Phosphorylation of nonhistone proteins during the HeLa cell cycle. Relationship to DNA synthesis and mitotic chromosome condensation

Cell cycle variations in the phosphorylation of chromatin-associated nonhistones were determined. Cells were radiolabeled with [32P]orthophosphate and chromatin was obtained by mild digestion of nuclei with micrococcal nuclease. The experiments were performed in the presence of a substrate inhibitor of alkaline phosphatase, beta-glycerophosphate. The results show that, while similar molecular weight species of phosphorylated nonhistones are associated with interphase chromatin through the HeLa cell cycle, the incorporation (32P cpm/micrograms of protein) profiles of selected major phosphononhistones show substantial changes. The most prominent peaks of specific radioactivity occur in the DNA synthesis phase (S phase). The phosphorylation states of the proteins of isolated metaphase chromosomes were also determined. Nonhistone proteins of isolated metaphase chromosomes are strikingly dephosphorylated, especially in comparison to histone H1. The phosphorylation of the major phosphononhistone of chromatin, which has a molecular weight of 55,000, was further characterized by techniques that included one-dimensional peptide mapping in sodium dodecyl sulfate-polyacrylamide gels and nonequilibrium pH gradient slab gel electrophoresis. Phosphoproteins are also components of the nuclear scaffold, and cell cycle variations in these proteins were investigated. The primary phosphorylated species has a molecular weight of 119,000. As with chromatin-associated nonhistones, this nuclear scaffold protein shows substantial incorporation of 32P in S phase, and a high level of incorporation also occurs close to mitosis.     

Cooperative regulation of GSK-3 by muscarinic and PDGF receptors is associated with airway myocyte proliferation

Muscarinic receptors and platelet-derived growth factor (PDGF) receptors synergistically induce proliferation of airway smooth muscle (ASM), but the pathways that regulate these effects are not yet completely identified. We hypothesized that glycogen synthase kinase-3 (GSK-3), a kinase that represses several promitogenic signaling pathways in its unphosphorylated form, is cooperatively inhibited by PDGF and muscarinic receptors in immortalized human ASM cell lines. PDGF or methacholine alone induced rapid GSK-3 phosphorylation. This phosphorylation was sustained only for PDGF; however, methacholine potentiated PDGF-induced sustained GSK-3 phosphorylation. Synergistic effects of methacholine also were observed on PDGF-induced retinoblastoma protein (Rb) phosphorylation and cell proliferation. Suppression of GSK-3 inhibitory function using SB 216763 also augmented PDGF-induced Rb phosphorylation and cell cycle progression; this synergy was similar in magnitude to that seen for methacholine with PDGF. GSK-3 phosphorylation induced by methacholine required PKC, since it was abolished by GF 109203X and G? 6976; however, inhibition of PKC had no effect on cell responses to PDGF. PKC inhibition also specifically abolished the synergistic effect of methacholine on PDGF-induced GSK-3 phosphorylation and cell proliferation. Collectively, these results show that GSK-3 plays a key repressive role in ASM cell proliferation. Moreover, muscarinic receptors mediate PKC-dependent GSK-3 inhibition, and this appears to be a primary mechanism underpinning augmentation of PDGF-induced cell growth.     

Regulation of Chk1 includes chromatin association and 14-3-3 binding following phosphorylation on Ser-345

Checkpoints are biochemical pathways that provide the cell with mechanisms to detect DNA damage and respond by arresting the cell cycle to allow DNA repair. The conserved checkpoint kinase Chk1 regulates mitotic progression in response to DNA damage and replication interference by blocking the activation of Cdk1/cyclin B. Chk1 is phosphorylated on Ser-317 and Ser-345 following a checkpoint signal, a process that is regulated by Atr, and by the sensor complexes containing Rad17 and Hus1. We show that Chk1 is associated with chromatin in cycling cells and that the chromatin-associated Chk1 is phosphorylated in the absence of exogenous DNA damage. The UV-induced Ser-345-phosphorylated forms of Chk1 that appear minutes after treatment are predominantly associated with chromatin. The Ser-345 site is in a 14-3-3 consensus binding motif and is required for nuclear retention of Chk1 following an hydroxyurea-induced checkpoint signal; nonetheless, Ser-345 or Ser-317 are not required for the chromatin association of Chk1. Hus1, a member of the proliferating cell nuclear antigen-like damage recognition complex plays a role in the phosphorylation of Chk1 on Ser-345, however, Hus1 is not required for phosphorylation on Ser-317 or for Chk1 localization to chromatin. These results indicate that there is more than one step in Chk1 activation and that the regulation of this checkpoint signaling is achieved at least in part through phosphorylation of Ser-345, which serves to localize Chk1 in the nucleus presumably by blocking Crm1-dependent nuclear export.     

