d-alpha-tocopherol inhibition of vascular smooth muscle cell proliferation occurs at physiological concentrations, correlates with protein kinase C inhibition, and is independent of its antioxidant properties.

d-alpha-Tocopherol, but not d-beta-tocopherol, negatively regulates proliferation of vascular smooth muscle cells at physiological concentrations. d-alpha-Tocopherol inhibits protein kinase C (PKC) activity, whereas d-beta-tocopherol is ineffective. Furthermore d-beta-tocopherol prevents the inhibition of cell growth and of PKC activity caused by d-alpha-tocopherol. The negative regulation by d-alpha-tocopherol of PKC activity appears to be the cause and not the effect of smooth muscle cell growth inhibition. d-alpha-Tocopherol does not act by binding to PKC directly but presumably by preventing PKC activation. It is concluded that, in vascular smooth muscle cells, d-alpha-tocopherol acts specifically through a nonantioxidant mechanism and exerts a negative control on a signal transduction pathway regulating cell proliferation.

Point mutation of the autophosphorylation site or in the nuclear location signal causes protein kinase A RII beta regulatory subunit to lose its ability to revert transformed fibroblasts.

The RII beta regulatory subunit of cAMP-dependent protein kinase (PKA) contains an autophosphorylation site and a nuclear location signal, KKRK. We approached the structure-function analysis of RII beta by using site-directed mutagenesis. Ser114 (the autophosphorylation site) of human RII beta was replaced with Ala (RII beta-P) or Arg264 of KKRK was replaced with Met (RII beta-K). ras-transformed NIH 3T3 (DT) cells were transfected with expression vectors for RII beta, RII beta-P, and RII beta-K, and the effects on PKA isozyme distribution and transformation properties were analyzed. DT cells contained PKA-I and PKA-II isozymes in a 1:2 ratio. Over-expression of wild-type or mutant RII beta resulted in an increase in PKA-II and the elimination of PKA-I. Only wild-type RII beta cells demonstrated inhibition of both anchorage-dependent and -independent growth and phenotypic change. The growth inhibitory effect of RII beta overexpression was not due to suppression of ras expression but was correlated with nuclear accumulation of RII beta. DT cells demonstrated growth inhibition and phenotypic change upon treatment with 8-Cl-cAMP. RII beta-P or RII beta-K cells failed to respond to 8-Cl-cAMP. These data suggest that autophosphorylation and nuclear location signal sequences are integral parts of the growth regulatory mechanism of RII beta.

Double-stranded-RNA-dependent protein kinase and TAR RNA-binding protein form homo- and heterodimers in vivo.

The yeast two-hybrid system and far-Western protein blot analysis were used to demonstrate dimerization of human double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in vivo and in vitro. A catalytically inactive mutant of PKR with a single amino acid substitution (K296R) was found to dimerize in vivo, and a mutant with a deletion of the catalytic domain of PKR retained the ability to dimerize. In contrast, deletion of the two dsRNA-binding motifs in the N-terminal regulatory domain of PKR abolished dimerization. In vitro dimerization of the dsRNA-binding domain required the presence of dsRNA. These results suggest that the binding of dsRNA by PKR is necessary for dimerization. The mammalian dsRNA-binding protein TRBP, originally identified on the basis of its ability to bind the transactivation region (TAR) of human immunodeficiency virus RNA, also dimerized with itself and with PKR in the yeast assay. Taken together, these results suggest that complexes consisting of different combinations of dsRNA-binding proteins may exist in vivo. Such complexes could mediate differential effects on gene expression and control of cell growth.

Hippocampal long-term depression and depotentiation are defective in mice carrying a targeted disruption of the gene encoding the RI beta subunit of cAMP-dependent protein kinase.

The cAMP-dependent protein kinase (PKA) has been shown to play an important role in long-term potentiation (LTP) in the hippocampus, but little is known about the function of PKA in long-term depression (LTD). We have combined pharmacologic and genetic approaches to demonstrate that PKA activity is required for both homosynaptic LTD and depotentiation and that a specific neuronal isoform of type I regulatory subunit (RI beta) is essential. Mice carrying a null mutation in the gene encoding RI beta were established by use of gene targeting in embryonic stem cells. Hippocampal slices from mutant mice show a severe deficit in LTD and depotentiation at the Schaffer collateral-CA1 synapse. This defect is also evident at the lateral perforant path-dentate granule cell synapse in RI beta mutant mice. Despite a compensatory increase in the related RI alpha protein and a lack of detectable changes in total PKA activity, the hippocampal function in these mice is not rescued, suggesting a unique role for RI beta. Since the late phase of CA1 LTP also requires PKA but is normal in RI beta mutant mice, our data further suggest that different forms of synaptic plasticity are likely to employ different combinations of regulatory and catalytic subunits.

