Kinase-dependent Mechanism Research Articles

Brain-Derived Neurotrophic Factor Regulation of Retinal Growth Cone Filopodial Dynamics Is Mediated through Actin Depolymerizing Factor/Cofilin

The molecular mechanisms by which neurotrophins regulate growth cone motility are not well understood. This study investigated the signaling involved in transducing BDNF-induced increases of filopodial dynamics. Our results indicate that BDNF regulates filopodial length and number through a Rho kinase-dependent mechanism. Additionally, actin depolymerizing factor (ADF)/cofilin activity is necessary and sufficient to transduce the effects of BDNF. Our data indicate that activation of ADF/cofilin mimics the effects of BDNF on filopodial dynamics, whereas ADF/cofilin inactivity blocks the effects of BDNF. Furthermore, BDNF promotes the activation of ADF/cofilin by reducing the phosphorylation of ADF/cofilin. Although inhibition of myosin II also enhances filopodial length, our results indicate that BDNF signaling is independent of myosin II activity and that the two pathways result in additive effects on filopodial length. Thus, filopodial extension is regulated by at least two independent mechanisms. The BDNF-dependent pathway works via regulation of ADF/cofilin, independently of myosin II activity.

Mechanisms of Resistance to the FLT3-Tyrosine Kinase Inhibitor PKC412 in Patients with AML.

The FLT3 receptor tyrosine kinase is expressed in 70-90% of cases of AML. Up to 35% of patients with AML show mutations in the JM-region or kinase domain of FLT3. These lead to autophosphorylation promoting ligand-independent cell proliferation and inhibition of apoptosis. Treatment with FLT3 tyrosine kinase inhibitors (TKI) is a promising tool in therapy of AML. Preliminary results investigating the FLT3-TKI PKC412 in patients with relapsed/refractory AML revealed that 11/15 patients (73%) with mutated FLT3 and 16/46 patients (35%) with WT FLT3 showed a >50% blast response in peripheral blood (Estey E et al. Blood . 2003; 102:919a). Despite its remarkable efficacy in reducing the leukemic clone, remissions upon single agent therapy using PKC412 tend to be short and secondary resistance occurs rather rapidly. So far, very little is known on the mechanisms of resistance to therapy using FLT3-TKIs. Therefore, we sought to characterize the molecular mechanisms involved. Overall PB and BM samples from 5 patients undergoing single agent therapy with PKC412 were analyzed. Three patients revealed reactivation of FLT3-kinase at relapse. Unchanged protein levels of Flt3 as detected by Western blotting before therapy and at relapse showed that amplification of FLT3 does not appear to play a role in development of secondary resistance. PKC412 plasma levels, as measured by HPLC, were similar in patients with and without reactivation of the kinase. Therefore, we examined the bioactivity of plasma obtained at relapse to inhibit phosphorylation of FLT3 in MV4-11 cells harboring FLT3-ITD. In two patients, plasma inhibited FLT3-tyrosine phosphorylation at the time of remission as well as at relapse, while in one patient, plasma was not able to inhibit FLT3-phosphorylation. Therefore, in this patient, an inhibitory activity in serum appeared to be operating. A potential candidate for this inhibitory activity is alpha1-glycoprotein (AGP) that was shown previously to mediate resistance to STI571 treatment. Our experiments showed that AGP is indeed able to inhibit bioactivity of PKC412 in-vitro. However, AGP-levels monitored in PB samples from patients included in this analysis did not show elevated plasma levels at relapse. Ex-vivo treatment of primary blasts with PKC412 at relapse showed that in one patient, blasts were still sensitive to PKC412. In two other patients, blasts showed resistance using a wide dose range of PKC412. Therefore, in these patients, cDNA sequencing of FLT3 was performed. In one patient, we identified a point mutation leading to an aminoacid exchange within the N-lobe of the FLT3-kinase domain at relapse. This mutation was not present at start of PKC412 therapy. Experiments analyzing the functional role of the novel mutation in the hematopoietic cell line 32D are in progress and will be presented. In conclusion, we have preliminarily identified examples of kinase-dependent and independent mechanisms operating in clinical resistance to PKC412.