Chromatin-associated protein phosphokinases of rat ventral prostate. Characteristics and effects of androgenic status.

Protein phosphokinase activity endogenous to rat ventral prostate chromatin was assayed by using edphosphophosvitin as an exogenous substrate. For maximal activity of the kinase reaction, the presence of 200 mM NaCl, 5 mM MgCl2, and 1 mM dithiothreitol was essential. Two apparent pH optima were observed, a broad one between pH 7 and 7.4, and one at pH 7.89. At pH 7.4 the apparent Km for 31% dephosphophosvitin was 0.3 mg per ml. With respect to ATP, two apparent Km values (0.04 and 0.41 mM) were found. The kinase activity was minimal toward exogenous histones when used as substrates (3% for lysine-rich and 0.3% for arginine-rich (f3) histones, compared with dephosphophosvitin controls). The protein phosphokinases were not significantly stimulated by cyclic adenosine 3':5'-monophosphate (cyclic AMP) when histones used as substrate. With dephosphophosvitin as substrate, cyclic AMP produced a small inhibition (5 to 15%). Orchiectomy of adult rats resulted in a rapid decline in the chromatin-associated protein phosphokinase activity assayed using optimal experimental condition described above. At 9 hours postorchiectomy, a 30% decline in the activity was observed; this was further reduced to about 50% of the control by 18 hours. This decrease in the kinase activity (e.g. at 9 hours postorchiectomy) appears to precede measurable changes in the protein and RNA complements of chromatin. Testosterone replacement following orchiectomy abolished this decline in the chromatin-associated activity. The chromatin-associated protein phosphokinase activity toward lysine-rich and arginine-rich histones was also sensitive to androgenic status of the animals and declined rapidly postorchiectomy. The results suggest the presence of multiple and androgen-sensitive protien phosphokinases associated with rat ventral prostate chromatin, which may modulate the phosphorylation of nuclear nonhistone phosphoproteins with changing gene action mediated by testosterone in this target tissue.     

Phospholipase C-gamma1 is required for survival in heat stress: involvement of protein kinase C-dependent Bcl-2 phosphorylation

The consequences of heat-induced phospholipase C-gamma1 (PLC-gamma1) phosphorylation are not known. We investigated the role of PLC-gamma1 activation and its downstream targets during the cellular response to heat stress using mouse embryonic fibroblasts genetically deficient in PLC-gamma1 (Plcg1 null MEF) and its wild type (wt MEF) as models. Treatment of wt MEF with heat resulted in temperature- and heating duration-dependent tyrosine phosphorylation of PLC-gamma1. HSP70 synthesis and the activation of extracellular signal-regulated kinases 1/2 (ERK1/2) and c-Jun N-terminal protein kinase (JNK) increased equally following heat treatment in both cell lines. However, heat-induced protein kinase C (PKC) activation was dramatically reduced in Plcg1 null MEF compared with wt MEF. Importantly, the mitochondrial localization of PKCalpha, PKC-dependent phosphorylation of Bcl-2, and cell viability in Plcg1 null MEF following heat treatment, were significantly decreased compared with the wild type. Furthermore, pretreatment with bryostatin-1, a PKC activator, enhanced Bcl-2 phosphorylation and cellular resistance to heat-induced apoptosis in Plcg1 null MEF. Taken together, these results suggest that PLC-gamma1 activation enhances cell survival through the PKC-dependent phosphorylation of Bcl-2 during the cellular response to heat stress.     

Protein kinase C and membrane transport: divergent responses of Na+/K+/Cl- cotransport and sugar transport to exogenous diacylglycerol

