The cytoplasmic domain of the hyaluronan receptor for endocytosis (HARE) contains multiple endocytic motifs targeting coated pit-mediated internalization

The hyaluronic acid (HA) receptor for endocytosis (HARE) is the primary scavenger receptor for HA and chondroitin sulfates in mammals. The two human isoforms of HARE (full-length 315-kDa and a 190-kDa proteolytic cleavage product), which are type I single-pass membrane proteins, are highly expressed in sinusoidal endothelial cells of lymph nodes, liver, and spleen. Their identical HARE cytoplasmic domains contain four candidate AP-2/clathrin-mediated endocytic signaling motifs as follows: YSYFRI(2485), FQHF(2495), NPLY(2519), and DPF(2534) (315-HARE numbering). Stably transfected cells expressing 190-HARE(DeltaYSYFRI), 190-HARE(DeltaFQHF), or 190-HARE(DeltaNPLY) (lacking Motifs 1, 2, or 3) had decreased (125)I-HA endocytosis rates of approximately 49, approximately 39, and approximately 56%, respectively (relative to wild type). In contrast, 190-HARE(DeltaDPF) cells (lacking Motif 4) showed no change in HA endocytic rate. Deletions of motifs 1 and 2 or of 1, 2, and 4 decreased the rate of HA endocytosis by only approximately 41%. Endocytosis was approximately 95% decreased in mutants lacking all four motifs. Cells expressing a 190-HARE(Y2519A) mutant of the NPLY motif retained 85-90% of wild type endocytosis, whereas this mutation in the triple motif deletant decreased endocytosis to approximately 7% of wild type. Tyr in NPLY(2519) is thus important for endocytosis. All HARE mutants showed similar HA binding and degradation of the internalized HA, indicating that altering endocytic motifs did not affect ectodomain binding of HA or targeting of internalized HA to lysosomes. We conclude that, although NPLY may be the most important motif, it functions together with two other endocytic motifs; thus three signal sequences (YSYFRI, FQHF, and NPLY) provide redundancy to mediate coated pit targeting and endocytosis of HARE.     

Expression, processing, and glycosaminoglycan binding activity of the recombinant human 315-kDa hyaluronic acid receptor for endocytosis (HARE)

The hyaluronic acid (HA) receptor for endocytosis (HARE; also designated stabilin-2 and FEEL-2) mediates systemic clearance of glycosaminoglycans from the circulatory and lymphatic systems via coated pit-mediated uptake. HARE is primarily found as two isoforms (315- and 190-kDa) in sinusoidal endothelial cells of the liver, lymph node, and spleen. Here we characterize the ligand specificity and function of the large stably expressed 315-HARE isoform in Flp-In 293 cell lines. Like human spleen sinusoidal endothelial cells, Flp-In 293 cell lines transfected with a single cDNA encoding the full-length 315-HARE express both the 315-kDa and the proteolytically truncated 190-kDa isoforms in a ratio of approximately 3-4:1. The 190-kDa HARE isoform generated from the 315-kDa HARE and the 315-kDa HARE specifically bound 125I-HA. Like the 190-kDa HARE expressed alone (Harris, E. N., Weigel, J. A., and Weigel, P. H. (2004) J. Biol. Chem. 279, 36201-36209), the 190- and 315-kDa HARE isoforms expressed in 315-HARE cell lines were recognized by anti-HARE monoclonal antibodies 30, 154, and 159. All 315-HARE cell lines could endocytose and degrade 125I-HA. Competition studies with live cells indicate that 190-HARE and 315-HARE bind HA with higher apparent affinity (Kd approximately 10-20 nM) than chondroitin sulfate (CS) types A, C, D, or E. Only slight competition of HA endocytosis was observed with CS-B (dermatan sulfate) and chondroitin. Direct binding assays with the 315-HARE ectodomain revealed high affinity HA binding, and lower binding affinities for CS-C, CS-D, and CS-E. A majority of each HARE isoform was intracellular, within the endocytic system, suggesting transient surface residency typical of an active endocytic recycling receptor.     

