Changes in activities of modulatiors of cyclic AMP-dependent and cyclic GMP-dependent protein kinases in pancreas and adipose tissue from alloxan-induced diabetic rats.
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Adipose Tissue↗
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Protein kinases in brown adipose tissue of developing rats: substrate specificities, separation, and changes in activity during development.
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Adipose Tissue, Brown↗
Cyclic AMP-binding proteins and protein kinase during regression of Walker 256 mammary carcinoma.
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Animals↗
Cyclin is a component of the sea urchin egg M-phase specific histone H1 kinase.
A so-called 'growth-associated' or 'M-phase specific' histone H1 kinase (H1K) has been described in a wide variety of eukaryotic cell types; p34cdc2 has previously been shown to be a catalytic subunit of this protein kinase. In fertilized sea urchin eggs the activity of H1K oscillates during the cell division cycle and there is a striking temporal correlation between H1K activation and the accumulation of a phosphorylated form of cyclin. H1K activity declines in parallel with proteolytic cyclin destruction of the end of the first cell cycle. By virtue of the high affinity of the fission yeast p13suc1 for the p34cdc2 protein, H1K strongly binds to p13-Sepharose beads. Cyclin, p34cdc2 and H1K co-purify on this affinity reagent as well as through several conventional chromatographic procedures. Anticyclin antibodies immunoprecipitate the M-phase specific H1K in crude extracts or in purified fractions. Sea urchin eggs appear to contain much less cyclin than p34cdc2, suggesting that p34cdc2 may interact with other proteins. These results demonstrate that cyclin and p34cdc2 are major components of the M-phase specific H1K.
Animals↗
Cdc2 H1 kinase is negatively regulated by a type 2A phosphatase in the Xenopus early embryonic cell cycle: evidence from the effects of okadaic acid.
In Xenopus embryos, the cell cycle is abbreviated to a rapid alternation between interphase and mitosis. The onset of each M phase is induced by the periodic activation of the cdc2 kinase which is triggered by a threshold level of cyclins and apparently involves dephosphorylation of p34cdc2. We have prepared post-ribosomal supernatants from eggs sampled during interphase (interphase extracts) and just before the first mitosis of the early embryonic cell cycle (prophase extracts). In 'interphase extracts', the cdc2 kinase never activates spontaneously upon incubation at room temperature whereas in 'prophase extracts' it does. We show here that in 'interphase extracts', specific inhibition of type 2A phosphatase by okadaic acid induces cdc2 kinase activation. This requires a subthreshold level of cyclin and the presence of a particulate factor in the extract. Inhibition of type 1 phosphatases by inhibitor 1 and inhibitor 2 never results in cdc2 kinase activation. These results demonstrate that during the period of cyclin accumulation, cdc2 kinase activation is inhibited by a type 2A phosphatase. In 'prophase extracts', spontaneous activation of the cdc2 kinase is inhibited by beta-glycerophosphate and NaF, but not by okadaic acid, inhibitor 1 and inhibitor 2 or divalent cation chelation. This demonstrates that when enough cyclin has accumulated, cdc2 kinase activation involves a protein phosphatase which must be distinct from the type 1 and 2A phosphatases, and from the calcium-dependent (type 2B) and magnesium-dependent (type 2C) phosphatases.
Animals↗
Regulation of M-phase promoting factor activity during development of mouse male germ cells.
While the role of M-phase promoting factor (MPF) in controlling meiosis in oocytes has been well documented, very little is known about its function in male germ cells. Previous studies have localized transcripts for cyclin B1, the regulatory subunit of MPF, in male germ cells, with highest levels in postmeiotic, early round spermatids and much reduced levels in the meiotically dividing pachytene spermatocytes. The present study describes the localization of the regulatory and the catalytic subunits of MPF, CycB1 and Cdc2, respectively, to specific cell types within the testis. Immunoblotting revealed that both CycB1 and Cdc2 were present at highest levels in pachytene spermatocytes, with lower levels observed in the postmeiotic compartment. To assay for MPF activity, kinase complexes were isolated from lysates of testicular cells using p13suc1 agarose and antibodies directed against Cdc2 and CycB1. Activity of these kinase complexes was analyzed using histone H1 as an exogenous substrate. Cdc2 and CycB1-associated kinase activities were localized to the meiotically dividing pachytene spermatocytes, but not to postmeiotic spermatids.