Even though the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) is known to bind to and activate protein kinase C (PKC), it is still not certain that all cellular responses to phorbol esters are necessarily mediated by PKC. In BALB/c 3T3 preadipose cells, TPA has previously been shown to rapidly inhibit Na+K+Cl- -cotransport activity, stimulate 2-deoxyglucose uptake and induce ornithine decarboxylase activity. The cell-permeable diacylglycerol sn-1,2-dioctanoylglycerol (DiC8) was used in order to distinguish between PKC-dependent and -independent responses of BALB/c 3T3 cells. DiC8 modulated 86Rb+ fluxes in BALB/c 3T3 cells in the same manner as TPA: furosemide-sensitive 86Rb+ influx and efflux was inhibited, while in cotransport-defective cells no effect was observed. In contrast, DiC8 did not stimulate 2-deoxyglucose uptake in either parental or cotransport-defective cell lines, even though TPA is a very effective inducer of this transport system in both cell types. Pretreatment of cells with DiC8 did not substantially alter the subsequent induction of 2-deoxyglucose uptake by TPA, although a slight but reproducible reduction in the magnitude of the response was observed in DiC8-pretreated cells. The PKC-dependent phosphorylation of an acidic 80-kDa protein was stimulated by both TPA and DiC8 in parental and cotransport-defective cell lines, suggesting that a gross defect in the primary effector system used by both TPA and diacylglycerols cannot explain any of our results. Ornithine decarboxylase was induced by DiC8 and the K1/2 was approximately the same as that for inhibition of Na+/K+/Cl- cotransport in these cells. Thus, our results suggest that PKC is clearly essential for some phorbol ester membrane transport responses (such as inhibition of Na+/K+/Cl- cotransport), but our results do not allow us to conclude that other responses (such as stimulation of 2-deoxyglucose uptake) necessarily require PKC activation.     

Eicosapentaenoic acid and docosahexaenoic acid modulate MAP kinase (ERK1/ERK2) signaling in human T cells.

This study was conducted on human Jurkat T cell lines to elucidate the role of EPA and DHA, n-3 PUFA, in the modulation of two mitogen-activated protein (MAP) kinases, that is, extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2). The n-3 PUFA alone failed to induce phosphorylation of ERK1/ERK2. We stimulated the MAP kinase pathway with anti-CD3 antibodies and phorbol 12-myristate 13-acetate (PMA), which act upstream of the MAP kinase (MAPK)/ERK kinase (MEK) as U0126, an MEK inhibitor, abolished the actions of these two agents on MAP kinase activation. EPA and DHA diminished the PMA- and anti-CD3-induced phosphorylation of ERK1/ERK2 in Jurkat T cells. In the present study, PMA acts mainly via protein kinase C (PKC) whereas anti-CD3 antibodies act via PKC-dependent and -independent mechanisms. Furthermore, DHA and EPA inhibited PMA-stimulated PKC enzyme activity. EPA and DHA also significantly curtailed PMA- and ionomycin-stimulated T cell blastogenesis. Together these results suggest that EPA and DHA modulate ERK1/ERK2 activation upstream of MEK via PKC-dependent and -independent pathways and that these actions may be implicated in n-3 PUFA-induced immunosuppression.     

Enhancement of lysophosphatidic acid-induced ERK phosphorylation by phospholipase D1 via the formation of phosphatidic acid

We made stable cell lines overexpressing PLD1 (GP-PLD1) from GP+envAm12 cell, a derivative of NIH 3T3 cell. PLD1 activity and extracellular signal-regulated kinase (ERK) phosphorylation were enhanced in GP-PLD1 cells by the treatment of lysophosphatidic acid (LPA). In contrast, these LPA-induced effects were attenuated with the pretreatment of pertussis toxin (PTX) or protein kinase C (PKC) inhibitor. Moreover, accumulation of phosphatidic acid (PA), a product of PLD action, potentiated the LPA-induced ERK activation in GP-PLD1 cells while blocking of PA production with the treatment of 1-butanol attenuated LPA-induced ERK phosphorylation. From these results, we suggest that LPA activate PLD1 through pertussis toxin-sensitive G protein and PKC-dependent pathways, then PA produced from PLD1 activation facilitate ERK phosphorylation.     

Studies on chromatin-associated protein phosphokinase of submandibular gland from isoproterenol-treated rats

