Pamela H. Scott

Stress-activated Protein Kinases: Activation, Regulation and Function

The response of cells to extracellular stimuli is mediated in part by a number of intracellular kinase and phosphatase enzymes. Within this area of research the activation of the p42 and p44 isoforms of mitogen-activated protein (MAP) kinases have been extensively described and characterised as central components of the signal transduction pathways stimulated by both growth factors and G-protein-coupled receptor agonists. Signaling events mediated by these kinases are fundamental to cellular functions such as proliferation and differentiation. More recently, homologues of the p42 and p44 isoforms of MAP kinase have been described, namely the stress-activated protein kinases (SAPKs) or alternatively the c-jun N-terminal kinases (JNKs) and p38 MAP kinase (the mammalian homologue of yeast HOG1). These MAP kinase homologues are integral components of parallel MAP kinase cascades activated in response to a number of cellular stresses including inflammatory cytokines (e.g., Interleukin-1 (Il-1) and tumour necrosis factor-alpha (TNF-alpha), heat and chemical shock, bacterial endotoxin and ischaemia/cellular ATP depletion. Activation of these MAP kinase homologues mediates the transduction of extracellular signals to the nucleus and are pivotal events in the regulation of the transcription events that determine functional outcome in response to such stresses. In this review we highlight the identification and characterisation of the stress-activated MAP kinase homologues, their role as components of parallel MAP kinase pathways and the regulation of cellular responses following exposure to cellular stress.

Phosphatidylinositol 3-kinase mediates mitogen-induced human airway smooth muscle cell proliferation.

Hypertrophy and hyperplasia of airway smooth muscle (ASM) are important pathological features that contribute to airflow obstruction in chronic severe asthma. Despite considerable research effort, the cellular mechanisms that modulate ASM growth remain unknown. Recent evidence suggests that mitogen-induced activation of phosphoinositide (PI)-specific phospholipase C (PLC) and PI-dependent calcium mobilization are neither sufficient nor necessary to stimulate human ASM proliferation. In this study, we identify phosphatidylinositol (PtdIns) 3-kinase as a key regulator of human ASM proliferation. Pretreatment of human ASM with the PtdIns 3-kinase inhibitors wortmannin and LY-294002 significantly reduced thrombin- and epidermal growth factor (EGF)-induced DNA synthesis (IC(50) approximately 10 nM and approximately 3 microM, respectively). In separate experiments, wortmannin and LY-294002 markedly inhibited PtdIns 3-kinase and 70-kDa S6 protein kinase (pp70(S6k)) activation induced by stimulation of human ASM cells with EGF and thrombin but had no effect on EGF- and thrombin-induced p42/p44 mitogen-activated protein kinase (MAPK) activation. The specificity of wortmannin and LY-294002 was further suggested by the demonstrated inability of these compounds to alter thrombin-induced calcium transients, total PI hydrolysis, or basal cAMP levels. Transient expression of constitutively active PtdIns 3-kinase (p110*) activated pp70(S6k), whereas a dominant-negative PtdIns 3-kinase (Deltap85) blocked EGF- and thrombin-stimulated pp70(S6k) activity. Collectively, these data suggest that activation of PtdIns 3-kinase is required for the mitogenic effect of EGF and thrombin in human ASM cells. Further investigation of the role of PtdIns 3-kinase may offer new therapeutic approaches in the treatment of diseases characterized by smooth muscle cell hyperplasia such as asthma and chronic bronchitis.Hypertrophy and hyperplasia of airway smooth muscle (ASM) are important pathological features that contribute to airflow obstruction in chronic severe asthma. Despite considerable research effort, the cellular mechanisms that modulate ASM growth remain unknown. Recent evidence suggests that mitogen-induced activation of phosphoinositide (PI)-specific phospholipase C (PLC) and PI-dependent calcium mobilization are neither sufficient nor necessary to stimulate human ASM proliferation. In this study, we identify phosphatidylinositol (PtdIns) 3-kinase as a key regulator of human ASM proliferation. Pretreatment of human ASM with the PtdIns 3-kinase inhibitors wortmannin and LY-294002 significantly reduced thrombin- and epidermal growth factor (EGF)-induced DNA synthesis (IC50 ∼10 nM and ∼3 μM, respectively). In separate experiments, wortmannin and LY-294002 markedly inhibited PtdIns 3-kinase and 70-kDa S6 protein kinase (pp70S6k) activation induced by stimulation of human ASM cells with EGF and thrombin but had no effect on EGF- and thrombin-induced p42/p44 mitogen-activated protein kinase (MAPK) activation. The specificity of wortmannin and LY-294002 was further suggested by the demonstrated inability of these compounds to alter thrombin-induced calcium transients, total PI hydrolysis, or basal cAMP levels. Transient expression of constitutively active PtdIns 3-kinase (p110*) activated pp70S6k, whereas a dominant-negative PtdIns 3-kinase (Δp85) blocked EGF- and thrombin-stimulated pp70S6kactivity. Collectively, these data suggest that activation of PtdIns 3-kinase is required for the mitogenic effect of EGF and thrombin in human ASM cells. Further investigation of the role of PtdIns 3-kinase may offer new therapeutic approaches in the treatment of diseases characterized by smooth muscle cell hyperplasia such as asthma and chronic bronchitis.