BCR/ABL Oncogenic Kinase Stimulates DNA Oxidative Damage To Induce Self-Mutagenesis Causing Resistance to Imatinib Mesylate.

Imatinib mesylate (IM) [Gleevec, STI571], a selective inhibitor of ABL kinase activity, revolutionized the treatment of BCR/ABL-positive leukemias. Unfortunately, resistance to IM has become a rising clinical problem. Although the resistance could be achieved by various mechanisms, mutations in the BCR/ABL kinase domain seem to play a major role. Identification of the mechanisms causing these mutations may have a significant impact on the effectiveness of IM therapy. Mutations usually result from enhanced DNA damage and/or deregulated mechanisms responsible for DNA repair. Much endogenous DNA damage arises from intermediates of oxygen reduction generating potentially damaging molecules called reactive oxygen species (ROS). We report here that BCR/ABL-mediated transformation is associated with the kinase-dependent stimulation of ROS production and elevation of DNA oxidative damage. Immunofluorescence visualized more 8-oxoG nuclear lesions and enzymatic assays detected more oxidized bases in DNA in BCR/ABL-transformed cells (CML patient cells and leukemic cell lines) in comparison to normal cells. To examine the potential effect of ROS on mutations in BCR/ABL kinase freshly transformed 32D-BCR/ABL cells (no mutations in BCR/ABL) were cultured in the absence (control) or presence of IM (to inhibit BCR/ABL kinase and ROS production), PDTC (ROS scavenger) or IM+BSO (to inhibit BCR/ABL kinase and elevate ROS because BSO prevents its decay) for 8 weeks in medium supplemented with IL-3; then IM-resistance was examined. About 0.1% of control cells displayed IM-resistance, which was dramatically reduced in the presence of IM or PDTC, implicating BCR/ABL kinase-dependent ROS-mediated mechanism. IM+BSO treatment resulted in partial inhibition of IM-resistance, suggesting that another BCR/ABL-induced mechanism may collaborate with ROS in mutagenesis. IM-resistance was associated with mutations in the sequence encoding bcr/abl kinase. The majority of detected mutations were GC→AT transitions causing amino acid substitutions; most of them are already proven to be clinically relevant. Similar conditions were applied to study the mutagenesis in Na+K+ ATPase (mutations may cause the resistance to ouabain) in 32D parental and freshly transformed 32D-BCR/ABL cells. BCR/ABL increased the ouabain resistance rate ~12-times, which was associated with mutations in the alpha1 subunit of Na+K+ ATPase, essential for ouabain resistance. Again the majority of mutations were GC→AT transitions. To confirm that similar mechanisms of BCR/ABL-dependent mutagenesis operate in living organisms, IM-resistance was examined in SCID mice injected with freshly transformed 32D-BCR/ABL cells and fed with normal or vitamin E-rich (VE) diet (to decrease ROS) for 8 weeks. Leukemia cells harvested from bone marrow and spleen of the mice fed with VE diet displayed ~2 -fold reduction in the frequency of IM-resistance in comparison to the animals obtaining regular food. Mutations in the BCR/ABL kinase were detected in IM-resistant clones. Altogether, these studies suggest that BCR/ABL kinase facilitates ROS-dependent mutagenesis of in different genes, including bcr/abl gene, and that this process is essential for IM-resistance. Early treatment with IM can prevent mutagenesis, but if mutations occur it will select the outgrowth of mutant IM-resistant cells. In addition, reduction of ROS should greatly diminish the mutagenesis in BCR/ABL kinase associated with IM-resistance.