Water-soluble chromatin from rat submandibular gland nuclei was isolated, and had a DNA: RNA:protein ratio of 8:1:20. The spectral properties of this preparation were similar to those described for chromatins from other tissues. The rat submandibular gland chromatin possessed protein phosphokinase activity. It was able to incorporate ³²P from [γ-³²P]-ATP into chromatin proteins, and into dephospho-phosvitin. The chromatin-associated protein phosphokinase activity (measured with dephospho-phosvitin as substrate) required Mg²⁺, Na⁺ or K⁺ and dithiothreitol for optimal activity. A single injection of isoproterenol influenced the activity of this enzyme system, so that it was decreased at 2 h, showed a transient increase at 4 h, and a large increase at 10–16 h after the injection. This event appears to precede the increase in ribosomal RNA induced by Ipr [13]. By 48 h the chromatin-associated protein kinase returned to the normal control levels. These changes appeared to be commensurate with the corresponding alterations in the non-histone acidic protein complement of these chromatins. Actinomycin D or cycloheximide, when administered 30 min prior to isoproterenol, blocked the increase in chromatin-associated protein kinase at 4 as well as 10 h after the injection of isoproterenol. Injection of pilocarpine did not influence the chromatin-associated protein phosphokinase activity. Dichloroisoproterenol appeared to be antagonistic to the influence of isoproterenol in mediating changes in chromatin-associated protein kinase. The results suggest that the isoproterenol-induced increase in chromatin-bound protein phosphokinase which precedes the increase in RNA synthesis is related to the eventual onset of DNA synthesis in rat submandibular gland stimulated by isoproterenol. The chromatin-bound protein phosphokinase activity (or activities) may have a regulatory role on gene action, mediated through the control of phosphorylation of nuclear non-histone acidic proteins [26].     

Stimulation in vitro of prostatic ribonucleic acid polymerase by 5 -dihydrotestosterone-receptor complexes

In a reconstituted $\text{in vitro}$ system, stimulation of RNA polymerase activities by 5α-DHT-receptor complexes prepared from prostatic supernatant and nuclear fractions has been observed. Stimulation of the nucleolar enzyme rather than the nucleoplasmic enzyme was noted. Higher levels of stimulation were observed in the presence of native chromatin as template than when purified exogenous DNA was used. The involvement of chromatin-associated proteins in the system was apparent.     

MARCKS dephosphorylation is involved in bradykinin-induced neurite outgrowth in neuroblastoma SH-SY5Y cells

Bradykinin (BK) plays a major role in producing peripheral sensitization in response to peripheral inflammation and in pain transmission in the central nerve system (CNS). Because BK activates protein kinase C (PKC) through phospholipase C (PLC)-β and myristoylated alanine-rich C kinase substrate (MARCKS) has been found to be a substrate of PKC, we explored the possibility that BK could induce MARCKS phosphorylation and regulate its function. BK stimulation induced transient MARCKS phosphorylation on Ser159 with a peak at 1 min in human neuroblastoma SH-SY5Y cells. By contrast, PKC activation by the phorbol ester phorbol 12,13-dibutyrate (PDBu) elicited MARCKS phosphorylation which lasted more than 10 min. Western blotting analyses and glutathione S-transferase (GST) pull-down analyses showed that the phosphorylation by BK was the result of activation of the PKC-dependent RhoA/Rho-associated coiled-coil kinase (ROCK) pathway. Protein phosphatase (PP) 2A inhibitors calyculin A and fostriecin inhibited the dephosphorylation of MARCKS after BK-induced phosphorylation. Moreover, immunoprecipitation analyses showed that PP2A interacts with MARCKS. These results indicated that PP2A is the dominant PP of MARCKS after BK stimulation. We established SH-SY5Y cell lines expressing wild-type MARCKS and unphosphorylatable MARCKS, and cell morphology changes after cell stimulation were studied. PDBu induced lamellipodia formation on the neuroblastoma cell line SH-SY5Y and the morphology was sustained, whereas BK induced neurite outgrowth of the cells via lamellipodia-like actin accumulation that depended on transient MARCKS phosphorylation. Thus these findings show a novel BK signal cascade-that is, BK promotes neurite outgrowth through transient MARCKS phosphorylation involving the PKC-dependent RhoA/ROCK pathway and PP2A in a neuroblastoma cell line.     

Overexpression of ORC subunits and increased ORC-chromatin association in transformed mammalian cells

