Molecular effectors and modulators of hypericin-mediated cell death in bladder cancer cells.

Photodynamic therapy (PDT) is an anticancer approach utilizing a light-absorbing molecule and visible light irradiation to generate, in the presence of O(2), cytotoxic reactive oxygen species, which cause tumor ablation. Given that the photosensitizer hypericin is under consideration for PDT treatment of bladder cancer we used oligonucleotide microarrays in the T24 bladder cancer cell line to identify differentially expressed genes with therapeutic potential. This study reveals that the expression of several genes involved in various metabolic processes, stress-induced cell death, autophagy, proliferation, inflammation and carcinogenesis is strongly affected by PDT and pinpoints the coordinated induction of a cluster of genes involved in the unfolded protein response pathway after endoplasmic reticulum stress and in antioxidant response. Analysis of PDT-treated cells after p38(MAPK) inhibition or silencing unraveled that the induction of an important subset of differentially expressed genes regulating growth and invasion, as well as adaptive mechanisms against oxidative stress, is governed by this stress-activated kinase. Moreover, p38(MAPK) inhibition blocked autonomous regrowth and migration of cancer cells escaping PDT-induced cell death. This analysis identifies new molecular effectors of the cancer cell response to PDT opening attractive avenues to improve the therapeutic efficacy of hypericin-based PDT of bladder cancer.

Deficiency in apoptotic effectors Bax and Bak reveals an autophagic cell death pathway initiated by photodamage to the endoplasmic reticulum.

Efficient exploitation of cell death mechanisms for therapeutic purpose requires the identification of the molecular events committing cancer cells to death and the intracellular elements of the pro-death and pro-survival machinery activated in response to the anticancer therapy. Photodynamic therapy (PDT) is a paradigm of anticancer therapy utilizing the generation of reactive oxygen species to kill the cancer cells. In this study we have identified the photodamage to the sarco(endo)plasmic-reticulum Ca(2+)-ATPase (SERCA) pump and consequent loss in the ER-Ca(2+) homeostasis as the most apical molecular events leading to cell death in hypericin-photosensitized cells. Downstream of the ER-Ca(2+) emptying, both caspase-dependent and -independent pathways are activated to ensure cell demise. The induction of apoptosis as a cell death modality is dependent on the availability of proapopototic Bax and Bak proteins, which are essential effectors of the mitochondrial outer membrane permeabilization (MOMP) and subsequent caspase activation. In Bax(-/-)/Bak(-/-) cells a nonapoptotic pathway dependent on sustained autophagy commits the oxidatively damaged cells to death. These results argue that the decision to die in this paradigm of oxidative stress is taken upstream of Bax-dependent MOMP and that the irreversible photodamage to the ER induced by hypericin-PDT acts as a trigger for an autophagic cell death pathway in apoptosis-deficient cells.

Elucidation of the tumoritropic principle of hypericin.

Hypericin is a potent agent in the photodynamic therapy of cancers. To better understand its tumoritropic behaviour, we evaluated the major determinants of the accumulation and dispersion of hypericin in subcutaneously growing mouse tumours. A rapid exponential decay in tumour accumulation of hypericin as a function of tumour weight was observed for each of the six tumour models investigated, and a similar relationship was found between tumour blood flow and tumour weight. Moreover, there was a close correlation between the higher hypericin uptake in RIF-1 tumours compared to R1 tumours and tumour vessel permeability. To define the role of lipoproteins in the transport of hypericin through the interstitial space, we performed a visual and quantitative analysis of the colocalization of hypericin and DiOC18-labelled lipoproteins in microscopic fluorescent overlay images. A coupled dynamic behaviour was found early after injection (normalised fluorescence intensity differences were on the whole less than 10%), while a shifted pattern in localisation of hypericin and DiOC18 was seen after 24 h, suggesting that during its migration through the tumour mass, hypericin is released from the lipoprotein complex. In conclusion, we were able to show that the tumour accumulation of hypericin is critically determined by a combination of biological (blood flow, vessel permeability) and physicochemical elements (affinity for interstitial constituents).

Neonatal neuronal overexpression of glycogen synthase kinase-3 beta reduces brain size in transgenic mice.

