Showing papers by "Erich A. Nigg published in 1992"
TL;DR: It is shown that cyclin B2 is cytoplasmic during interphase of the cell cycle, but undergoes an abrupt translocation to the cell nucleus at the onset of mitotic prophase, and the phenotypic consequences of expressing wild-type and mutated versions of avian cyclinB2 in HeLa cells are examined.
Abstract: Cyclin proteins form complexes with members of the p34cdc2 kinase family and they are essential components of the cell cycle regulatory machinery. They are thought to determine the timing of activation, the subcellular distribution, and/or the substrate specificity of cdc2-related kinases, but their precise mode of action remains to be elucidated. Here we report the cloning and sequencing of avian cyclin B2. Based on the use of monospecific antibodies raised against bacterially expressed protein, we also describe the subcellular distribution of cyclin B2 in chick embryo fibroblasts and in DU249 hepatoma cells. By indirect immunofluorescence microscopy we show that cyclin B2 is cytoplasmic during interphase of the cell cycle, but undergoes an abrupt translocation to the cell nucleus at the onset of mitotic prophase. Finally, we have examined the phenotypic consequences of expressing wild-type and mutated versions of avian cyclin B2 in HeLa cells. We found that expression of cyclin B2 carrying a mutation at arginine 32 (to serine) caused HeLa cells to arrest in a pseudomitotic state. Many of the arrested cells displayed multiple mitotic spindles, suggesting that the centrosome cycle had continued in spite of the cell cycle arrest.
291 citations
TL;DR: The results strongly suggest that CK II functions primarily in regulating nuclear activities, and that the two catalytic subunits, alpha and alpha', may carry out overlapping functions.
Abstract: Casein kinase II (CK II) has been implicated in regulating multiple processes related to cell growth, proliferation, and differentiation. To better understand the function(s) and regulation of this ubiquitous kinase, it is important to know its subcellular distribution. However, this issue has been the subject of contradictory reports. In this study, we have used indirect immunofluorescence microscopy and cell fractionation to study the subcellular distribution of all three subunits of chicken CK II, alpha, alpha', and beta. We examined primary chick embryo fibroblasts, virally transformed chicken hepatoma cells, as well as HeLa cells transiently transfected with cDNAs encoding chicken CK II subunits. We found that each of the three CK II subunits was located predominantly in the cell nucleus, irrespective of the cell type analyzed or the procedure used for cell fixation. No major differences were detected in the subcellular distributions of individual CK II subunits, and no evidence was obtained for subunit redistributions during interphase of the cell cycle. During mitosis, the bulk of the enzyme was dispersed throughout the cell, though a fraction of all three subunits was associated with the mitotic spindle. Biochemical studies based on mechanical enucleation of chicken cells confirmed the predominantly nuclear location of all three CK II subunits. Finally, immunoblotting experiments were carried out to study the expression of CK II subunits. A survey of different adult chicken tissues revealed substantial tissue-specific differences in the levels of CK II protein, but no evidence was obtained for pronounced tissue specificity in the expression of individual CK II subunits. These results strongly suggest that CK II functions primarily in regulating nuclear activities, and that the two catalytic subunits, alpha and alpha', may carry out overlapping functions.
198 citations
TL;DR: Recently, progress in the study of lamins has been made in three areas: signals required for targetting newly synthesized lamins to the correct subnuclear compartment have been identified, and a mechanistic explanation for how the nuclear lamina is diassembled at the onset of mitosis is emerging.
128 citations
TL;DR: Whereas dimers made of the truncated B2 headless and rod lamins had lost their propensity to associate head-to-tail, tailless lamin B2 dimers revealed an enhanced head- to-tail association.
127 citations
TL;DR: Two distinct members of the mitogen-activated protein (MAP) kinase family are shown to phosphorylate chicken lamin B2 at a single site identified as Ser16, raising the possibility that some of the purported substrates of p34cdc2 may actually be physiological substrate of MAP kinases.
Abstract: Members of the mitogen-activated protein (MAP) kinase family are implicated in mediating entry of cells into the cell cycle, as well as passage through meiotic M phase. These kinases have attracted much interest because their activation involves phosphorylation on both tyrosine and threonine residues, but little is known about their physiological targets. In this study, two distinct members of the MAP kinase family (p44mpk and p42mapk) are shown to phosphorylate chicken lamin B2 at a single site identified as Ser16. Moreover, these MAP kinases cause depolymerization of in-vitro-assembled longitudinal lamin head-to-tail polymers. Ser16 was previously shown to be phosphorylated during mitosis in vivo, and to be a target of the mitotic protein kinase p34cdc2 in vitro. Accordingly, lamins were proposed to be direct in vivo substrates of p34cdc2. This proposal is supported by quantitative analyses indicating that lamin B2, when assayed in vitro, is a substantially better substrate for p34cdc2 than for MAP kinases. Nevertheless, a physiological role of MAP kinases in lamin phosphorylation is not excluded. The observation that members of the MAP kinase family display sequence specificities overlapping that of p34cdc2 raises the possibility that some of the purported substrates of p34cdc2 may actually be physiological substrates of MAP kinases.