Association of mitogen-activated protein kinase with the microtubule cytoskeleton.

Using indirect immunofluorescence microscopy and biochemical techniques, we have determined that approximately one-third of the total mitogen-activated protein kinase (MAPK) is associated with the microtubule cytoskeleton in NIH 3T3 mouse fibroblasts. This population of enzyme can be separated from the soluble form that is found distributed throughout the cytosol and is also present in the nucleus after mitogen stimulation. The microtubule-associated enzyme pool constitutes half of all detectable MAPK activity after mitogenic stimulation. These findings extend the known in vivo associations of MAPK with microtubules to include the entire microtubule cytoskeleton of proliferating cells, and they suggest that a direct association of MAPK with microtubules may be in part responsible for the observed correlations between MAPK activities and cytoskeletal alteration.

The interferon-inducible double-stranded RNA-activated protein kinase self-associates in vitro and in vivo.

The interferon-inducible double-stranded (ds) RNA-activated protein kinase (PKR) exhibits antiviral, anticellular, and antitumor activities. The mechanisms of its enzymatic activation by autophosphorylation and of the observed transdominant inhibitory phenotype of enzymatically inactive mutants have invoked PKR dimerization. Here we present direct evidence in support of PKR-PKR interaction. We show that radiolabeled PKR can specifically interact with matrix-bound unlabeled PKR in the absence of dsRNA. The self-association activity resides, in part, in the N-terminal region of 170 residues, which also constitutes the dsRNA-binding domain (DRBD). DRBD can bind to matrix-bound PKR or to matrix-bound DRBD. Dimerization of DRBD was directly demonstrated by chemical crosslinking. Affinity chromatography and electrophoretic mobility supershift assays demonstrated that mutants that fail to bind dsRNA can still exhibit protein-protein interaction. The PKR-PKR interaction could also be observed in a two-hybrid transcriptional activation assay in mammalian cells and consequently is likely to be an important feature of PKR activity in vivo.

Basal expression of the cystic fibrosis transmembrane conductance regulator gene is dependent on protein kinase A activity.

The cystic fibrosis transmembrane conductance regulator (CFTR) functions as a Cl- channel that becomes activated after phosphorylation by cAMP-dependent protein kinase (PKA). We demonstrate that PKA also plays a crucial role in maintaining basal expression of the CFTR gene in the human colon carcinoma cell line T84. Inhibition of PKA activity by expression of a dominant-negative regulatory subunit or treatment with the PKA-selective inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) caused a complete suppression of CFTR gene expression without affecting other constitutively active genes. Basal expression of a 2.2-kb region of the CFTR promoter linked to a luciferase reporter gene (CFTR-luc) exhibited the same dependence on PKA. The ability of cAMP to induce CFTR over basal levels is cell-type specific. In T84 cells, both the endogenous CFTR gene and CFTR-luc exhibited only a modest inducibility (approximately 2-fold), whereas in the human choriocarcinoma cell line JEG-3, CFTR-luc could be induced at least 4-fold. A variant cAMP-response element is present at position -48 to -41 in the CFTR promoter, and mutation of this sequence blocks basal expression. We conclude that cAMP, acting through PKA, is an essential regulator of basal CFTR gene expression and may mediate an induction of CFTR in responsive cell types.

Phosphorylation of the human leukemia inhibitory factor (LIF) receptor by mitogen-activated protein kinase and the regulation of LIF receptor function by heterologous receptor activation.