Glycogen synthase kinase-3beta (GSK-3beta) is important in neurogenesis. Here we demonstrate that the kinase influenced post-natal maturation and differentiation of neurons in vivo in transgenic mice that overexpress a constitutively active GSK-3beta[S9A]. Magnetic resonance imaging revealed a reduced volume of the entire brain, concordant with a nearly 20% reduction in wet brain weight. The reduced volume was most prominent for the cerebral cortex, without however, disturbing the normal cortical layering. The resulting compacted architecture was further demonstrated by an increased neuronal density, by reduced size of neuronal cell bodies and of the somatodendritic compartment of pyramidal neurons in the cortex. No evidence for apoptosis was obtained. The marked overall reduction in the level of the microtubule-associated protein 2 in brain and in spinal cord, did not affect the ultrastructure of the microtubular cytoskeleton in the proximal apical dendrites. The overall reduction in size of the entire CNS induced by constitutive active GSK-3beta caused only very subtle changes in the psychomotoric ability of adult and ageing GSK-3beta transgenic mice.

The somatostatin analogue TT-232 induces apoptosis in A431 cells: sustained activation of stress-activated kinases and inhibition of signalling to extracellular signal-regulated kinases.

TT-232 is a somatostatin analogue containing a five-residue ring structure. The present report describes TT-232-induced signalling events in A431 cells, where a 4-h preincubation with the peptide irreversibly induced a cell death program, which involves DNA-laddering and the appearance of shrunken nuclei, but is unrelated to somatostatin signalling. Early intracellular signals of TT-232 include a transient two-fold activation of the extracellular signal-regulated kinase (ERK2) and a strong and sustained activation of the stress-activated protein kinases c-Jun NH(2)-terminal kinase (JNK)/SAPK and p38MAPK. Blocking the signalling to ERK or p38MAPK activation had no effect on the TT-232-induced cell killing. At the commitment time for inducing cell death, TT-232 decreased EGFR-tyrosine phosphorylation and prevented epidermial growth factor (EGF)-induced events like cRaf-1 and ERK2 activation. Signalling to ERK activation by FCS, phorbol 12-myristate 13-acetate (PMA) and platelet-derived growth factor (PDGF) was similarly blocked. Our data suggest that TT-232 triggers an apoptotic type of cell death, concomitant with a strong activation of JNK and a blockade of cellular ERK2 activation pathways.

Different pathways mediate cytochrome c release after photodynamic therapy with hypericin.

In this study we show that overexpression of Bcl-2 in PC60R1R2 cells reveals a caspase-dependent mechanism of cytochrome c release following photodynamic therapy (PDT) with hypericin. Bcl-2 overexpression remarkably delayed cytochrome c release, procaspase-3 activation and poly(adenosine diphosphate-ribose)polymerase cleavage during PDT-induced apoptosis while it did not protect against PDT-induced necrosis. PDT-treated cells showed a reduction in the mitochondrial membrane potential which occurred with similar kinetics in PC60R1R2 and PC60R1R2/Bcl-2 cells, and was affected neither by the permeability transition pore inhibitor cyclosporin A nor by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk). Hypericin-induced mitochondrial depolarization coincided with cytochrome c release in PC60R1R2 cells while it precedes massive cytochrome c efflux in PC60R1R2/Bcl-2 cells. Preincubation of PC60R1R2 cells with zVAD-fmk or cyclosporin A did not prevent the mitochondrial efflux of cytochrome c, and caspase inhibition only partially protected the cells from PDT-induced apoptosis. In contrast, in PC60R1R2/Bcl-2 cells cytochrome c release and apoptosis were suppressed by addition of zVAD-fmk or cyclosporin A. These observations suggest that the progression of the PDT-induced apoptotic process in Bcl-2-overexpressing cells involves a caspase-dependent feed-forward amplification loop for the release of cytochrome c.

Efficacy of antitumoral photodynamic therapy with hypericin: relationship between biodistribution and photodynamic effects in the RIF-1 mouse tumor model.

