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PubMed1998

Transient activation of cyclin B/Cdc2 during terminal differentiation of lens fiber cells.

Previous work has shown that postmitotic, differentiating fiber cells of the embryonic chicken lens express cyclin B and Cdc2. The present study explores the possible physiological role of these proteins in lens differentiation by examining the developmental regulation of cyclin B/Cdc2 expression and activity in lens fiber cells of embryonic and newborn rats. Cyclin B mRNA and protein were detected not only in the lens epithelium, which contains proliferating cells, but also in postmitotic, differentiating fiber cells. In contrast, cyclin A mRNA and protein were detected only in epithelial cells. Immunoprecipitation with cyclin B antibody coprecipitated Cdc2 from both epithelial and fiber cell extracts. Immunoprecipitates of cyclin B from both epithelial cells and fiber cells showed H1 kinase activity when assayed in vitro, but the developmental pattern of cyclin B-associated kinase activity in these two lens fractions was markedly different. In the epithelium, H1 kinase activity decreased gradually with developmental age in parallel with the decrease in epithelial cell proliferation, whereas, in the fiber cells, kinase activity peaked sharply at embryonic day 18 (E18) and E19. Microscopic examination of rat lenses indicated that peak cyclin B/Cdc2 activity was correlated with changes in chromatin structure and nuclear envelope breakdown in the terminally differentiating primary lens fiber cells. These findings suggest that cyclin B/Cdc2 activity may play an active role in nuclear changes leading to primary fiber cell denucleation.

Animals↗
PubMed1999

Effect of cyclin E overexpression on lovastatin-induced G1 arrest and RhoA inactivation in NIH3T3 cells.

The HMG-CoA reductase inhibitor, lovastatin, blocks targeting of the Rho and Ras families of small GTPases to their active sites by inhibiting protein prenylation. Control NIH3T3 cells, and those overexpressing human cyclin E protein were treated with lovastatin for 24 h to determine the effects of cyclin E overexpression on lovastatin-induced growth arrest and cell rounding. Lovastatin treatment (10 microM) of control 3T3 cells resulted in growth arrest at G1 accompanied by actin stress fiber disassembly, cell rounding, and decreased active RhoA from the membranous protein fraction. By contrast, in NIH3T3 cells overexpressing cyclin E, lovastatin did not cause loss of RhoA from the membrane (active) protein fraction, actin stress fiber disassembly, cell rounding or growth arrest within 24 h. Analysis of cell cycle proteins showed that 24 h of lovastatin treatment in the control cells caused an elevation in the levels of the cyclin-dependent kinase inhibitor p27(kip1), inhibition of both cyclin E- and cyclin A-dependent kinase activity, and decreased levels of hyperphosphorylated retinoblastoma protein (pRb). By contrast, lovastatin treatment of the cyclin E overexpressors did not suppress either cyclin E- or cyclin A-dependent kinase activity, nor did it alter the level of maximally phosphorylated pRb, despite increased levels of p27(kip1). However, by 72 h, the cyclin E overexpressors rounded up but remained attached to the substratum, indicating a delayed response to lovastatin. In contrast with lovastatin, inactivation of membrane-bound Rho proteins (i.e., GTP-bound RhoA, RhoB, RhoC) with botulinum C3 transferase caused cell rounding and G1 growth arrest in both cell types but did not inhibit cyclin E-dependent histone kinase activity in the cyclin E overexpressors. In addition, 24 h of cycloheximide treatment caused depletion of RhoA from the membrane (active) fraction in neo cells, but in the cells overexpressing cyclin E, RhoA remained in the active (membrane-associated) fraction. Our observations suggest that (1) RhoA activation occurs downstream of cyclin E-dependent kinase activation, and (2) overexpression of cyclin E decreased the turnover rate of active RhoA.

3T3 Cells↗
PubMed1991

Mutations of p34cdc2 phosphorylation sites induce premature mitotic events in HeLa cells: evidence for a double block to p34cdc2 kinase activation in vertebrates.