We used a bacterially expressed fusion protein containing the entire cytoplasmic domain of the human leukemia inhibitory factor (LIF) receptor to study its phosphorylation in response to LIF stimulation. The dose- and time-dependent relationships for phosphorylation of this construct in extracts of LIF-stimulated 3T3-L1 cells were superimposable with those for the stimulation of mitogen-activated protein kinase (MAPK). Indeed, phosphorylation of the cytoplasmic domain of the low-affinity LIF receptor alpha-subunit (LIFR) in Mono Q-fractionated, LIF-stimulated 3T3-L1 extracts occurred only in those fractions containing activated MAPK; Ser-1044 served as the major phosphorylation site in the human LIFR for MAPK both in agonist-stimulated 3T3-L1 lysates and by recombinant extracellular signal-regulated kinase 2 in vitro. Expression in rat H-35 hepatoma cells of LIFR or chimeric granulocyte-colony-stimulating factor receptor (G-CSFR)-LIFR mutants lacking Ser-1044 failed to affect cytokine-stimulated expression of a reporter gene under the control of the beta-fibrinogen gene promoter but eliminated the insulin-induced attenuation of cytokine-stimulated gene expression. Thus, our results identify the human LIFR as a substrate for MAPK and suggest a mechanism of heterologous receptor regulation of LIFR signaling occurring at Ser-1044.

Evidence for a role of endothelin 1 and protein kinase C in nitroglycerin tolerance.

We sought to examine mechanisms responsible for increased vasoconstriction that occurs during development of nitroglycerin tolerance. Rabbits were treated for 3 days with nitroglycerin patches (0.4 mg/hr), and their aortic segments were studied in organ chambers. This treatment resulted in attenuated in vitro relaxations to nitroglycerin and increased contractile sensitivity to angiotensin II, serotonin, phenylephrine, KCl, and a direct activator of protein kinase C, the phorbol ester phorbol 12,13-dibutyrate. The protein kinase C antagonists calphostin C (100 nM) and staurosporine (10 nM) corrected the hypersensitivity to constrictors in tolerant vessels, yet had minimal effects on constrictions in control vessels. Paradoxically, constrictions caused by endothelin 1 were decreased in nitrate-tolerant vessels. Immunocytochemical analysis revealed intense endothelin 1-like and big endothelin 1-like immunoreactivity in the media of nitroglycerin-tolerant but not of control aortas. The enhanced vasoconstriction to angiotensin II, serotonin, KCl, and phenylephrine could be mimicked in normal vessels by addition of subthreshold concentrations of endothelin 1, and this effect was prevented by calphostin C. We propose that increased autocrine production of endothelin 1 in nitrate tolerance sensitizes vascular smooth muscle to a variety of vasoconstrictors through a protein kinase C-mediated mechanism.

Activation of YRP kinase by v-Src and protein kinase C-mediated signal transduction pathways.

We have previously reported that a serine(threonine) protein kinase that phosphorylates histone H1 in vitro is activated by tyrosine phosphorylation in v-Src-transformed rat 3Y1 fibroblasts. We now refer to this kinase as YRP kinase, for tyrosine-regulated protein kinase. Since YRP kinase may play a role in mediating the growth-stimulatory and morphology-altering effects of v-Src, we have further examined the signal transduction involved in the activation of YRP kinase. Although YRP kinase is constitutively activated in fibroblasts transformed by v-Src, activation of protein kinase C was also found to lead to activation of YRP kinase. Activation of YRP kinase by protein kinase C was found to be potentiated by vanadate treatment or overexpression of c-Src. The activation of YRP kinase by v-Src, however, does not appear to be mediated by protein kinase C, suggesting that YRP kinase can be activated by two separate signal transduction pathways. Transformation of fibroblasts by v-Ras or v-Mil did not result in activation of YRP kinase, indicating that the MAP kinase pathway does not mediate the activation of YRP kinase by v-Src or protein kinase C.

Requirement of mitogen-activated protein kinase kinase 3 (MKK3) for tumor necrosis factor-induced cytokine expression

The p38 mitogen-activated protein kinase is activated by treatment of cells with cytokines and by exposure to environmental stress. The effects of these stimuli on p38 MAP kinase are mediated by the MAP kinase kinases (MKKs) MKK3, MKK4, and MKK6. We have examined the function of the p38 MAP kinase signaling pathway by investigating the effect of targeted disruption of the Mkk3 gene. Here we report that Mkk3 gene disruption caused a selective defect in the response of fibroblasts to the proinflammatory cytokine tumor necrosis factor, including reduced p38 MAP kinase activation and cytokine expression. These data demonstrate that the MKK3 protein kinase is a critical component of a tumor necrosis factor-stimulated signaling pathway that causes increased expression of inflammatory cytokines.