P38 Inhibition attenuates the pro-inflammatory response to C-reactive protein by human peripheral blood mononuclear cells

An active role for C-reactive protein (CRP) in inflammatory vascular diseases has been recently suggested. Monocytes play an important role in vascular pathology and are activated by p38 mitogen activated protein kinase (MAPK) dependent mechanisms in many inflammatory settings. Therefore, we investigated whether CRP directly promotes a pro-inflammatory phenotype in human peripheral blood mononuclear cells (HPBMC) via p38 MAPK signaling. CRP exposure leads to a rapid phosphorylation of p38 MAPK in HPBMC. CRP-induced p38 kinase activity in HPBMC was blocked by treatment with an inhibitor of p38 kinase, SD-282. CRP-induced the expression of tissue factor protein and the secretion of IL-6, IL-8, IL-1β, TNFα and PGE 2. Co-exposure to CRP and SD-282 blocked the secretion of these pro-inflammatory and pro-thrombotic mediators. CRP treatment elevated IL-6, IL-8, IL-1β, TNFα, COX-2 and TF mRNA expression. These effects of CRP also required p38 activity, since SD-282 blocked mRNA induction of each. Taken together these data suggest a mechanistic relationship between p38 MAPK signaling and CRP-induced pro-inflammatory and pro-thrombotic activities in HPBMC. Thus, p38 inhibition may represent a novel approach to attenuate inflammation and its consequences in cardiovascular disease.

HGF promotes survival and growth of maturing sympathetic neurons by PI-3 kinase- and MAP kinase-dependent mechanisms

Hepatocyte growth factor (HGF) is a pleiotrophic factor whose many functions include promoting neuronal survival and growth. Hitherto, these effects have been observed in the presence of other neurotrophic factors like NGF and CNTF, and this requirement for an accessory factor has made it difficult to elucidate the signaling pathways that mediate its survival and growth-enhancing effects. Here, we show that HGF promotes the survival of mature sympathetic neurons of the superior cervical ganglion (SCG) grown at low density in defined medium lacking other neurotrophic factors. This effect was first clearly observed in cultures established from postnatal day 20 (P20) mice and became maximal by P40. HGF also enhanced the growth of neurite arbors from neurons throughout postnatal development and in the adult. HGF treatment resulted in phosphorylation of Akt and ERK1/ERK2. Preventing Akt activation with the phosphatidylinositol-3 (PI-3) kinase inhibitor LY294002 blocked the HGF survival response, and inhibition of ERK activation with the MEK inhibitors PD98059 or U0126 reduced the HGF survival response and the neurite growth-promoting effects of HGF. These results indicate that HGF promotes the survival and growth of maturing sympathetic neurons by both PI-3 kinase- and MAP kinase-dependent mechanisms.

Lipid Rafts Regulate Lipopolysaccharide-induced Activation of Cdc42 and Inflammatory Functions of the Human Neutrophil

Lipid rafts are cholesterol-rich membrane microdomains that are thought to act as coordinated signaling platforms by regulating dynamic, agonist-induced translocation of signaling proteins. They have been described to play a role in multiple prototypical cascades, among them the lipopolysaccharide pathway, and to host multiple signaling proteins, including kinases and low molecular weight G-proteins. Here we report lipopolysaccharide-induced activation of the Rho family GTPase Cdc42, and we show its activation in the human neutrophil to be mediated by a p38 mitogen-activated protein kinase-dependent mechanism. Subcellular fractionation reveals that lipopolysaccharide induces translocation of Cdc42 to lipid rafts, where it and p38 are both found to be activated. By contrast, lipopolysaccharide causes translocation of Rac from the polymorphonuclear leukocyte (PMN) rafts and does not induce its activation. With the use of methyl-beta-cyclodextrin, a cholesterol-depleting agent that reversibly disrupts rafts, we confirm an important regulatory role for rafts in the activation state of p38 and Cdc42 and in the Rho GTPase-dependent functions superoxide anion production and actin polymerization. Methyl-beta-cyclodextrin induces activation of p38 and Cdc42, but not Rac, in the nonstimulated PMN, yet inhibits subsequent lipopolysaccharide-induced activation of p38 and Cdc42. In parallel, methyl-beta-cyclodextrin primes the human PMN for subsequent superoxide release triggered by the formylated bacterial tripeptide formyl-Met-Leu-Phe, and induces actin polymerization in a subcellular distribution distinct from that induced by lipopolysaccharide. In sum, these findings provide evidence for an important regulatory role of cholesterol in both transmission of the lipopolysaccharide signal and the inflammatory phenotype of the human neutrophil.