Animals↗
In isolated human centrosomes, the associated kinases phosphorylate a specific subset of centrosomal proteins.
Several studies have shown that kinases and phosphatases can interact with the centrosome during interphase and mitosis suggesting that centrosomal components might be the targets of these enzymes. The association of the cAMP-dependent protein kinase type II and the mitotic kinase p34cdc2 with centrosomes from human lymphoblast cells has previously been shown (Keryer et al, 1993, Exp Cell Res 204, 230-240; Bailly et al, 1989, EMBO J 8, 3985-3995). In this paper we demonstrate that isolated centrosomes are able to phosphorylate a few number of centrosomal proteins (M(r) 230-220000; 135000 and 50000) and also H1 histone. The phosphorylation of H1-histone is cell cycle dependent and modulated by phosphatases. The use of kinase and phosphatase inhibitors and the addition of the catalytic subunit of cAMP-dependent kinase or of cyclinB-p34cdc2 kinase showed that both kinases phosphorylate the same centrosomal substrates. In addition two centrosomal proteins (M(r) 100000 and 37000) were phosphorylated only by p34cdc2 kinase. Although the low amount of centrosomal proteins precluded a full characterization of these substrates we discuss the identity of the major centrosomal phosphoproteins by comparison with proteins known to associate with microtubule-organizing centres or mitotic spindles. Our results raise also the intriguing possibility that the cAMP-dependent protein kinase could be regulated by the mitotic kinase at the entry of mitosis.
Amino Acid Sequence↗
p34cdc2 kinase activity is maintained upon activation of the replication checkpoint in Schizosaccharomyces pombe.
All eukaryotes use feedback controls to order and coordinate cell cycle events. In Schizosaccharomyces pombe, several classes of checkpoint genes serve to ensure that DNA replication is complete and free of error before the onset of mitosis. Wild-type cells normally arrest upon inhibition of DNA synthesis or in response to DNA damage, although the exact mechanisms controlling this arrest are unclear. Genetic evidence in fission yeast suggests that the dependence of mitosis upon completion of DNA replication is linked to the regulation of the p34cdc2 cyclin-dependent kinase. It has been hypothesized that inhibition of DNA synthesis triggers down-regulation of p34cdc2 kinase activity, although this has never been shown biochemically. We analyzed the activity of p34cdc2 in wild-type and checkpoint-defective cells treated with a DNA synthesis inhibitor. Using standard in vitro assays we demonstrate that p34cdc2 kinase activity is maintained in wild-type cells arrested at the replication checkpoint. We also used a novel in vivo assay for p34cdc2 kinase activity, in which we expressed a fragment of the human retinoblastoma tumor suppressor protein in fission yeast. Phosphorylation of this fragment of the human retinoblastoma tumor suppressor protein is dependent on p34cdc2 kinase activity, and this activity is also maintained in cells arrested at the replication checkpoint. These data suggest that the mechanism for cell-cycle arrest in response to incomplete DNA synthesis is not dependent on the attenuation of p34cdc2 activity.
CDC2 Protein Kinase↗
pp39mos is associated with p34cdc2 kinase in c-mosxe-transformed NIH 3T3 cells.