The mitogen‐activated protein (MAP) kinase ERK induces tRNA synthesis by phosphorylating TFIIIB

RNA polymerase (pol) III transcription increases within minutes of serum addition to growth‐arrested fibroblasts. We show that ERK mitogen‐activated protein kinases regulate pol III output by directly binding and phosphorylating the BRF1 subunit of transcription factor TFIIIB. Blocking the ERK signalling cascade inhibits TFIIIB binding to pol III and to transcription factor TFIIIC2. Chromatin immunoprecipitation shows that the association of BRF1 and pol III with tRNALeu genes in cells decreases when ERK is inactivated. Furthermore, mutation of an ERK docking domain or phosphoacceptor site in BRF1 prevents serum induction of pol III transcription. These data identify a novel target for ERK, and suggest that its ability to stimulate biosynthetic capacity and growth involves direct transcriptional activation of tRNA and 5S rRNA genes.

CK2 Forms a Stable Complex with TFIIIB and Activates RNA Polymerase III Transcription in Human Cells

ABSTRACT CK2 is a highly conserved protein kinase with growth-promoting and oncogenic properties. It is known to activate RNA polymerase III (PolIII) transcription in Saccharomyces cerevisiae and is shown here to also exert a potent effect on PolIII in mammalian cells. Peptide and chemical inhibitors of CK2 block PolIII transcription in human cell extracts. Furthermore, PolIII transcription in mammalian fibroblasts is decreased significantly when CK2 activity is compromised by chemical inhibitors, antisense oligonucleotides, or kinase-inactive mutants. Coimmunoprecipitation and cofractionation show that endogenous human CK2 associates stably and specifically with the TATA-binding protein-containing factor TFIIIB, which brings PolIII to the initiation site of all class III genes. Serum stimulates TFIIIB phosphorylation in vivo, an effect that is diminished by inhibitors of CK2. Binding to TFIIIC2 recruits TFIIIB to most PolIII promoters; this interaction is compromised specifically by CK2 inhibitors. The data suggest that CK2 stimulates PolIII transcription by binding and phosphorylating TFIIIB and facilitating its recruitment by TFIIIC2. CK2 also activates PolI transcription in mammals and may therefore provide a mechanism to coregulate the output of PolI and PolIII. CK2 provides a rare example of an endogenous activity that operates on the PolIII system in both mammals and yeasts. Such evolutionary conservation suggests that this control may be of fundamental importance.

Overlapping Functions of the pRb Family in the Regulation of rRNA Synthesis

ABSTRACT The “pocket” proteins pRb, p107, and p130 are a family of negative growth regulators. Previous studies have demonstrated that overexpression of pRb can repress transcription by RNA polymerase (Pol) I. To assess whether pRb performs this role under physiological conditions, we have examined pre-rRNA levels in cells from mice lacking either pRb alone or combinations of the three pocket proteins. Pol I transcription was unaffected in pRb-knockout fibroblasts, but specific disruption of the entire pRb family deregulated rRNA synthesis. Further analysis showed that p130 shares with pRb the ability to repress Pol I transcription, whereas p107 is ineffective in this system. Production of rRNA is abnormally elevated in Rb−/−p130−/− fibroblasts. Furthermore, overexpression of p130 can inhibit an rRNA promoter both in vitro and in vivo. This reflects an ability of p130 to bind and inactivate the upstream binding factor, UBF. The data imply that rRNA synthesis in living cells is subject to redundant control by endogenous pRb and p130.

Retinoblastoma Protein Disrupts Interactions Required for RNA Polymerase III Transcription

ABSTRACT The retinoblastoma protein (RB) has been shown to suppress RNA polymerase (Pol) III transcription in vivo (R. J. White, D. Trouche, K. Martin, S. P. Jackson, and T. Kouzarides, Nature 382:88–90, 1996). This regulation involves interaction with TFIIIB, a multisubunit factor that is required for the expression of all Pol III templates (C. G. C. Larminie, C. A. Cairns, R. Mital, K. Martin, T. Kouzarides, S. P. Jackson, and R. J. White, EMBO J. 16:2061–2071, 1997; W.-M. Chu, Z. Wang, R. G. Roeder, and C. W. Schmid, J. Biol. Chem. 272:14755–14761, 1997). However, it has not been established why RB binding to TFIIIB results in transcriptional repression. For several Pol II-transcribed genes, RB has been shown to inhibit expression by recruiting histone deacetylases, which are thought to decrease promoter accessibility. We present evidence that histone deacetylases exert a negative effect on Pol III activity in vivo. However, RB remains able to regulate Pol III transcription in the presence of the histone deacetylase inhibitor trichostatin A. Instead, RB represses by disrupting interactions between TFIIIB and other components of the basal Pol III transcription apparatus. Recruitment of TFIIIB to most class III genes requires its binding to TFIIIC2, but this can be blocked by RB. In addition, RB disrupts the interaction between TFIIIB and Pol III that is essential for transcription. The ability of RB to inhibit these key interactions can explain its action as a potent repressor of class III gene expression.