Fibroblast growth factor-1 improves cardiac functional recovery and enhances cell survival after ischemia and reperfusion: A fibroblast growth factor receptor, protein kinase c, and tyrosine kinase-dependent mechanism

We sought to investigate the role of fibroblast growth factor (FGF)-1 during acute myocardial ischemia and reperfusion. The FGFs display cardioprotective effects during ischemia and reperfusion. We investigated FGF-1-induced cardioprotection during ischemia and reperfusion and the intracellular signaling pathways responsible for these effects in an ex vivo murine setup of myocardial ischemia and reperfusion. Cardiac-specific human FGF-1 overexpression was associated with enhanced post-ischemic hemodynamic recovery and decreased lactate dehydrogenase release during reperfusion. Inhibition of the FGF receptor, protein kinase C (PKC), and tyrosine kinase (TK) resulted in blockade of FGF-1-induced protective effects on cardiac functional recovery and cell death. The overexpression of FGF-1 induces cardioprotection through a pathway that involves the FGF receptor, PKC, and TK.

Control of reproductive processes by growth hormone: Extra- and intracellular mechanisms

Recent data on the association between growth hormone (GH) and male and female reproductive processes, as well as the effects of GH on these processes and on some reproductive and non-reproductive disorders, and possible extra- and intracellular mediators of its action are reviewed. The available data suggest that GH is an important endocrine and autocrine/paracrine regulator of reproduction. It controls proliferation, apoptosis, growth and differentiation and the secretory and generative activities of different reproductive organs. It also regulates their response to gonadotrophin-releasing hormone (GnRH) and gonadotropins. Despite the effects of GH on the IGF/IGFBP (insulin-like growth factor binding protein) system, oxytocin, steroids, activin, gonadotropin and gonadotropin receptors, the majority of GH’s actions on the reproductive processes are probably mediated not by these substances but by specific GH receptors acting through cAMP/protein kinase A, protein kinase G, tyrosine kinase-, MAP kinase and CDC2 kinase-dependent intracellular mechanisms. Although GH treatments can increase the risk of some reproductive and non-reproductive disorders, they may be useful in improving gonadal function, inducing superovulation and in embryo production.

Mechanisms regulating the constitutive activation of the extracellular signal-regulated kinase (ERK) signaling pathway in ovarian cancer and the effect of ribonucleic acid interference for ERK1/2 on cancer cell proliferation.

The ERK1/2 MAPK pathway is a critical signaling system that mediates ligand-stimulated signals for the induction of cell proliferation, differentiation, and cell survival. Studies have shown that this pathway is constitutively active in several human malignancies and may be involved in the pathogenesis of these tumors. In the present study we examined the ERK1/2 pathway in cell lines derived from epithelial and granulosa cell tumors, two distinct forms of ovarian cancer. We show that ERK1 and ERK2 are constitutively active and that this activation results from both MAPK kinase-dependent and independent mechanisms and is correlated with elevated BRAF expression. MAPK phosphatase 1 (MKP-1) expression, which is involved in ERK1/2 deactivation, is down-regulated in the cancer cells, thus further contributing to ERK hyperactivity in these cells. Treatment of these cancer cell lines with the proteasome inhibitor ZLLF-CHO increased MKP-1 but not MKP-2 expression and decreased ERK1/2 phosphorylation. More importantly, silencing of ERK1/2 protein expression using RNA interference led to the complete suppression of tumor cell proliferation. These results provide evidence that the ERK pathway plays a major role in ovarian cancer pathogenesis and that down-regulation of this master signaling pathway is highly effective for the inhibition of ovarian tumor growth.