Purification and molecular identification of the human hyaluronan receptor for endocytosis

The clearance of hyaluronan (HA) and chondroitin sulfates from the circulating blood and lymph in the body is mediated by the membrane-bound HA receptor for endocytosis (HARE). Previously, we found that two HARE species of approximately 175 kDa and approximately 300 kDa are abundant in the sinusoidal endothelial cells in rat liver, spleen, and lymph nodes (Zhou et al. [2000], J. Biol. Chem., 275, 37733-37741). In the present study, immunocytochemical analysis of human tissues showed a similar pattern with abundant expression of HARE in the sinusoidal endothelial cells of human liver, spleen, and lymph nodes. The two human HARE proteins were immunoaffinity-purified from human spleen. Each protein was recognized in western blots using several anti-rat HARE monoclonal antibodies and was able to bind 125I-HA specifically. In nonreducing SDS-PAGE, these two human HARE species migrated at approximately 190 kDa and approximately 315 kDa; both proteins are approximately 15 kDa larger than the corresponding rat HAREs, although the de-N-glycosylated core proteins are essentially the same mass. After reduction, the human 190-kDa HARE gave a single 196-kDa species, which was not seen in the approximately 315-kDa HARE after reduction. The reduced approximately 315-kDa HARE yielded two major proteins at approximately 250 kDa and approximately 220 kDa. We determined the sequence of the human 190-kDa HARE cDNA based on analysis of internal tryptic peptides, as well as RT-PCR and 5' RACE analyses using human spleen and lymph node cDNA libraries. The human gene that encodes HARE is on chromosome 12.     

The human hyaluronan receptor for endocytosis (HARE/Stabilin-2) is a systemic clearance receptor for heparin

The hyaluronic acid receptor for endocytosis (HARE; also designated Stabilin-2) mediates systemic clearance of hyaluronan and chondroitin sulfates from the vascular and lymphatic circulations. The internalized glycosaminoglycans are degraded in lysosomes, thus completing their normal turnover process. Sinusoidal endothelial cells of human liver, lymph node, and spleen express two HARE isoforms of 315 and 190 kDa. Here we report that the 190- and 315-kDa HARE isoforms, expressed stably either in Flp-In 293 cell lines or as soluble ectodomains, specifically bind heparin (Hep). The K(d) for Hep binding to purified 190- and 315-kDa HARE ectodomains was 17.2 +/- 4.9 and 23.4 +/- 5.3 nm, respectively. Cells expressing HARE readily and specifically internalized (125)I-streptavidin-biotin-Hep complexes, which was inhibited >70% by hyperosmolar conditions, confirming that uptake is mediated by the clathrin-coated pit pathway. Internalization of Hep occurred for many hours with an estimated HARE recycling time of approximately 12 min. Internalized fluorescent streptavidin-biotin-Hep was present in a typical endocytic vesicular pattern and was delivered to lysosomes. We conclude that HARE in the sinusoidal endothelial cells of lymph nodes and liver likely mediates the efficient systemic clearance of Hep and many different Hep-binding protein complexes from the lymphatic and vascular circulations.     

Endocytic function, glycosaminoglycan specificity, and antibody sensitivity of the recombinant human 190-kDa hyaluronan receptor for endocytosis (HARE)