In vertebrates, entry into mitosis is accompanied by dephosphorylation of p34cdc2 kinase on threonine 14 (Thr14) and tyrosine 15 (Tyr15). To examine the role of these residues in controlling p34cdc2 kinase activation, and hence the onset of mitosis, we replaced Thr14 and/or Tyr15 by non-phosphorylatable residues and transfected wild-type and mutant chicken p34cdc2 cDNAs into HeLa cells. While expression of wild-type p34cdc2 did not interfere with normal cell cycle progression, p34cdc2 carrying mutations at both Thr14 and Tyr15 displayed increased histone H1 kinase activity and rapidly induced premature mitotic events, including chromosome condensation and lamina disassembly. No phenotype was observed in response to mutation of only Thr14, and although single-site mutation at Tyr15 did induce premature mitotic events, effects were partial and their onset was delayed. These results identify both Thr14 and Tyr15 as sites of negative regulation of vertebrate p34cdc2 kinase, and they suggest that dephosphorylation of p34cdc2 represents the rate-limiting step controlling entry of vertebrate cells into mitosis.

Amino Acid Sequence↗
PubMed1980

Membrane-bound protein kinase activity in acetylcholine receptor-enriched membranes.

Membrane protein phosphorylation may be a general regulatory mechanism mediating the response of cells to exogenous metabolic and physical signals. We have determined that the membrane-bound acetylcholine receptor is the major substrate phosphorylated in situ by a nearby membrane protein kinase. Moreover, these same membranes also contain phosphoprotein phosphatase activity which dephosphorylates the membrane-bound receptor. These findings suggest that reversible phosphorylation of the actylcholine receptor may be critical for receptor function at the synapse. Therefore, it is necessary to define the properties of the enzymes which mediate this phosphorylation-dephosphorylation mechanism. In this report we describe the properties of the first component of this system, the membrane-bound protein kinase in receptor-enriched membranes from the electric organ of Torpedo californica. Only ATP is effective as a phosphate donor for this cyclic AMP-independent membrane kinase; GTP does not support phosphorylation of the receptor. Both casein and histone can also be phosphorylated by the membrane protein kinase, but casein is a better substrate. Although phosphorylation of the receptor appears to be regulated by cholinergic ligands and K+, casein phosphorylation is not specifically affected by these agents. Moreover, while phosphorylation of the acetylcholine receptor is maximal in receptor=enriched membranes, casein phosphorylation is similar in all membrane fractions prepared from the electric organ. Taken together, these findings suggest that the membrane protein kinase activity in receptor-enriched membranes is similar to most other membrane kinases. Therefore, the unique characteristics of membrane-bound acetylcholine receptor phosphorylation appear to be determined by the receptor and its availability as a substrate for the membrane kinase.

Acetylcholine↗
PubMed1979

Amino acid sequence of rat thymus histone H2B and identification of the in vitro phosphorylation sites.

The amino acid sequence of rat thymus histone obtained in highly purified form by preparative electrophoresis, was determined. This sequence is identical to the sequence of calf thymus histone H2B. The in vitro phosphorylation of the rat histone with a cyclic AMP-dependent protein kinase isolated from rat pancreas led to the identification of four sites of phosphorylation: two major ones, at serine residues 32 and 36, and two minor ones, specific of the rat protein kinase, at serine residues 87 and 91.

Amino Acid Sequence↗
PubMed1977

Studies on the mechanism of action of histone kinase dependent on adenosine 3':5'-monophosphate. Evidence for involvement of histidine and lysine residues in the phosphotransferase reaction.

The reaction of the phosphate residue transfer catalysed by histone kinase dependent on adenosine 3':5'-monophosphate (cyclic AMP) was studied. The phosphotransferase reaction was shown to obey the mechanism of ping-pong bi-bi type. After incubation of the catalytic subunit of histone kinase with [gamma-32P]ATP the incorporation of one mole of [32P]phosphage per mole of protein was observed. The tryptic [32P]phosphohistidine-containing peptide was isolated and its N-terminus and amino acid composition were determined. The 2',3'-dialdehyde derivative of ATP (oATP) was used as the affinity label for the catalytic subunit of cyclic-AMP-dependent histone kinase. The inhibitor formed an alidmine bond with epsilon-amino group of the lysine residue of the active site and was irreversibly bound to the enzyme after reduction by sodium borohydride with concurrent irreversible inactivation of the enzyme. After inactivation, about one mole of 14C-labelled inhibitor was incorporated per mole of the enzyme. ATP effectively protected the catalytic subunit of histone kinase against inactivation by oATP. Tryptic digestion of the enzyme-inhibitor complex led to the isolation of the 14C-labelled peptide of the active site of histone kinase. Basing on these results, the role of histidine and lysine residues in the active site of the catalytic subunit of histone kinase was suggested.

Amino Acids↗
PubMed1992

Association of cdk2 kinase with the transcription factor E2F during S phase.