Testosterone enhances calcium reabsorption by the kidney

The kidney is a target tissue for androgens, but the role of these hormones in the regulation of calcium (Ca 2+) reabsorption remains unclear. The present study examines the effects of testosterone on Ca 2+ transport by the luminal membranes of proximal and distal nephrons of the rabbit kidney. Tubule suspensions were pre-incubated in the presence or absence of the hormone, and 45 Ca 2+ uptake by the luminal membranes was measured using the rapid filtration technique. In the proximal tubules, testosterone did not influence Ca 2+ uptake. In the distal tubules, a 5 min incubation with the hormone increased this uptake with a maximal response at 10 −10 M. Ca 2+ transport by the distal membranes shows a dual kinetics. Testosterone enhanced the V max value of the low affinity component. In an attempt to identify the underlying mechanisms involved in this action, several messenger inhibitors were introduced in the tubule suspension. PD 98059 and U0 126 as well as AG 99 and genistein interfered with the hormone action suggesting the implication of a MEK kinase and a tyrosine kinase. To determine the type of the channels involved in this effect, Ca 2+ uptake was measured in the presence of diltiazem, ω-conotoxin MVIIC and mibefradil, i.e. selective inhibitors of the L-type, P/Q type and T-type channels. An inhibition of Ca 2+ transport was observed exclusively with mibefradil. These results indicate that testosterone enhances Ca 2+ transport by opening a T-type Ca 2+ channel in the distal luminal membrane, via MEK kinase and tyrosine kinase dependent mechanisms.

Streptozotocin directly impairs cardiac contractile function in isolated ventricular myocytes via a p38 map kinase-dependent oxidative stress mechanism

Streptozotocin (STZ) has long been used to induce experimental diabetes mellitus to study diabetic complications such as diabetic cardiomyopathy. However, direct impact of STZ on cardiac function is unknown. This study was designed to evaluate the cardiac contractile effect of STZ in isolated adult rat ventricular myocytes. Contractile properties were assessed with an IonOptix MyoCam system including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR 90 ), and maximal velocities of shortening/relengthening (± d L /d t ). Intracellular Ca 2+ handling was evaluated with the fluorescent dye fura-2. Acute exposure of STZ (10 −9 –10 −5 M) depressed PS, prolonged TR 90 , and decreased electrically stimulated intracellular Ca 2+ rise in a concentration-dependent manner. TPS, ± d L /d t , resting intracellular Ca 2+ level, and intracellular Ca 2+ clearing rate were unaffected. The STZ-induced mechanical alterations were alleviated by the antioxidant vitamin C (100 μM) and the p38 MAP kinase inhibitor SB203580 (1 μM). 2 ′ ,7 ′ -Dichlorofluorescein diacetate staining revealed enhanced production of reactive oxygen species following STZ treatment, which was prevented by either vitamin C or SB203580. Collectively, our data provided convincing evidence that the tool drug for experimental diabetes STZ may itself cause deleterious cardiac contractile dysfunction via an oxidative stress and p38 MAP kinase-dependent mechanism. Thus, caution should be taken when assessing diabetic heart complications using STZ-induced diabetic models.

Streptozotocin directly impairs cardiac contractile function in isolated ventricular myocytes via a p38 map kinase-dependent oxidative stress mechanism