The human hyaluronan receptor for endocytosis (hHARE) mediates the endocytic clearance of hyaluronan (HA) and chondroitin sulfate from lymph fluid and blood. Two hHARE isoforms (190 and 315 kDa) are present in sinusoidal endothelial cells of liver, spleen, and lymph nodes (Zhou, B., McGary, C. T., Weigel, J. A., Saxena, A., and Weigel, P. H. (2003) Glycobiology 13, 339-349). Here we report the specificity and function of the 190-kDa HARE, expressed without the larger isoform, in Flp-In 293 cell lines (190hHARE cells). Like the native protein, recombinant hHARE contains approximately 25 kDa of N-linked oligosaccharides, binds HA in a ligand blot assay, cross-reacts with three anti-rat HARE monoclonal antibodies, and is inactivated by reduction. The 190hHARE cell lines mediated rapid, continuous (125)I-HA endocytosis and degradation for >1 day. About 30-50% of the total cellular receptors were on the cell surface, and their recycling time for reutilization was approximately 8.5 min. The average K(d) for the binding of HA to the 190-kDa hHARE at 4 degrees C was 7 nm with 118,000 total HA binding sites per cell. Competition studies at 37 degrees C indicated that the 190-kDa hHARE binds HA and chondroitin better than dermatan sulfate and chondroitin sulfates A, C, D, and E, but it does not bind to heparin, heparan sulfate, or keratan sulfate. Although competition was observed at 37 degrees C, none of the glycosaminoglycans tested, except HA, competed for (125)I-HA binding by 190hHARE cells at 4 degrees C. Anti-HARE monoclonal antibodies #30 and #154, which do not inhibit (125)I-HA uptake mediated by the 175-kDa rat HARE, partially blocked HA endocytosis by the 190-kDa hHARE. We conclude that the 190-kDa hHARE can function independently of other hHARE isoforms to mediate the endocytosis of multiple glycosaminoglycans. Furthermore, the rat and human small HARE isoforms have different glycosaminoglycan specificities and sensitivities to inhibition by cross-reacting antibodies.     

Characterization of the recombinant rat 175-kDa hyaluronan receptor for endocytosis (HARE)

Hyaluronan (HA) and chondroitin sulfate (CS) clearance from lymph and blood in mammals is mediated by the HA receptor for endocytosis (HARE), which is present as two isoforms in rat and human (175/300 kDa and 190/315 kDa, respectively) in the sinusoidal endothelial cells of liver, spleen, and lymph nodes (Zhou, B., McGary, C. T., Weigel, J. A., Saxena, A., and Weigel, P. H. (2003) Glycobiology 13, 339-349). The small rat and human HARE proteins are not encoded directly by mRNA but are derived from larger precursors. Here we characterize the specificity and function of the 175-kDa HARE, expressed in the absence of the 300-kDa species, in stably transfected SK-Hep-1 cells. The HARE cDNA was fused with a leader sequence to allow correct orientation of the membrane protein. The recombinant rHARE contained approximately 25 kDa of N-linked oligosaccharides and, like the native protein, was able to bind HA in a ligand blot assay, even after de-N-glycosylation. SK-HARE cell lines demonstrated specific 125I-HA endocytosis, receptor recycling, and delivery of HA to lysosomes for degradation. The Kd for the binding of HA (number-average molecular mass approximately 133 kDa) to the 175-kDa HARE at 4 degrees C was 4.1 nm with 160,000 to 220,000 HA-binding sites per cell. The 175-kDa rHARE binds HA, dermatan sulfate, and chondroitin sulfates A, C, D, and E, but not chondroitin, heparin, heparan sulfate, or keratan sulfate. Surprisingly, recognition of glycosaminoglycans (GAGs) other than HA by native or recombinant HARE was temperature-dependent. Although competition was observed at 37 degrees C, none of the other GAGs competed for 125I-HA binding to SK-HARE cells at 4 degrees C. Anti-HARE monoclonal antibody-174 showed a similar temperature-dependence in its ability to block HA endocytosis. These data suggest that temperature-induced conformational changes may alter the GAG specificity of HARE. The results confirm that the 175-kDa rHARE does not require the larger HARE isoform to mediate endocytosis of multiple GAGs.     