We investigated the possible interactions between pp39mos and p34cdc2 kinase in NIH 3T3 cells transformed by c-mosxe. pp39mos is coprecipitated with p34cdc2 when using either anti-PSTAIR antibody or p13suc1-Sepharose beads. Likewise, p34cdc2 is coprecipitated with pp39mos when using anti-mos antibody. However, pp39mos was not present in histone H1 kinase-active p34cdc2 complexes precipitated with anti-p34cdc2 C-terminal peptide antibody even during metaphase of the cell cycle. The molar ratio of p34 to pp39mos in the p13suc1 complex is approximately 2:1. Consistent with the tight association between pp39mos and tubulin, tubulin was also present in equivalent amounts with pp39mos and p34 in the p13suc1 complex. This pp39mos-p34cdc2-tubulin complex may be important in transformation by the mos oncogene.
3T3 Cells↗
Responses of pertussis toxin-treated microvascular endothelial cells to transforming growth factor beta 1. No evidence for pertussis-sensitive G-protein involvement in TGF-beta signal transduction.
Responses of bovine adrenal capillary endothelial cells (BACE) on treatment with transforming growth factor beta 1 (TGF-beta 1) have been characterized and tested for sensitivity to inactivation of pertussis toxin-sensitive G-proteins. TGF-beta 1 elicited growth inhibition, monolayer remodeling, elevation of steady state mRNA levels for collagen type 1 (alpha 1(1) and alpha 2(1)) and TGF-beta 1, and inhibition of p34cdc2 histone H1 kinase activity in BACE cells. Pertussis toxin treatment enhanced both inhibition of BACE cell [3H]methylthymidine uptake and remodeling of BACE monolayers by TGF-beta 1. These findings contrast with studies of mink lung epithelial cells, in which TGF-beta 1 growth inhibition has been shown to be pertussis-sensitive. Further investigation revealed that pertussis toxin treatment of BACE cells had no effect on TGF-beta 1-stimulated elevation of steady state mRNA levels for collagen type 1 (alpha 1(1) or alpha 2(1)) or for TGF-beta 1. Analysis of p34cdc2 activity in BACE cells revealed potent inhibition of p34cdc2 histone H1 kinase activity by TGF-beta 1. Pertussis toxin treatment also abolished the increase in p34cdc2 activity, however, precluding the determination of the pertussis toxin sensitivity of this response to TGF-beta 1. Consistent with suppression of p34cdc2 activation, pertussis toxin also caused substantial inhibition of mitogen-stimulated BACE cell [3H]methylthymidine uptake. It is concluded that TGF-beta 1 signal transduction in this cell type does not involve G-proteins of the pertussis toxin-sensitive class and that, in view of its potent effects on DNA synthesis and p34cdc2 activation, the use of pertussis toxin to determine G-protein involvement in cytokine signalling pathways should be approached with caution.
Animals↗
Dissociation of p34cdc2 complex formation from phosphorylation and histone H1 kinase activity.
The cell cycle inhibitor mimosine was used to examine the activation of the p34cdc2 protein kinase in S phase of the cell cycle. Addition of mimosine to cycling epithelial cells halted cell cycle traverse in S phase, coincident with an inhibition of p34cdc2 histone H1 kinase activity. Mimosine treatment did not alter p34cdc2 synthesis or turnover; however, overall phosphorylation of p34cdc2 was decreased to near undetectable levels. Although activity of p34cdc2 was inhibited, the ability of the protein to form high molecular weight complexes, a phenomenon associated with kinase activation in vivo, was not affected. These results indicate that p34cdc2 complex formation can occur in the absence of phosphorylation and that phosphorylation of p34cdc2 is then required to activate these preformed complexes.
Animals↗
Overexpression of cyclin A in the mammary glands of transgenic mice results in the induction of nuclear abnormalities and increased apoptosis.