Streptozotocin (STZ) has long been used to induce experimental diabetes mellitus to study diabetic complications such as diabetic cardiomyopathy. However, direct impact of STZ on cardiac function is unknown. This study was designed to evaluate the cardiac contractile effect of STZ in isolated adult rat ventricular myocytes. Contractile properties were assessed with an IonOptix MyoCam system including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR 90), and maximal velocities of shortening/relengthening (± d L/d t). Intracellular Ca 2+ handling was evaluated with the fluorescent dye fura-2. Acute exposure of STZ (10 −9–10 −5 M) depressed PS, prolonged TR 90, and decreased electrically stimulated intracellular Ca 2+ rise in a concentration-dependent manner. TPS, ± d L/d t, resting intracellular Ca 2+ level, and intracellular Ca 2+ clearing rate were unaffected. The STZ-induced mechanical alterations were alleviated by the antioxidant vitamin C (100 μM) and the p38 MAP kinase inhibitor SB203580 (1 μM). 2 ′,7 ′-Dichlorofluorescein diacetate staining revealed enhanced production of reactive oxygen species following STZ treatment, which was prevented by either vitamin C or SB203580. Collectively, our data provided convincing evidence that the tool drug for experimental diabetes STZ may itself cause deleterious cardiac contractile dysfunction via an oxidative stress and p38 MAP kinase-dependent mechanism. Thus, caution should be taken when assessing diabetic heart complications using STZ-induced diabetic models.

Bacterial DNA induced iNOS expression through MyD88-p38 MAP kinase in mouse primary cultured glial cells

To study the role of bacterial DNA in the immune function of the brain, we examined the effect of CpG–DNA on the inducible nitric oxide synthase (iNOS) expression in mouse primary cultured glial cells. The expression of Toll-like receptor 9 (TLR9), the receptor of bacterial DNA, was detected by RT-PCR. We observed an increase in iNOS mRNA 6 h after CpG–DNA application. The expression of iNOS protein peaked at 12 h and declined thereafter. CpG–DNA increased p38 mitogen-activated protein kinase (MAPK) activation in primary cultured glial cells. SB203580, a specific inhibitor of p38 MAP kinase, inhibited the CpG–DNA-induced iNOS expression. Moreover, CpG–DNA failed to activate p38 MAP kinase and iNOS induction in the primary cultured glial cells prepared from myeloid differentiation factor 88 (MyD88) deficient mice. Therefore, it is suggested that functional receptor for bacterial DNA exists in primary cultured glial cells and CpG–DNA induces iNOS expression via the MyD88-p38 MAP kinase-dependent mechanisms. Thus, the present results point to the important role of bacterial DNA by acting on glial cells to operate brain immune function.

Fatty acid synthase expression is induced by the Epstein-Barr virus immediate-early protein BRLF1 and is required for lytic viral gene expression.

The Epstein-Barr virus (EBV) immediate-early (IE) protein BRLF1 (R) is a transcription factor that induces the lytic form of EBV infection. R activates certain early viral promoters through a direct binding mechanism but induces transcription of the other EBV IE gene, BZLF1 (Z), indirectly through cellular factors binding to a CRE motif in the Z promoter (Zp). Here we demonstrate that R activates expression of the fatty acid synthase (FAS) cellular gene through a p38 stress mitogen-activated protein kinase-dependent mechanism. B-cell receptor engagement of Akata cells also increases FAS expression. The FAS gene product is required for de novo synthesis of the palmitate fatty acid, and high-level FAS expression is normally limited to liver, brain, lung, and adipose tissue. We show that human epithelial tongue cells lytically infected with EBV (from oral hairy leukoplakia lesions) express much more FAS than uninfected cells. Two specific FAS inhibitors, cerulenin and C75, prevent R activation of IE (Z) and early (BMRF1) lytic EBV proteins in Jijoye cells. In addition, cerulenin and C75 dramatically attenuate IE and early lytic gene expression after B-cell receptor engagement in Akata cells and constitutive lytic viral gene expression in EBV-positive AGS cells. However, FAS inhibitors do not reduce lytic viral gene expression induced by a vector in which the Z gene product is driven by a strong heterologous promoter. In addition, FAS inhibitors do not reduce R activation of a naked DNA reporter gene construct driven by the Z promoter (Zp). These results suggest that cellular FAS activity is important for induction of Z transcription from the intact latent EBV genome, perhaps reflecting the involvement of lipid-derived signaling pathways or palmitoylated proteins. Furthermore, using FAS inhibitors may be a completely novel approach for blocking the lytic form of EBV replication.