We investigated the hypericin-mediated PDT effects on the tumor and normal skin and in correlation with its biodistribution. These studies were carried out on C3H mice bearing RIF-1 tumors. The hypericin distribution and PDT effects were recorded at different intervals (0.5-24 hr) after intravenous injection of a 5-mg/kg dose of hypericin. After administration, rapid biphasic exponential decay was observed in the plasma drug concentration. It was found that hypericin was preferentially bound to the plasma lipoproteins. The tumor drug levels increased rapidly over the first few hours and reached a maximum around 6 hr after injection. In contrast, PDT efficacy was maximal when irradiation was performed at 0.5 hr after hypericin administration, which led to 100% cure. The PDT efficacy decreased rapidly as the administration-irradiation interval was prolonged. No tumor cure was obtained at the 6-hr interval, even though it was at this time that the tumor drug level peaked. Fluorescence microscopic studies showed that hypericin was mainly confined within the tumor vasculature at 0.5 hr after injection, whereupon it rapidly diffused to the surrounding tumor tissue. At 6 hr, a strong hypericin fluorescence was observed in the tumor tissue with only faint fluorescence within the vasculature, whereas at 24 hr the fluorescence in the tumor also decreased and became more diffused, and no fluorescence could be seen in the tumor vasculature. Like the tumor response, skin reactions were also found to be much more dramatic at short administration-irradiation intervals. Hypericin distribution and PDT response studies revealed a close correlation between the plasma drug level and the PDT effects, which suggests that vascular damage is the primary effect of hypericin-mediated PDT in this tumor model.

Regulation of protein kinase D by multisite phosphorylation. Identification of phosphorylation sites by mass spectrometry and characterization by site-directed mutagenesis.

Activation of the serine/threonine kinase, protein kinase D (PKD/PKC mu) via a phorbol ester/PKC-dependent pathway involves phosphorylation events. The present study identifies five in vivo phosphorylation sites by mass spectrometry, and the role of four of them was investigated by site-directed mutagenesis. Four sites are autophosphorylation sites, the first of which (Ser(916)) is located in the C terminus; its phosphorylation modifies the conformation of the kinase and influences duration of kinase activation but is not required for phorbol ester-mediated activation of PKD. The second autophosphorylation site (Ser(203)) lies in that region of the regulatory domain, which in PKC mu interacts with 14-3-3tau. The last two autophosphorylation sites (Ser(744) and Ser(748)) are located in the activation loop but are only phosphorylated in the isolated PKD-catalytic domain and not in the full-length PKD; they may affect enzyme catalysis but are not involved in the activation of wild-type PKD by phorbol ester. We also present evidence for proteolytic activation of PKD. The fifth site (Ser(255)) is transphosphorylated downstream of a PKC-dependent pathway after in vivo stimulation with phorbol ester. In vivo phorbol ester stimulation of an S255E mutant no longer requires PKC-mediated events. In conclusion, our results show that PKD is a multisite phosphorylated enzyme and suggest that its phosphorylation may be an intricate process that regulates its biological functions in very distinct ways.

p38 mitogen-activated protein kinase regulates a novel, caspase-independent pathway for the mitochondrial cytochrome c release in ultraviolet B radiation-induced apoptosis.

The mechanisms of UVB-induced apoptosis and the role of p38 mitogen-activated protein kinase (MAPK) were investigated in HaCaT cells. UVB doses that induced apoptosis also produced a sustained activation of p38 MAPK and mitochondrial cytochrome c release, leading to pro-caspase-3 activation. Late into the apoptotic process, UVB also induced a caspase-mediated cleavage of Bid. Caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone substantially blocked the UVB-induced apoptosis without preventing the release of mitochondrial cytochrome c and the p38 MAPK activation. The inhibition of p38 MAPK counteracted both apoptosis and cytochrome c release as well as the DEVD-amino-4-methylcoumarin cleavage activity without affecting the processing of pro-caspase-8. These results indicate that UVB induces multiple and independent apoptotic pathways, which culminate in pro-caspase-3 activation, and that the initial cytochrome c release is independent of caspase activity. Importantly, we show that a sustained p38 MAPK activation contributes to the UVB-induced apoptosis by mediating the release of mitochondrial cytochrome c into the cytosol.

JNK/SAPK activation by platelet-derived growth factor in A431 cells requires both the phospholipase C-gamma and the phosphatidylinositol 3-kinase signaling pathways of the receptor.

Wild-type or mutant betaPDGF receptors were introduced into A431 cells that lack endogenous PDGF receptors. PDGF stimulates JNK1 activity in a dose- and time-dependent manner in cells expressing the wild-type receptor. A receptor mutant lacking all the binding sites for SHP-2, GAP, PI3K, and PLC-gamma fails to activate JNK1. Receptor mutants with no binding site for either SHP-2 or GAP can fully activate JNK1 but those which do not bind either PI3K or PLC-gamma are unable to induce JNK1 activation. PDGF-dependent JNK1 activation was reduced upon cell pretreatment with wortmannin or GF109203X and is completely abrogated by chronic PMA stimulation. Altogether, these results indicate that PDGF activates JNK1 through a pathway that involves both PI3K and PLC-gamma and subsequent activation of protein kinase C.