A Hyaluronan Receptor for Endocytosis (HARE) Link Domain N-Glycan Is Required for Extracellular Signal-regulated Kinase (ERK) and Nuclear Factor-κB (NF-κB) Signaling in Response to the Uptake of Hyaluronan but Not Heparin,Dermatan Sulfate,or Acetylated Low Density Lipoprotein (LDL)

The human hyaluronan (HA) receptor for endocytosis (HARE; the 190-kDa C terminus of Stab2) is a major clearance receptor for multiple circulating ligands including HA, heparin (Hep), acetylated LDL (AcLDL), dermatan sulfate (DS), apoptotic debris, and chondroitin sulfate types A, C, D, and E. We previously found that HARE contains an N-glycan in the HA binding Link domain (at Asn²²⁸⁰), and cells expressing membrane-bound HARE(N2280A) bind and endocytose HA normally (Harris, E. N., Parry, S., Sutton-Smith, M., Pandey, M. S., Panico, M., Morris, H. R., Haslam, S. M., Dell, A., and Weigel, P. H. (2010) Glycobiology 20, 991–1001). Also, NF-κB-mediated signaling is activated by HARE-mediated endocytosis of HA, Hep, AcLDL, or DS but not by chondroitin sulfates (Pandey, M. S., and Weigel, P. H. (2014) J. Biol. Chem. 289, 1756–1767). Here we investigated the role of Link N-glycans in ligand uptake and NF-κB and ERK1/2 signaling. HA·HARE-mediated ERK1/2 activation was HA size- dependent, as found for NF-κB activation. HARE(N2280A) cells internalized HA, Hep, AcLDL, and DS normally. No ERK1/2 activation occurred during HA endocytosis by HARE(N2280A) cells, but activation did occur with Hep. Dual-luciferase recorder assays showed that NF-κB-mediated gene expression occurred normally in HARE(N2280A) cells endocytosing Hep, AcLDL, or DS but did not occur with HA. Activation of NF-κB by endogenous degradation of IκB-α was observed for HARE(N2280A) cells endocytosing Hep, AcLDL, or DS but not HA. We conclude that a Link domain complex N-glycan is required specifically for HARE·HA-mediated activation of ERK1/2 and NF-κB-mediated gene expression and that this initial activation mechanism is different from and independent of the initial mechanisms for HARE-mediated signaling in response to Hep, AcLDL, or DS uptake.     

The ligand-binding profile of HARE: hyaluronan and chondroitin sulfates A, C, and D bind to overlapping sites distinct from the sites for heparin, acetylated low-density lipoprotein, dermatan sulfate, and CS-E

The hyaluronic acid receptor for endocytosis (HARE)/ Stabilin-2 is the primary systemic scavenger receptor for hyaluronan (HA), the chondroitin sulfates (CS), dermatan sulfate (DS), and nonglycosaminoglycan (GAG) ligands such as acetylated low-density lipoprotein (AcLDL), pro-collagen propeptides, and advanced glycation end products. We recently discovered that HARE is also a systemic scavenger receptor for heparin (Hep) (Harris EN, Weigel JA, Weigel PH. 2008. The human hyaluronan receptor for endocytosis [HARE/Stabilin-2] is a systemic clearance receptor for heparin. J Biol Chem. 283:17341-17350). Our goal was to map the binding sites of eight different ligands within HARE. We used biotinylated GAGs and radio-iodinated streptavidin or AcLDL to assess the binding activities of ligands directly or indirectly (by competition with unlabeled ligands) in endocytosis assays using stable cell lines expressing the 315 or 190 kDa HA receptor for endocytosis (315- or 190-HARE) isoforms, and ELISA-like assays, with purified recombinant soluble 190-HARE ecto-domain. For example, Hep binding to HARE was competed by DS, CS-E, AcLDL, and dextran sulfate, but not by other CS types, HA, dextran, or heparosan. (125)I-AcLDL binding to HARE was partially competed by Hep and dextran sulfate, but not competed by HA. Two ligands, DS and CS-E, competed with both Hep and HA to some degree. Hep and HA binding or endocytosis is mutually inclusive; binding of these two GAGs occurs with functionally separate, noncompetitive, and apparently noninteracting domains. Thus, HARE binds to HA and Hep simultaneously. Although the domain(s) responsible for Hep binding remains unknown, the Link domain was required for HARE binding to HA, CS-A, CS-C, and CS-D. These results enable us to outline, for the first time, a binding activity map for multiple ligands of HARE.     