1002-100 Tyrosine kinase-dependent mechanism occurs downstream of protein kinase C activation in the pharmacological preconditioning by alpha-1 adrenoceptor stimulation

1002-100 Tyrosine kinase-dependent mechanism occurs downstream of protein kinase C activation in the pharmacological preconditioning by alpha-1 adrenoceptor stimulation

A small molecular activator of cardiac hypertrophy uncovered in a chemical screen for modifiers of the calcineurin signaling pathway.

The calcium, calmodulin-dependent phosphatase calcineurin, regulates growth and gene expression of striated muscles. The activity of calcineurin is modulated by a family of cofactors, referred to as modulatory calcineurin-interacting proteins (MCIPs). In the heart, the MCIP1 gene is activated by calcineurin and has been proposed to fulfill a negative feedback loop that restrains potentially pathological calcineurin signaling, which would otherwise lead to abnormal cardiac growth. In a high-throughput screen for small molecules capable of regulating MCIP1 expression in muscle cells, we identified a unique 4-aminopyridine derivative exhibiting an embedded partial structural motif of serotonin (5-hydroxytryptamine, 5-HT). This molecule, referred to as pyridine activator of myocyte hypertrophy, acts as a selective agonist for 5-HT(2A/2B) receptors and induces hypertrophy of cardiac muscle cells through a signaling pathway involving calcineurin and a kinase-dependent mechanism that inactivates class II histone deacetylases, which act as repressors of cardiac growth. These findings identify MCIP1 as a downstream target of 5-HT(2A/2B) receptor signaling in cardiac muscle cells and suggest possible uses for 5-HT(2A/2B) agonists and antagonists as modulators of cardiac growth and gene expression.

Arginine stimulates intestinal cell migration through a focal adhesion kinase dependent mechanism

Arginine stimulates intestinal cell migration through a focal adhesion kinase dependent mechanism

Dissection of tumor-necrosis factor-α inhibition of long-term potentiation (LTP) reveals a p38 mitogen-activated protein kinase-dependent mechanism which maps to early—but not late—phase LTP

The pro-inflammatory cytokine tumor-necrosis factor-α (TNF-α) is elevated in several neuropathological states that are associated with learning and memory deficits. Previous work has reported that TNF-α inhibits the induction of LTP in areas CA1 [Neurosci Lett 146 (1992) 176] and dentate gyrus [Neurosci Lett 203 (1996) 17]. The mechanism(s) underlying this process of inhibition have not to date been addressed. Here, we show that perfusion of TNF-α prior to long-term potentiation (LTP) inducing stimuli inhibited LTP, and that in late-LTP (3 h post-tetanus) a depression in synaptic field recordings was observed (68±5%, n=6 versus control 175±7%, n=6, P<0.001). We investigated the involvement of the mitogen-activated protein kinase (MAPK) p38 in the inhibition of LTP by TNF-α as p38 MAPK has previously been shown to be involved in interleukin-1β inhibition of LTP in the dentate gyrus [Neuroscience 93 (1999b) 57]. Perfusion of TNF-α led to an increase in the levels of phosphorylated p38 MAPK detectable in the granule cells of the dentate gyrus. The p38 MAPK inhibitor SB 203580 (1 μM) was found by itself to have no significant effect on either early or late phase LTP in the dentate gyrus. SB 203580 was found to significantly reverse the inhibition of early LTP by TNF-α (SB/TNF-α 174±5%, n=6 versus TNF-α 120±7%, n=6, P<0.001, 1 h post-tetanus) to values comparable to control LTP (control 175±7%, n=6). Interestingly however, the depressive effects of TNF-α on late LTP (2–3 h) were clearly not attenuated by p38 MAPK inhibition (SB/TNF-α 132±5%, n=6 versus control LTP 175±7%, n=6, P<0.001, 3 h post-tetanus). This work suggests that TNF-α inhibition of LTP represents a biphasic response, a p38 MAPK-dependent phase that coincides with the early phase of LTP and a p38 MAPK independent phase that temporally maps to late LTP.