Gbetagamma-mediated regulation of Golgi organization is through the direct activation of protein kinase D.

We have shown previously that the betagamma subunits of the heterotrimeric G proteins regulate the organization of the pericentriolarly localized Golgi stacks. In this report, evidence is presented that the downstream target of Gbetagamma is protein kinase D (PKD), an isoform of protein kinase C. PKD, unlike other members of this class of serine/threonine kinases, contains a pleckstrin homology (PH) domain. Our results demonstrate that Gbetagamma directly activates PKD by interacting with its PH domain. Inhibition of PKD activity through the use of pharmacological agents, synthetic peptide substrates, and, more specifically, the PH domain of PKD prevents Gbetagamma-mediated Golgi breakdown. Our findings suggest a possible mechanism by which the direct interaction of Gbetagamma with PKD regulates the dynamics of Golgi membranes and protein secretion.

The activation of the c-Jun N-terminal kinase and p38 mitogen-activated protein kinase signaling pathways protects HeLa cells from apoptosis following photodynamic therapy with hypericin.

In this study, we elucidate signaling pathways induced by photodynamic therapy (PDT) with hypericin. We show that PDT rapidly activates JNK1 while irreversibly inhibiting ERK2 in several cancer cell lines. In HeLa cells, sustained PDT-induced JNK1 and p38 mitogen-activated protein kinase (MAPK) activations overlap the activation of a DEVD-directed caspase activity, poly(ADP-ribose) polymerase (PARP) cleavage, and the onset of apoptosis. The caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (zDEVD-fmk) protect cells against apoptosis and inhibit DEVD-specific caspase activity and PARP cleavage without affecting JNK1 and p38 MAPK activations. Conversely, stable overexpression of CrmA, the serpin-like inhibitor of caspase-1 and caspase-8, has no effect on PDT-induced PARP cleavage, apoptosis, or JNK1/p38 activations. Cell transfection with the dominant negative inhibitors of the c-Jun N-terminal kinase (JNK) pathway, SEK-AL and TAM-67, or pretreatment with the p38 MAPK inhibitor PD169316 enhances PDT-induced apoptosis. A similar increase in PDT-induced apoptosis was observed by expression of the dual specificity phosphatase MKP-1. The simultaneous inhibition of both stress kinases by pretreating cells with PD169316 after transfection with either TAM-67 or SEK-AL produces a more pronounced sensitizing effect. Cell pretreatment with the p38 inhibitor PD169316 causes faster kinetics of DEVD-caspase activation and PARP cleavage and strongly oversensitizes the cells to apoptosis following PDT. These observations indicate that the JNK1 and p38 MAPK pathways play an important role in cellular resistance against PDT-induced apoptosis with hypericin.

Control of glycogen synthesis in cultured human muscle cells.

The regulation of glycogen synthesis and associated enzymes was studied in human myoblasts and myotubes maintained in culture. Both epidermal growth factor (EGF) and insulin stimulated glycogen synthesis approximately 2-fold, this stimulation being accompanied by a rapid and stable activation of the controlling enzyme glycogen synthase (GS). EGF also caused inhibition of glycogen synthase kinase 3 (GSK-3) and activation of the alpha isoform of protein kinase B (PKB) with the time-course and magnitude of its effects being similar to those induced by insulin. An inhibitor of the mitogen-activated protein (MAP) kinase pathway did not prevent stimulation of GS by EGF, suggesting that this pathway is not essential for the effect. A partial decrease in the fold activation of GS was, however, observed when p70(S6k) activation was blocked with rapamycin, suggesting a contribution of this pathway to the control of GS by either hormone. Wortmannin, a selective inhibitor of phosphatidylinositol 3'-kinase (PI-3 kinase) completely blocked the effects of both EGF and insulin in these cells. These results demonstrate that EGF, like insulin, activates glycogen synthesis in muscle, acting principally via the PKB/GSK-3 pathway but with a contribution from a rapamycin-sensitive component that lies downstream of PI-3 kinase.

Hypericin-induced photosensitization of HeLa cells leads to apoptosis or necrosis. Involvement of cytochrome c and procaspase-3 activation in the mechanism of apoptosis.