The origin recognition complex (ORC) is a conserved heterohexamer required for the formation of pre-replication (pre-RC) complexes at origins of DNA replication. Many studies of ORC subunits have been carried out in transformed human cell lines but the properties of ORC in primary cells have not been addressed. Here, we compare the expression levels and chromatin-association of ORC subunits in HeLa cells to the primary human cell line, WI38, and a virally transformed derivative of WI38, VA13. ORC subunits 2 and 4 were highly overexpressed in both HeLa and VA13, whereas ORC1 levels were elevated in VA13 but considerably higher in HeLa cells. Cellular extraction revealed that the proportion of ORC2 and ORC4 subunits bound to chromatin was similar in all three cell lines throughout the cell-cycle. In contrast, very little ORC1 was associated with chromatin after extraction of primary WI38 cells, whereas the majority of overexpressed ORC1 in both HeLa and VA13 co-fractionated with chromatin throughout the cell-cycle. Although none of the cell lines displayed significant changes in the levels or chromatin-association of ORC during the cell-cycle, the chromatin-associated fraction of ORC1 displayed an increase in apparent molecular weight during S-phase. Similar experiments comparing immortalized CHO cells to an isogenic virally transformed derivative revealed no changes in levels of ORC subunits but an increase in the proportion of all three ORC subunits associated with chromatin. These results demonstrate a complex influence of cellular immortalization and transformation properties on the expression and regulation of ORC subunits. These results extend the potential link between cancer and deregulation of pre-RC proteins, and underscore the importance of considering the transformation status of cell lines when working with these proteins.     

p90(RSK) blocks bad-mediated cell death via a protein kinase C-dependent pathway

Although activation of protein kinase C (PKC) is known to promote cell survival and protect against cell death, the PKC targets and pathways that serve this function have remained elusive. Here we demonstrate that two potent activators of PKC, 12-O-tetradecanoylphorbol-13-acetate and bryostatin, both stimulate phosphorylation of Bad at Ser(112), a site known to regulate apoptotic cell death by interleukin-3. PKC inhibitors but not PI 3-kinase/Akt inhibitors block 12-O-tetradecanoylphorbol-13-acetate-stimulated Bad phosphorylation. PKC isoforms tested in vitro were unable to phosphorylate Bad at Ser(112), suggesting that PKC acts indirectly to activate a downstream Bad kinase. p90(RSK) and family members RSK-2 and RSK-3 are activated by phorbol ester and phosphorylate Bad at Ser(112) both in vitro and in vivo. p90(RSK) stimulates binding of Bad to 14-3-3 and blocks Bad-mediated cell death in a Ser(112)-dependent manner. These findings suggest that p90(RSK) can function in a PKC-dependent pathway to promote cell survival via phosphorylation and inactivation of Bad-mediated cell death.     

Engagement of beta2 integrins recruits 14-3-3 proteins to c-Cbl in human neutrophils

We found that engagement of beta2 integrins on human neutrophils triggered both tyrosine and serine phosphorylation of c-Cbl. Pretreatment of the neutrophils with the broad range protein kinase C (PKC) inhibitor GF-109203X blocked the serine but not the tyrosine phosphorylation of c-Cbl. Moreover, the Src kinase inhibitor PP1 prevented the beta2 integrin-induced tyrosine phosphorylation of c-Cbl but not the simultaneous serine phosphorylation. These results indicate that Src family kinases and PKC can separately modulate the properties of c-Cbl. Indeed, tyrosine kinase-dependent phosphorylation of c-Cbl regulated the ubiquitin ligase activity of that protein, whereas PKC-dependent phosphorylation of c-Cbl had no such effect. Instead, c-Cbl that underwent PKC-induced serine phosphorylation associated with the scaffolding and anti-apoptotic 14-3-3 proteins. Consequently, c-Cbl can independently target proteins for degradation or intracellular localization and may initiate an anti-apoptotic signal in neutrophils.     

Temporal profiling of the chromatin proteome reveals system-wide responses to replication inhibition

Although the replication, expression, and maintenance of DNA are well-studied processes, the way that they are coordinated is poorly understood. Here, we report an analysis of the changing association of proteins with chromatin (the chromatin proteome) during progression through interphase of the cell cycle. Sperm nuclei were incubated in Xenopus egg extracts, and chromatin-associated proteins were analyzed by mass spectrometry at different times. Approximately 75% of the proteins varied in abundance on chromatin by more than 15%, suggesting that the chromatin proteome is highly dynamic. Proteins were then assigned to one of 12 different clusters on the basis of their pattern of chromatin association. Each cluster contained functional groups of proteins involved in different nuclear processes related to progression through interphase. We also blocked DNA replication by inhibiting either replication licensing or S phase CDK activity. This revealed an unexpectedly broad system-wide effect on the chromatin proteome, indicating that the response to replication inhibition extends to many other functional modules in addition to the replication machinery. Several proteins that respond to replication inhibition (including nuclear pore proteins) coprecipitated with the Mcm2-7 licensing complex on chromatin, suggesting that Mcm2-7 play a central role in coordinating nuclear structure with DNA replication.