Surface Expression of the Netrin Receptor UNC5H1 Is Regulated through a Protein Kinase C-Interacting Protein/Protein Kinase-Dependent Mechanism

Netrin-1 is a bifunctional guidance cue that directs migrating neurons and axons based on specific receptors expressed on the cell surface. Attraction occurs through the receptor Deleted in Colorectal Cancer (DCC) and repulsion occurs through a receptor complex of DCC and UNC5H, the vertebrate homolog to Caenorhabditis elegans UNC-5, but how the specific surface expression of these receptors is achieved remains unknown. Here, we demonstrate that surface expression of UNC5H1 is regulated in neurons by protein interacting with C kinase-1 (PICK1) and protein kinase C (PKC), and show that one mechanism by which cells control their response to netrin-1 is by changing the surface availability of receptors. We identified PICK1 as a binding partner for UNC5H1 using the yeast two-hybrid system and found that the extreme three C-terminal amino acids of UNC5H1 interact with the PSD-95/Dlg/ZO-1 (PDZ) domain of PICK1. Coexpression of UNC5H1 and PICK1 in heterologous cells results in the recruitment of PICK1 to UNC5H1 clusters. Endogenous UNC5H1 and PICK1 coimmunoprecipitate from extracts of cultured hippocampal neurons and P4 cortices, and immunohistochemistry shows that UNC5H1, PICK1, and PKC are all present in growth cones. PKC activation induces the formation of UNC5H1/PICK1/PKC complexes and leads to the specific removal of UNC5H1, but not DCC, from the surface of neurons and growth cones via a PICK1/PKC-dependent mechanism. Lastly, we demonstrate that activating PKC, which decreases surface expression of UNC5H1, inhibits netrin-1-dependent collapse of hippocampal growth cones. Together, our results suggest that by regulating the surface expression of UNC5Hs, an axon can modulate its repellent response to netrin-1.

A Degrading Job

Checkpoint responses lead to cell cycle arrest following DNA damage, which allows time for DNA repair. In Saccharomyces cerevisiae , Rad53 kinase is critical to this arrest and also for deoxyribonucleotide triphosphate production during S phase. Most of Rad53's functions depend on its activation by the upstream checkpoint kinase Mec1; however, rad53 deletion mutants show a slow-growth phenotype, whereas mec1 deletion mutants do not. During S phase, histone synthesis is coordinated with DNA replication, and newly synthesized histones are deposited behind the replication fork by chaperones (see Quivy and Almouzni). Gunjan and Verreault investigated the role of Rad3, which can interact with the histone chaperone Asf1, in histone metabolism in wild-type and mutant strains of S. cerevisiae . Yeast lacking--or expressing kinase-deficient--Rad3 were hypersensitive to histone overexpression (which is toxic), whereas yeast lacking Mec1 were not. Western analysis of tagged overexpressed histones or of fractionated or chaperone-bound endogenous histones indicated that degradation of excess histones that were not packaged into chromatin was impaired in rad53 mutants. Therefore, rad53 mutant cells accumulated excess histones. Histone overexpression increased sensitivity to DNA damage, without directly damaging DNA, and reducing histone gene dosage suppressed the rad53 mutant phenotype of sensitivity to DNA damage, slow growth, and chromosome loss. Rad53 formed a complex with histones that did not include Asf1 or any other known histone chaperone proteins; complex formation was affected by Rad53 kinase activity. Thus, Rad53 appears to have a novel function of monitoring and maintaining histone levels that is independent of Mec1 and its activation by DNA damage. A. Gunjan, A. Verreault, A Rad53 kinase-dependent surveillance mechanism that regulates histone protein levels in S. cerevisiae . Cell 115 , 537-549 (2003). [Online Journal] J.-P. Quivy, G. Almouzni, Rad53: A controller ensuring the fine-tuning of histone levels. Cell 115 , 508-510 (2003). [Online Journal]