Here we report that photoactivated hypericin can induce either apoptosis or necrosis in HeLa cells. Under apoptotic conditions the cleavage of poly(ADP-ribose) polymerase (PARP) into the 85-kDa product is blocked by the caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (z-DEVD-fmk). Both inhibitors protect cells from apoptosis but cannot prevent hypericin-induced necrosis. Conversely, HeLa cells overexpressing the viral cytokine response modifier A (CrmA), which inhibits caspase-1 and -8, still undergo hypericin-induced apoptosis and necrosis. Evidence is provided for the release of mitochondrial cytochrome c in the cytosol and for procaspase-3 activation in the hypericin-induced cell killing.

Platelet-derived growth factor stimulates protein kinase D through the activation of phospholipase Cgamma and protein kinase C.

Platelet-derived growth factor (PDGF) stimulates protein kinase D (PKD) in a time- and dose-dependent manner. We have used a series of PDGF receptor mutants that display a selective impairment of the binding of SH2-containing proteins (GTPase-activating protein, SHP-2, phospholipase Cgamma (PLCgamma), or phosphatidylinositol 3'-kinase (PI3K)) to show that Tyr-1021, the PLCgamma-binding site, is essential for PKD stimulation by PDGF in A431 cells. We next investigated whether any one of these four binding sites could mediate PKD activation in the absence of the other three sites. F5, a receptor mutant that lacks all four binding sites for GTPase-activating protein, PLCgamma, PI3K, and SHP-2, fails to activate PKD. A panel of single add-back mutants was used to investigate if any one of these four sites could restore signaling to PKD. Of the four sites, only the PLCgamma+ single add-back receptor restored PDGF-mediated activation of PKD, and only this add-back receptor produced diacylglycerol (DAG) in a PDGF-dependent manner. 1,2-Dioctanoyl-sn-glycerol, a membrane-permeant DAG analog, was found to be sufficient for activation of PKD. Taken together, these data indicate that PLCgamma activation is not only necessary, but also sufficient to mediate PDGF-induced PKD activation. Although the presence of a pleckstrin homology domain makes PKD a potential PI3K target, PKD was not stimulated by selective PI3K activation, and wortmannin, an inhibitor of PI3K, did not inhibit PDGF signaling to PKD. The activation of PKD by DAG or by the wild-type and PLCgamma+ add-back PDGF receptors was inhibited by GF109203X, suggesting a role for protein kinase C in the stimulation of PKD by PDGF. PDGF induced a time-dependent phosphorylation of PKD that closely correlated with activation. The PDGF-induced activation and phosphorylation of PKD were reversed by in vitro incubation of PKD with protein phosphatase 1 or 2A, indicating that PDGF signaling to PKD involves the Ser/Thr phosphorylation of PKD. Taken together, these results conclusively show that PDGF activates PKD through a pathway that involves activation of PLCgamma and, subsequently, protein kinase C.

Identification and characterization of an auto-activating MEK kinase from bovine brain: phosphorylation of serine-298 in the proline-rich domain of the mammalian MEKs.

Mitogen-activated protein kinase kinases (MKKs or MEKs) are dual specificity tyrosine/threonine protein kinases that are activated by phosphorylation at two closely spaced serine residues (serines-218 and -222) by the c-mos and raf proto-oncogenes. This double phosphorylation is both necessary and sufficient for MEKs to activate the MAP kinase enzymes in vitro. The specificity or regulation of in vivo signaling to the mammalian MEKs (MEK1 and MEK2) was recently reported also to involve the differential phosphorylation of a proline-rich peptide located between the MEK kinase-subdomains IX and X. Here we report the purification and characterization of an auto-activating protein kinase from bovine brain that phosphorylates serine-298 of the MEK1 and MEK2 proline-rich insert peptides. The auto-activation of the MEK-S298 peptide kinase is the result of an intermolecular phosphorylation event that can be prevented by the peptide substrates. The inactive kinase migrates on gel filtration as a 90 kDa protein, and after activation as a 43 kDa phosphoprotein. Incorporation of 32P[phosphate] into 40-42 kDa proteins on SDS-PAGE parallels the activation of the enzyme, and dephosphorylation by protein phosphatase 2Ac reverses the activation. SDS-PAGE renaturation assays show that the 40 kDa protein has the capacity to autophosphorylate, and exhibits kinase activity towards myelin basic protein after activation. Phosphorylation of purified bovine brain MEK or recombinant MEK1 by the auto-activated kinase does not activate the enzyme, and does not interfere with the in vitro raf-mediated MEK activation. We conclude that still unknown kinases may control the MAP kinase pathway by targeting MEK.

