosphorylations of the nuclear lamin proteins. In particular, we will emphasize the evidence leading to the conclusion that the mitotic lamina dissassembly might be triggered by direct phosphorylation of the nuclear lamins by cdc2 kinase, a major regulator of the eukaryotic cell-cycle (for review see Draetta 1990; Hunt 1991; Norbury and Nurse 1992; Kirschner 1992).