A blocking antibody to the hyaluronan receptor for endocytosis (HARE) inhibits hyaluronan clearance by perfused liver

Hyaluronan (HA) and chondroitin sulfate clearance from lymph and blood is mediated by the hyaluronan receptor for endocytosis (HARE). The purification and molecular cloning (Zhou, B., Weigel, J. A., Saxena, A., and Weigel, P. H. (2002) Mol. Biol. Cell 13, 2853-2868) of this cell surface receptor were finally achieved after we developed monoclonal antibodies (mAbs) against HARE. There are actually two independent isoreceptors for HA, which in rat are designated the 175-kDa HARE and 300-kDa HARE. Only one mAb (number 174) effectively and completely blocked the specific uptake of 125I-HA at 37 degrees C by rat liver sinusoidal endothelial cells. 125I-HA binding to both the 175-kDa and 300-kDa HARE proteins in a ligand blot assay was almost completely inhibited by <1 microg/ml mAb-174, whereas mouse IgG had little or no effect. MAb-174 also performed very well in Western analysis, indirect fluorescence microscopy, and a variety of immuno-procedures. Immunohistochemistry using mAb-174 localized HARE to the sinusoidal cells of rat liver, spleen, and lymph node. Western analysis using mAb-174 revealed that the sizes of both HARE glycoproteins were the same in these three tissues. 125I-HA was taken up and degraded by excised rat livers that were continuously perfused ex vivo with a recirculating medium. This HA clearance and metabolism by liver, which is a physiological function of HARE, was very effectively blocked by mAb-174 but not by mouse IgG. The results indicate that mAb-174 will be a useful tool to study the functions of HARE and the physiological significance of HA clearance.     

ERKs and p38 kinases mediate ultraviolet B-induced phosphorylation of histone H3 at serine 10

Histone H3 is the core protein of the nucleosome. Phosphorylation of H3 involves immediate early gene expression, chromatin remodeling, and chromosome condensation during mitosis. Very recently, Rsk2 or MSK1 kinase-mediated phosphorylation of H3 at serine 10 was reported. In the present study, we show that both ERKs and p38 kinase may mediate ultraviolet B-induced phosphorylation of H3 at serine 10. PD 98059, a MEK1 inhibitor, and SB 202190, a p38 kinase inhibitor, efficiently inhibited ultraviolet B-induced phosphorylation of H3. Phosphorylation of H3 was also inhibited in cells expressing dominant negative mutant (DNM) ERK2 and DNM p38 kinase. In contrast, no inhibition of H3 phosphorylation in Jnk1 or Jnk2 knockout cells (Jnk1(-/-) or Jnk2(-/-)) and cells expressing DNM JNK1 was observed. More importantly, incubation of active ERK2 or p38 kinase with H3 protein resulted in phosphorylation of H3 at serine 10 in vitro. These results suggest that ERK and p38 kinase are at least two important mediators of phosphorylation of H3 at serine 10.     

A delayed gonadotropin-dependent and growth factor-mediated activation of the extracellular signal-regulated kinase 1/2 cascade negatively regulates aromatase expression in granulosa cells