Ganglioside GM1 activates the mitogen-activated protein kinase Erk2 and p70 S6 kinase in U-1242 MG human glioma cells.

Gangliosides are implicated in the regulation of cellular proliferation as evidenced by differences in ganglioside composition associated with malignant transformation and density of cells in culture, as well as their inhibitory effects when added to cells growing in culture. Exogenously added gangliosides have a bimodal effect on proliferation in U-1242 MG glioma cells, inhibiting DNA synthesis in growing cells and stimulating it in quiescent cells. We investigated the mechanisms involved in stimulation of DNA synthesis using [3H]thymidine incorporation and immune complex kinase assays to identify responsible signal transduction pathways. Treatment of quiescent U-1242 MG cells with GM1 caused activation of the mitogen-activated protein (MAP) kinase isoform Erk2. Pretreatment with the specific MAP kinase kinase inhibitor PD98059 prevented the GM1-stimulated Erk2 activation and GM1-stimulated DNA synthesis. GM1 treatment stimulated another distinct signaling pathway leading to activation of p70 S6 kinase (p70s6k), and this was prevented by pretreatment with rapamycin. Rapamycin also inhibited GM1-stimulated DNA synthesis. Activation of both pathways and stimulation of DNA synthesis were inhibited by forskolin treatment; however, GM1 had no effect on cyclic AMP levels. Platelet-derived growth factor also activated both Erk2 and p70s6k but did not cause DNA synthesis, suggesting that GM1 may stimulate additional cascades, which also contribute to GM1-mediated DNA synthesis.

Differential stimulation of ERK and JNK activities by ultraviolet B irradiation and epidermal growth factor in human keratinocytes.

Exposure of mammalian cells to solar ultraviolet (UV) radiation leads to the expression of several genes, and UV has been recognized as a major initiator and promoter of skin cancer. The component of the solar radiation that contributes most to human skin malignancy is UVB (280-320 nm) and, to a lesser extent, UVA (320-400 nm), whereas the high-energy UVC (100-280 nm) is absorbed by the earth's upper atmosphere. Sublethal doses of UVB produce strong induction of c-jun and c-fos transcripts in several cells including human primary keratinocytes. The present report confirms that this is also the case in the HaCaT cell line and shows that similar UVB doses are potent inducers of the JNK/SAPK family of mitogen-activated protein kinases but only weak activators of ERKs. Epidermal growth factor (EGF) caused rapid induction of both JNK- and ERK-signaling pathways, and the downmodulation of the EGF-signaling pathway by EGF pre-treatment inhibited the UVB-induced JNK1 activation. Prior UVB irradiation of the cells decreased the level of the ERK2 activation by a subsequent EGF treatment, but this sensitized the cells and allowed for the super-activation of JNK1 after a rechallenge with either UVB or EGF. The antioxidant N-acetylcysteine impaired the UVB- and EGF-induced activation of JNK1. Our data suggest the presence of shared signaling component(s) in the UVB- and EGF-induced cellular response pathways and imply that oxidative stress plays a significant role in the activation of JNK1 by UVB and EGF.

Phosphorylation of yeast plasma membrane H+-ATPase by casein kinase I.

The plasma membrane H+-ATPase of Saccharomyces cerevisiae is subject to phosphorylation by a casein kinase I activity in vitro. We show this casein kinase I activity to result from the combined function of YCK1 and YCK2, two highly similar and plasma membrane-associated casein kinase I homologues. First, H+-ATPase phosphorylation is severely impaired in the plasma membrane of YCK-deficient yeast strains. Furthermore, the wild-type level of the phosphoprotein is restored by the addition of purified mammalian casein kinase I to the mutant membranes. We used the H+-ATPase as well as a synthetic peptide substrate that contains a phosphorylation site for casein kinase I to compare kinase activity in membranes prepared from yeast cells grown in the presence or absence of glucose. The addition of glucose results in increased H+-ATPase activity which is associated with a decline in the phosphorylation level of the enzyme. Mutations in both YCK1 and YCK2 affect this regulation, suggesting that H+-ATPase activity is modulated by glucose via a combination of a "down-regulating" casein kinase I activity and another, yet uncharacterized, "up-regulating" kinase activity. Biochemical mapping of phosphorylated H+-ATPase identifies a major phosphopeptide that contains a consensus phosphorylation site (Ser-507) for casein kinase I. Site-directed mutagenesis of this consensus sequence indicates that Glu-504 is important for glucose-induced decrease in the apparent Km for ATP.