TFIIIB is phosphorylated, disrupted and selectively released from tRNA promoters during mitosis in vivo

Mitosis involves a generalized repression of gene expression. In the case of RNA polymerase III transcription, this is due to phosphorylation‐mediated inactivation of TFIIIB, an essential complex comprising the TATA‐binding protein TBP and the TAF subunits Brf1 and Bdp1. In HeLa cells, this repression is mediated by a mitotic kinase other than cdc2–cyclin B and is antagonized by protein phosphatase 2A. Brf1 is hyperphosphorylated in metaphase‐arrested cells, but remains associated with promoters in condensed chromosomes, along with TBP. In contrast, Bdp1 is selectively released. Repression can be reversed by raising the concentration of Brf1 or Bdp1. The data support a model in which hyperphosphorylation disrupts TFIIIB during mitosis, compromising its ability to support transcription.

Regulation of RNA polymerase III transcription during hypertrophic growth.

The cell division‐independent growth of terminally differentiated cardiomyocytes is commonly associated with cardiovascular disease. We demonstrate that it is accompanied by a substantial rise in transcription by RNA polymerase (pol) III, which produces essential components of the biosynthetic apparatus, including 5S rRNA and tRNAs. This increase in transcription is achieved by changes in both the activity and level of the essential pol III‐specific transcription factor TFIIIB. Erk and c‐Myc, which directly activate TFIIIB in proliferating fibroblasts, also induce pol III transcription in growing cardiomyocytes. Furthermore, hypertrophic stimulation increases expression of the essential TFIIIB subunit Brf1, an effect not seen when fibroblasts proliferate. Erk mediates this induction of Brf1 expression and therefore contributes in at least two ways to pol III transcriptional activation during hypertrophy. Increased production of tRNA and 5S rRNA will contribute to the enhanced translational capacity required to sustain hypertrophic growth.

RNA polymerase III transcription: its control by tumor suppressors and its deregulation by transforming agents.

The level of RNA polymerase (pol) III transcription is tightly linked to the rate of growth; it is low in resting cells and increases following mitogenic stimulation. When mammalian cells begin to proliferate, maximal pol III activity is reached shortly before the G1/S transition; it then remains high throughout S and G2 phases. Recent data suggest that the retinoblastoma protein RB and its relatives p107 and p130 may be largely responsible for this pattern of expression. During G0 and early G1 phase, RB and p130 bind and repress the pol III-specific factor TFIIIB; shortly before S phase they dissociate from TFIIIB, allowing transcription to increase. At the end of interphase, when cells enter mitosis, pol III transcription is again suppressed; this mitotic repression is achieved through direct phosphorylation of TFIIIB. Thus, pol III transcription levels fluctuate as mammalian cells cycle, being high in S and G2 phases and low during mitosis and early G1. In addition to this cyclic regulation, TFIIIB can be bound and repressed by the tumor suppressor p53. Conversely, it is a target for activation by several viruses, including SV40, HBV, and HTLV-1. Some viruses also increase the activity of a second pol III-specific factor called TFIIIC. A large proportion of transformed and tumor cell types express abnormally high levels of pol III products. This may be explained, at least in part, by the very high frequency with which RB and p53 become inactivated during neoplastic transformation; loss of function of these cardinal tumor suppressors may release TFIIIB from key restraints that operate in normal cells.

Hypoxic stress suppresses RNA polymerase III recruitment and tRNA gene transcription in cardiomyocytes

RNA polymerase (pol) III transcription decreases when primary cultures of rat neonatal cardiomyocytes are exposed to low oxygen tension. Previous studies in fibroblasts have shown that the pol III-specific transcription factor IIIB (TFIIIB) is bound and regulated by the proto-oncogene product c-Myc, the mitogen-activated protein kinase ERK and the retinoblastoma tumour suppressor protein, RB. The principal function of TFIIIB is to recruit pol III to its cognate gene template, an activity that is known to be inhibited by RB and stimulated by ERK. We demonstrate by chromatin immunoprecipitation (ChIP) that c-Myc also stimulates pol III recruitment by TFIIIB. However, hypoxic conditions cause TFIIIB dissociation from c-Myc and ERK, at the same time as increasing its interaction with RB. Consistent with this, ChIP assays indicate that the occupancy of tRNA genes by pol III is significantly reduced, whereas promoter binding by TFIIIB is undiminished. The data suggest that hypoxia can inhibit pol III transcription by altering the interactions between TFIIIB and its regulators and thus compromising its ability to recruit the polymerase. These effects are independent of cell cycle changes.