101 citations
TL;DR: Electron microscopic analyses have begun to provide insights into the principles that govern lamina assembly in vitro, and sequence motifs required for targeting newly synthesized lamins to the nuclear envelope have been identified.
88 citations
TL;DR: The subcellular distribution and regulation of MAP kinase isoforms in chicken hepatoma DU249 cells was investigated with antibodies directed against peptides patterned after sequences in the mitogen-activated protein (MAP) kinases, sea star p44mpk, and rat p44erk1.
Abstract: The subcellular distribution and regulation of MAP kinase isoforms in chicken hepatoma DU249 cells was investigated with antibodies directed against peptides patterned after sequences in the mitogen-activated protein (MAP) kinases, sea star p44mpk, and rat p44erk1. MonoQ chromatography of cytosol from these cells afforded the resolution of at least four peaks of myelin basic protein (MBP) phosphotransferase activity, but only one of these (peak II) was stimulated in extracts from phorbol ester-treated cells. A 40- to 41-kDa (p41) doublet on Western blots detected with three different MAP kinase antibodies was coincident with peak II, and it probably corresponded to the avian homolog of p42mapk/erk2. Immunofluorescent studies with DU249 cells and chicken embryo fibroblasts revealed that most of the cross-reactive protein with at least two different MAP kinase antibodies was distributed in the nucleus. Subcellular fractionation studies confirmed a predominantly nuclear localization for p41 MAP kinase. Nocodazole arrest of DU249 cells was exploited for the detection of an M-phase-activated MBP kinase that was resolved from p41 MAP kinase by phenyl-Superose chromatography. Western blotting analysis with antibodies for the cdc2-encoded protein kinase and p13suc1-agarose binding studies allowed positive identification of this MBP kinase as p34cdc2.
84 citations
TL;DR: It is shown that the isoprenoid transferase which modifies rab termini is different from the enzyme which farnesylates nuclear lamins and ras proteins in vitro and in vivo.
Abstract: rab proteins are low molecular weight GTP-binding proteins highly related to Ypt1p and Sec4p, which are involved in the control of secretion in yeast Saccharomyces cerevisiae. Morphological and biochemical studies have shown that rab proteins are membrane associated and are localized to specific subcompartments along the exocytic and endocytic pathway. Membrane association requires the presence of C-terminal cysteine residues. The present report indicates that the structurally distinct cysteine motifs of rab proteins are subjected to isoprenylation both in vitro and in vivo. Studies on deletion mutants suggest that an intact C-terminal end is required for the association of rab proteins with the membrane and is necessary for the post-translational modification. Finally, we show that the isoprenoid transferase which modifies rab termini is different from the enzyme which farnesylates nuclear lamins and ras proteins in vitro.
55 citations
TL;DR: The nuclear lamina is a protein network lining the nucleoplasmic surface of the inner nuclear menibrane presumed to represent a karyoskeletal element important for nuclear envelope integrity and interphase chromatin organization.
Abstract: Introduction ‘I’he nuclear lamina is a protein network lining the nucleoplasmic surface of the inner nuclear menibrane. I t is presumed to represent a karyoskeletal element important for nuclear envelope integrity and interphase chromatin organization (for reviews see [ 1, 21). Its major constituents. the nuclear lamins, are members of the intermediate filament (IF) protein family [3-51. On the basis of biochemical properties, structural criteria, and expression patterns, vertebrate lamins have been classified as either Aor t proteins a tripartite organization in that they display a central a-helical rod domain. flanked by non-a-helical domains at the Nand C-terminal ends. I lowever, lamins are readily distinguished from cytoplasmic members of the IF Family by the presence of a nuclear localization signal (N1,S) and a C-terminal CaaX box (reviewed in [6, 15 1). ‘I’he former motif is necessary for nuclear transport 161, while the CaaX box is modified by isopreny1;ition. proteolytic trimming, and carboxyl methylation [ 1710 1. Such modifications occur not only on lamins, but also on certain yeast mating factors, rus proteins and many other (;TI’-hydrolysing (G-) proteins (reviewed in [ 20-23 I). ‘I’hese hydrophobic modifications appear t o represent a general mechanism for increasing the
49 citations
Journal Article•
TL;DR: The identification of threonine 161 as an additional in vivo phosphorylation site in vertebrate p34cdc2 is described, which is cell cycle dependent and likely to be required for p34CDc2 activity.