Aberrant expression of several cyclin genes has been demonstrated to be associated with many types of tumors. To determine the capacity of cyclin A to function as an oncogene in vivo, wild-type and mutant cyclin A proteins were specifically overexpressed in the mammary glands of transgenic mice using regulatory sequences from the ovine beta-lactoglobulin gene. Several lines of transgenic mice were generated that expressed human cyclin A or a nondegradable mutant version of human cyclin A, in which the amino-terminal 89 amino acids encompassing the cyclin destruction box were removed. The cyclin A transgene products were localized in the nuclei of mammary epithelial cells, and the transgenic mammary glands had an increase in cyclin A- and cdk2-associated H1 kinase activity. Many mammary epithelial cells in the transgenic glands exhibited nuclear abnormalities, including multinucleation and karyomegaly, which were suggestive of preneoplastic alterations. The abnormalities were more severe in mammary glands of the mutant cyclin A transgenics, which expressed a stabilized cyclin A protein. In situ analysis of mid-lactation mammary gland sections revealed increased numbers of apoptotic cells in the transgenic glands. Double transgenic animals were generated that expressed both the mutant human cyclin A and human cdk2 transgenes, and a more pronounced phenotype resulted. The bigenic mammary glands exhibited focal areas of hyperplasia, as well as a greater incidence of apoptosis than observed in the single transgenic glands, demonstrating in vivo cooperation between these genes in transformation and apoptotic signaling pathways.
Amino Acid Sequence↗
Resolution and general properties of different types of ribosomal protein kinases in mouse plasmocytoma.
Three different types of protein kinases (ATP: protein phosphotransferase, EC 2.7.1.37) were isolated and partially purified from a mouse plasmacytoma microsomal KCl wash fraction, then chromatographed on DEAE cellulose and phosphocellulose. The three protein kinase activities designated by protein kinase I, II and III were characterized with respect to their capacity to utilize [gamma-32P]ATP and [gamma-32P]GTP, to interact with cyclic AMP, stimulation by cyclic AMP, substrate specificity and sedimentation behaviour on glycerol gradient centrifugation. Protein kinase I was found to be cyclic AMP dependent and preferentially phosphorylated histones. Protein kinase II and III were insensitive to cyclic AMP, protein kinase II preferentially phosphorylated histones and the protein(s) of a ribosomal KCl wash fraction eluted from DEAE cellulose between 0.2 and 0.35 M KCl and termed "PPx". Protein kinase III phosphorylated casein and ribosomal proteins to a great extent. Studies with glycerol density gradient centrifugation indicated that protein kinase I sediments as a component of about 4.4 S, protein kinase II of 4.3 S and protein kinase III of 3 S. Chromatography on phosphocellulose of the protein kinases isolated from purified free polysomes showed the same type of protein kinases as those from microsomes. So it appears unlikely that protein kinase I and II were contaminants from the cytosol.
Animals↗
The metaphase specific phosphorylation of HMG I.
In vivo labelling of HeLa cells arrested in metaphase with [32P]-phosphate and in vitro phosphorylation of HMG I with the partially purified growth associated H1 kinase was used to study metaphase specific phosphorylation of HMG I. It was found that threonine 53 and 78 became phosphorylated. These amino acids are embedded in respectively the sequence PTPKR and TPGRK which are similar to the sequences phosphorylated by the growth associated H1 kinase.
Amino Acid Sequence↗
Purification and characterization of a specific histone H1 protein kinase from mouse plasmacytoma.
A protein kinase with high specificity for histone H1 was purified from a plasmacytoma microsomal fraction by a high-salt wash, ammonium sulfate precipitation, chromatography on DEAE-cellulose, hydroxyapatite and Sephadex G-200 columns, and the main properties of this kinase were studied. A sulfhydryl compound, such as 2-mercaptoethanol or dithiothreitol, was necessary for full activity. The optimum pH was 7.4-7.8. After purification, the histone H1 kinase was not stimulated by cAMP or cGMP. It was not inhibited by the heat-stable cAMP-dependent protein kinase inhibitor from beef heart. It utilized preferentially GTP over ATP as phosphate donor. Km values for ATP and GTP were 58 microM and 1.4 microM respectively; the Km for histone H1 was 14 microgram ml-1. The molecular weight was approximately 90 000 by gel-exclusion chromatography. Analysis of the purified H1-specific protein kinase by polyacrylamide gel electrophoresis in dodecylsulfate showed two bands having molecular weights of approximately 64 000 and 54 000. Many characteristics of this kinase were similar to those of the chromatin-bound protein kinase reported by other workers in rapidly proliferating cells.