The transcription factor E2F controls the expression of several proliferation-related genes and is a target of the adenovirus E1A oncogene. In human cells, both cyclin A and the cdk2 protein kinase were found in complexes with E2F. Although the total amounts of cdk2 were constant in the cell cycle, binding to E2F was detected only when cells entered S phase, a time when the cdk2 kinase is activated. These data suggest that the interaction between cdk2 and E2F requires an active kinase that has cyclin A as a targeting component.

Adenovirus Early Proteins↗
PubMedDate not supplied

A new screening test for antimitotic compounds using the universal M phase-specific protein kinase, p34cdc2/cyclin Bcdc13, affinity-immobilized on p13suc1-coated microtitration plates.

A universal intracellular factor, the "M phase-Promoting Factor" (MPF), triggers the G2/M transition of the cell cycle in all organisms. This factor displays an easily assayable histone H1 kinase (H1K) activity and is composed of at least two subunits, p34cdc2 (catalytic) and cyclin Bcdc13 (regulatory). We describe here a microtitration plate assay using affinity-immobilized H1K-MPF as a cell cycle-specific target to screen for antimitotic compounds. First, meiotic starfish oocytes were selected as the most convenient and abundant source of M phase extracts containing high levels of H1K. Second, we used the strong and specific interaction between p34cdc2 and p13suc1 to affinity-immobilize H1K-MPF on p13suc1-coated microtitration plates. p13suc1-coated wells specifically retain the M phase kinase, the activity of which is assayed with histone H1 and gamma-32P-ATP. Among 10 microtitration plates, Maxisorp plates (Nunc) proved to be the most efficient at retaining H1K through p13suc1. Experimental conditions to coat the plates with p13suc1, to immobilize and to measure p34cdc2/cyclin Bcdc13 kinase activity, as well as to store p13suc1-precoated plates, have been optimized. Using this system we tested 18 currently used anticancer drugs and S or G2 inhibitors; none of them displayed any inhibitory activity. The microtitration assay has allowed the detection of two H1K inhibitors, isopentenyladenine (I50: 40 microM) and staurosporine (I50: 3.2 nM). This affinity-immobilized H1K-MPF can thus now be used as a simple screening system to detect inhibitors of a major cell cycle-regulating component. This method may prove useful to screen for antimitotic compounds of potential anticancer interest.

Acetylcholine↗
PubMed1985

Histone kinase activities in normal and transformed mouse cells.

Crude extracts from replicating normal and transformed cells were assayed for protein kinase activities specific for different sites in purified Hl histone in vitro. Extracts from normal cells favored the NH2-terminal region while extracts from transformed cells favored the COOH-terminal region. Analysis of phosphopeptides demonstrated that histone kinases from both normal and transformed cells catalyzed the phosphorylation of a number of sites in common, and these were typical of sites phosphorylated in replicating cells. The preference for the NH2-terminal region by extracts from normal cells was due to the extensive phosphorylation of a site previously shown to be phosphorylated by cyclic AMP-dependent protein kinase. This activity was very low in transformed cells.

Amino Acid Sequence↗
PubMed1987

Identification of insulin receptor tyrosine residues autophosphorylated in vitro.

To identify the autophosphorylation sites on the human insulin receptor (IR), partially purified human IR was incubated in vitro in the presence of insulin and manganese [gamma-32P]ATP so as to achieve near-maximal activation of the histone 2b kinase activity. Approximately 70% of all beta subunit [32P]phosphotyrosine resides on two tryptic peptide segments identified by microsequencing as IR precursor (Ullrich, A., Bell, J. R., Chen, E.-Y., Herrera, R., Petruzelli, L. M., Dull, T. J., Gray, A., Coussens, L., Liao, Y.-C., Tsubokawa, M., Mason, A., Seeburg, P. H., Grunfeld, C., Rosen, O. M., and Ramachandran, J. (1985) Nature 313, 756-761) 1144-1152 (tyrosine at 1146, 1150, 1151, designated peptide 5) and 1315-1329 (tyrosine at 1316, 1322, designated peptide 8), which were recovered in approximately equal amounts. Half of the remaining unidentified [32P]phosphotyrosine residues reside on another tryptic peptide of Mr 4000-5000. Assignment of [32P]phosphotyrosine to specific residues required subdigestion and Edman degradation of 32P peptides covalently coupled to solid supports. Peptide 5 was recovered in triple and double phosphorylated forms in a molar ratio of about 2:1. Tyr-1146 contained 32P in both forms of peptide 5; in the double phosphorylated form, phenylthiohydantoin-[32P]phosphotyrosine was recovered at both Tyr-1150 and Tyr-1151, in a ratio of about 1:2. Thus, the double phosphorylated peptide 5 is presumably a mixture of Tyr-P-1146/1150 and Tyr-P-1146/1151, predominantly the latter. Peptide 8 was recovered only as the double phosphorylated form. We conclude that autophosphorylation of human IR in vitro leads to the phosphorylation of at least 6 of the 13 tyrosine residues on the beta subunit intracellular extension. Five of these tyrosines are clustered in two domains; one domain is in the structurally unique C-terminal tail and contains Tyr-1316 and -1322 which are both phosphorylated. The second domain is located in the segment of the tyrosine kinase region homologous to the major in vitro autophosphorylation site of pp60 v-src and contains Tyr-1146, which is fully phosphorylated, and Tyr-1150 and -1151; although the majority of IR beta subunits exhibit phosphorylation of both tyrosine 1150 and 1151, up to 20-25% of Tyr-1150 remains unphosphorylated at complete kinase activation.