Abstract: The protein kinase p34cdc2 is a key regulator of the cell cycle in all eukaryotes. Its activity is controlled by cell cycle-dependent interactions with other proteins, notably cyclins, and by changes in its phosphorylation state. Two inhibitory phosphorylation sites in chicken p34cdc2 have previously been mapped to threonine 14 and tyrosine 15. Here we describe the identification of threonine 161 as an additional in vivo phosphorylation site in vertebrate p34cdc2. Phosphorylation of this site is cell cycle dependent and likely to be required for p34cdc2 activity.
48 citations
Journal Article•
TL;DR: In this paper, the effects of in vitro mutagenesis of the four known phosphorylation sites in the chicken p34(cdc2) protein, Thr 14, Tyr 15, Thr 161 and Ser 277, upon cell cycle progression were analyzed.
Abstract: We have used the fission yeast Schizosaccharomyces pombe to analyse the effects of in vitro mutagenesis of the four known phosphorylation sites in the chicken p34(cdc2) protein, Thr 14, Tyr 15, Thr 161 and Ser 277, upon cell cycle progression. We have studied both the effect of overexpression of mutant proteins in a cdc2+ background and assayed their ability to rescue null and temperature-sensitive alleles of cdc2. Mutations of Thr 14 and Tyr 15 within the ATP binding domain of p34(cdc2) that mimic constitutive phosphorylation cause dominant negative cell cycle arrest when overexpressed. In contrast, some substitutions that simulate permanent dephosphorylation of the corresponding sites advance dephosphorylation of the corresponding sites advance mitosis. These data confirm the model that p34(cdc2) function is negatively regulated by phosphorylation of residues in the ATP binding site. Mutagenesis of the conserved residue Thr 161 functionally inactivates p34(cdc2), and our data suggest that both phosphorylation and dephosphorylation events at Thr 161 are required for progression through the cell cycle. Mutations at the fourth site of phosphorylation. Ser 277, lead to cold-sensitive cell cycle arrest, in minimal but not rich growth medium, suggesting that this site is involved in monitoring the nutritional status of the cell.
01 Jan 1992
TL;DR: It is found that chicken cyclin B2 undergoes a striking redistribution from the cytoplasm to the nucleus just prior to the onset of mitosis, which causes HeLa cells to arrest in mitosis and cell cycle-dependent complex formation with cyclin proteins.
Abstract: Activation of the protein kinase p34cdc2 is required for entry into meiotic or mitotic M phase in all eukaryotic cells. One important mechanism regulating the activity of p34cdc2 during the cell cycle is based on phosphorylation/dephosphorylation. Avian p34cdc2 is phosphorylated on threonine 14 (Thr14), tyrosine 15 (Tyr15), threonine 161 (Thr161) and serine 277 (Ser277). Dephosphorylation of both Thr14 and Tyr15 is required for activation of p34cdc2 at the G2/M transition, indicating that phosphorylation of these residues negatively regulates p34cdc2 activity. Conversely, phosphorylation of Thr161 is required for kinase activity. Whether modification of this residue is due to intramolecular autophosphorylation or to the action of an as yet unidentified kinase remains unresolved. Likewise, the role of phosphorylation of p34cdc2 on Ser277 during G1 phase of the cell cycle remains to be determined. The function of p34cdc2 is regulated also by cell cycle-dependent complex formation with cyclin proteins. We found that chicken cyclin B2 undergoes a striking redistribution from the cytoplasm to the nucleus just prior to the onset of mitosis. Expression of a non-destructible cyclin B2 mutant causes HeLa cells to arrest in mitosis. Frequently, arrested cells displayed multiple mitotic spindles.
TL;DR: The chapter presents early events in signal transduction and basic aspects of nuclear transport, and considers specific examples of intracellular signaling such as protein kinase translocations to the nucleus, nuclear factor kappa B (dorsal/rel), the steroid hormone receptor family.
Abstract: Publisher Summary This chapter presents the evidence pertaining to possible mechanisms of information transfer between the cytoplasm and the nucleus Signal transduction from cytoplasm to nucleus is proposed to depend on the existence of equilibria in protein distributions across the nuclear envelope and on various mechanisms controlling the subcellular distribution of regulatory macromolecules Prominent roles in the control of nucleocytoplasmic protein distributions are attributed to cytoplasmic anchorage-release or nuclear localization signals-masking/unmasking mechanisms and to posttranslational modifications, notably phosphorylation The chapter presents early events in signal transduction and basic aspects of nuclear transport The concepts of signal transduction is illustrated by considering specific examples of intracellular signaling such as protein kinase translocations to the nucleus, nuclear factor kappa B (dorsal/rel), the steroid hormone receptor family