Animals↗
Adenosine 3',5'-monophosphate (cAMP)-binding proteins and cAMP-dependent protein kinases in bovine adrenal cortical cell plasma membrane.
A cAMP-binding protein and cAMP-dependent protein kinase have been identified and partially characterized in bovine adrenocortical plasma membranes. [3H]cAMP binding to the plasma membrane preparation demonstrates both high (Ka = 4.2 X 10(9) M-1) and low (Ka = 1.3 X 10(8) M-1) affinity binding sites. cAMP-dependent protein kinase was demonstrated using both bovine histone and endogenous substrates. The detergent-dependent solubilization characteristics of this cAMP-dependent protein kinase indicate that it is an integral membrane protein.
Adrenal Cortex↗
Membrane-associated protein kinase activities in seminal plasma from vasectomized men.
Differential centrifugation was used to prepare heavy and light membrane fractions from the seminal plasma of vasectomized men. The two membrane fractions combined contained half of the phosvitin and histone kinase activities but only 7% of the total protein content in vasectomy semen. These two kinase activities as well as phosphorylation of endogenous membrane proteins were optimally stimulated by Mg2+; Mn2+ could effectively substitute for Mg2+ only in endogenous phosphorylation reactions. Neither the phosvitin nor histone kinase responded to cAMP or cGMP, but the histone kinase was strongly inhibited by the heat-stable cAMP-dependent protein kinase inhibitor. The phosvitin kinase was not affected by this inhibitor. The phosphorylation of endogenous proteins in the heavy membrane fraction was not affected by the protein kinase inhibitor but protein phosphorylation in the light membrane fraction was partly (45%) inhibited. The differential effects of increased ionic strength, sulphydryl protecting agents, and the protein kinase inhibitor on protein kinase activity towards lysine-rich histones, phosvitin and endogenous proteins, as well as differential extractability and binding to an anion exchange column of histone kinase and phosvitin kinase activities, indicate that more than one kinase activity is present in these membrane subfractions. Electron microscopic examination showed that there are several kinds of membrane-limited components in vasectomy seminal fluid that vary in size, density, and ultrastructure. The association of type(s) of protein kinase to individual membrane components remains to be established.
Cell Membrane↗
Ionic inhibition of catalytic phosphorylation of histone by bovine brain protein kinase.
The effects of various ions commonly found in protein kinase assays upon the rate of histone phosphorylation catalyzed by the highly purified bovine brain enzyme, protein kinase I, have been investigated. Sodium, potassium, and magnesium were found to inhibit histone phosphorylation by protein kinase I in a similar manner. The degree of inhibition by any of these cations was demonstrated to be directly proportional to the square root of the ionic strength of the assay medium. The relationship between the ionic strength of the assay medium and the rate of histone phosphorylation catalyzed by protein kinase I was employed to correct the rate of histone phosphorylation at various magnesium acetate concentrations to a standard ionic strength. When this was done an analysis of the previously postulated rate law for histone phosphorylation c atalyzed by protein kinase I gave a binding constant for the magnesium-ATP complex which was in agreement with that expected for this complex on the basis of various binding constants available in the literature. These results demonstrate that it is unnecessary to postulate a specific ion inhibition process for protein kinase I by the ions employed in this study. They also support the reasonable assumption that magnesium ion binds to ATP at or prior to the rate-determining step in histone phosphorylation catalyzed by protein kinase I. The expression developed in this paper for the effect of ionic strength upon protein kinase I activity can now be used to correct activity measurements made under various assay conditions to a standard assay state, allowing facile comparisons of kinetic data. It should be possible to develop similar expressions for other protein kinases and substrates to permit useful interpretation of kinetic data.
Animals↗