Adenosine Triphosphate↗
PubMed1996

Activation of the proteasome during Xenopus egg activation implies a link between proteasome activation and intracellular calcium release.

Ubiquitin-dependent proteolysis is required for cell cycle progression. Here, we demonstrate that the proteasome is activated during in vivo Xenopus egg activation, induced by treatment with the calcium ionophore A23187. It was found that activation is due to the calcium-induced assembly of the 26 S proteasome from the 20 S proteasome. We propose that proteasome activation is regulated by cell cycle calcium transients, which are controlled upstream by an endogenous cell cycle oscillator that is independent of the cyclin-dependent kinase cycle.

Animals↗
PubMed1984

Cyclic AMP-stimulated protein kinase activity in rabbit peripheral myelin.

Cyclic AMP-sensitive protein kinase activity has been found in suspensions of purified rabbit peripheral myelin. The enzyme phosphorylated the P0, "Y", X, P1, and P2 myelin proteins. Kinase activity, which was maximal at physiological pH, 2.5 mM Mg2+, and 2 microM cAMP, was stimulated three-fold over basal levels by cyclic AMP. Addition of calcium or EGTA had no effect on the enzyme activity in the presence or absence of cyclic AMP. Cyclic GMP also did not stimulate endogenous or exogenous protein phosphorylation. Theophylline, an inhibitor of 3',5'-cyclic nucleotide phosphodiesterase activity, increased protein kinase activity in the presence of cyclic AMP. These data show that PNS myelin proteins can be phosphorylated in situ by a protein kinase system whose activity is stimulated selectively by cyclic AMP.

Animals↗
PubMed1984

Characterization of the protein kinase activities of human platelet supernatant and particulate fractions.

After human platelets were lysed by freezing and thawing in the presence of EDTA, about 35% of the total cyclic AMP-dependent protein kinase activity was specifically associated with the particulate fraction. In contrast, Ca2+-activated phospholipid-dependent protein kinase was found exclusively in the soluble fraction. Photoaffinity labelling of the regulatory subunits of cyclic AMP-dependent protein kinase with 8-azido-cyclic [32P]AMP indicated that platelet lysate contained a 4-fold excess of 49 000-Da RI subunits over 55 000-Da RII subunits. The RI and RII subunits were found almost entirely in the particulate and soluble fractions respectively. Chromatography of the soluble fraction on DEAE-cellulose demonstrated a single peak of cyclic AMP-dependent activity with the elution characteristics and regulatory subunits characteristic of the type-II enzyme. A major enzyme peak containing Ca2+-activated phospholipid-dependent protein kinase was eluted before the type-II enzyme, but no type-I cyclic AMP-dependent activity was normally observed in the soluble fraction. The particulate cyclic AMP-dependent protein kinase and associated RI subunits were solubilized by buffers containing 0.1 or 0.5% (w/v) Triton X-100, but not by extraction with 0.5 M-NaCl, indicating that this enzyme is firmly membrane-bound, either as an integral membrane protein or via an anchor protein. DEAE-cellulose chromatography of the Triton X-100 extracts demonstrated the presence of both type-I cyclic AMP-dependent holoenzyme and free RI subunits. These results show that platelets contain three main protein kinase activities detectable with histone substrates, namely a membrane-bound type-I cyclic AMP-dependent enzyme, a soluble type-II cyclic AMP-dependent enzyme and Ca2+-activated phospholipid-dependent protein kinase, which was soluble in lysates containing EDTA.

Blood Platelets↗