The RIIβ regulatory subunit of protein kinase A binds to cAMP response element: An alternative cAMP signaling pathway

cAMP, through the activation of cAMP-dependent protein kinase (PKA), is involved in transcriptional regulation. In eukaryotic cells, cAMP is not considered to alter the binding affinity of CREB/ATF to cAMP-responsive element (CRE) but to induce serine phosphorylation and consequent increase in transcriptional activity. In contrast, in prokaryotic cells, cAMP enhances the DNA binding of the catabolite repressor protein to regulate the transcription of several operons. The structural similarity of the cAMP binding sites in catabolite repressor protein and regulatory subunit of PKA type II (RII) suggested the possibility of a similar role for RII in eukaryotic gene regulation. Herein we report that RIIβ subunit of PKA is a transcription factor capable of interacting physically and functionally with a CRE. In contrast to CREB/ATF, the binding of RIIβ to a CRE was enhanced by cAMP, and in addition, RIIβ exhibited transcriptional activity as a Gal4-RIIβ fusion protein. These experiments identify RIIβ as a component of an alternative pathway for regulation of CRE-directed transcription in eukaryotic cells.

Role of p38 mitogen-activated protein kinase in HIV type 1 production in vitro

The proinflammatory cytokines interleukin (IL)-1 and tumor necrosis factor (TNF) promote HIV type 1 viral replication in vitro. In the present studies, HIV production was increased in the macrophagic U1 cell line expressing the HIV genome after exposure to IL-1β, osmotic stress, or surface adhesion, suggesting a confluence of signaling pathways for proinflammatory cytokines and cell stressors. The p38 mitogen-activated protein kinase (MAPK) mediates both cytokine and stress responses; thus the role of this kinase in HIV production was investigated. HIV production as measured by p24 antigen correlated with changes in the expression of a specific (non-alpha) isoform of p38 MAPK. In the presence of a specific p38 MAPK inhibitor (p38 inh), IL-1β-induced HIV production was suppressed by more than 90% and IL-1β-induced IL-8 production was suppressed completely, both with IC50 of 0.01 μM. p38 inhibition blocked cell-associated p24 antigen and secreted virus to a similar extent. The p38 inh also decreased constitutive HIV production in freshly infected peripheral blood mononuclear cells by up to 50% (P < 0.05). Interruption of p38 MAPK activity represents a viable target for inhibition of HIV.

Resistance gene N-mediated de novo synthesis and activation of a tobacco mitogen-activated protein kinase by tobacco mosaic virus infection

Salicylic acid-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK), two distinct members of the mitogen-activated protein (MAP) kinase family, are activated in tobacco resisting infection by tobacco mosaic virus (TMV). WIPK activation by TMV depends on the disease-resistance gene N because infection of susceptible tobacco not carrying the N gene failed to activate WIPK. Activation of WIPK required not only posttranslational phosphorylation but also a preceding rise in its mRNA and de novo synthesis of WIPK protein. The induction by TMV of WIPK mRNA and protein also occurred systemically. Its activation at the mRNA, protein, and enzyme levels was independent of salicylic acid. The regulation of WIPK at multiple levels by an N gene-mediated signal(s) suggests that this MAP kinase may be an important component upstream of salicylic acid in the signal-transduction pathway(s) leading to local and systemic resistance to TMV.

Protein kinase mutants of human ATR increase sensitivity to UV and ionizing radiation and abrogate cell cycle checkpoint control

In fission yeast, the rad3 gene product plays a critical role in sensing DNA structure defects and activating damage response pathways. A structural homologue of rad3 in humans (ATR) has been identified based on sequence similarity in the protein kinase domain. General information regarding ATR expression, protein kinase activity, and cellular localization is known, but its function in human cells remains undetermined. In the current study, the ATR protein was examined by gel filtration of protein extracts and was found to exist predominantly as part of a large protein complex. A kinase-inactivated form of the ATR gene was prepared by site-directed mutagenesis and was used in transfection experiments to probe the function of this complex. Introduction of this kinase-dead ATR into a normal fibroblast cell line, an ATM-deficient fibroblast line derived from a patient with ataxia–telangiectasia, or a p53 mutant cell line all resulted in significant losses in cell viability. Clones expressing the kinase-dead ATR displayed increased sensitivity to x-rays and UV and a loss of checkpoint control. We conclude that ATR functions as a critical part of a protein complex that mediates responses to ionizing and UV radiation in human cells. These responses include effects on cell viability and cell cycle checkpoint control.