Human chorionic gonadotropin and human FSH (hFSH) elicit a transient increase in ERK1/2 phosphorylation lasting less than 60 min in immature granulosa cells expressing a low density of gonadotropin receptors. In cells expressing a high density of receptors, human chorionic gonadotropin and human FSH elicit this fast transient increase in ERK1/2 phosphorylation and also a delayed and more sustained increase that is detectable after 6-9 h. Both the early and delayed increases in ERK1/2 phosphorylation can be blocked with inhibitors of protein kinase A, the epidermal growth factor receptor kinase, metalloproteases, and MAPK kinase. The delayed effect, but not the early effect, can also be blocked with an inhibitor of protein kinase C. Because the delayed increase in ERK1/2 phosphorylation correlates with low aromatase expression in response to gonadotropins, we tested the effects of these inhibitors on aromatase expression. These inhibitors had little or no effect on gonadotropin-induced aromatase expression in cells expressing a low density of receptors, but they enhanced gonadotropin-induced aromatase expression in cells expressing a high density of receptors. Phorbol esters also induced a prolonged increase in ERK1/2 phosphorylation and, when added together with hFSH, blocked the induction of aromatase expression by hFSH in cells expressing a low density of hFSH receptor. A MAPK kinase inhibitor reversed the inhibitory effect of the phorbol ester on aromatase induction. We conclude that the effects of gonadotropins on ERK1/2 phosphorylation are mediated by epidermal growth factor-like growth factors and that the delayed effect is partially mediated by protein kinase C and acts as a negative regulator of aromatase expression.     

Oxidative stress-induced apoptosis is mediated by ERK1/2 phosphorylation

Oxidative stress is known to induce apoptosis in a wide variety of cell types, apparently by modulating intracellular signaling pathways. High concentrations of H2O2 have been found to induce apoptosis in L929 mouse fibroblast cells. To elucidate the mechanisms of H2O2-mediated apoptosis, ERK1/2, p38-MAPK, and JNK1/2 phosphorylation was examined, and ERK1/2 and JNK1/2 were found to be activated by H2O2. Inhibition of ERK1/2 activation by treatment of L929 cells with PD98059 or dominant-negative ERK2 transfection blocked H2O2-induced apoptosis, while inhibition of JNK1/2 by dominant-negative JNK1 or JNK2 or MKK4 or MKK7 transfection did not affect H2O2-mediated apoptosis. H2O2-mediated ERK1/2 activation was not only Ras-Raf dependent, but also both tyrosine kinase (PDGFbeta receptor and Src) and PKCdelta dependent. H2O2-mediated PKCdelta-dependent and tyrosine kinase-dependent ERK1/2 activations were independent from each other. Based on the above results, we suggest for the first time that oxidative damage-induced apoptosis is mediated by ERK1/2 phosphorylation which is not only Ras-Raf dependent, but also both tyrosine kinase and PKCdelta dependent.     

Skeletal muscle cell activation by low-energy laser irradiation: a role for the MAPK/ERK pathway

Low-energy laser irradiation (LELI) has been shown to promote skeletal muscle regeneration in vivo and to activate skeletal muscle satellite cells, enhance their proliferation and inhibit differentiation in vitro. In the present study, LELI, as well as the addition of serum to serum-starved myoblasts, restored their proliferation, whereas myogenic differentiation remained low. LELI induced mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) phosphorylation with no effect on its expression in serum-starved myoblasts. Moreover, a specific MAPK kinase inhibitor (PD098059) inhibited the LELI- and 10% serummediated ERK1/2 activation. However, LELI did not affect Jun N-terminal kinase (JNK) or p38 MAPK phosphorylation or protein expression. Whereas a 3-sec irradiation induced ERK1/2 phosphorylation, a 12-sec irradiation reduced it, again with no effect on JNK or p38. Moreover, LELI had distinct effects on receptor phosphorylation: it caused phosphorylation of the hepatocyte growth factor (HGF) receptor, previously shown to activate the MAPK/ERK pathway, whereas no effect was observed on tumor suppressor necrosis alpha (TNF-alpha) receptor which activates the p38 and JNK pathways. Therefore, by specifically activating MAPK/ERK, but not JNK and p38 MAPK enzymes, probably by specific receptor phosphorylation, LELI induces the activation and proliferation of quiescent satellite cells and delays their differentiation.     

Characterization of OSR1, a member of the mammalian Ste20p/germinal center kinase subfamily

In examining the protein kinase components of mitogen-activated protein (MAP) kinase (MAPK) cascades that regulate the c-Jun N-terminal kinase (JNK) in Drosophila S2 cells, we previously found that distinct upstream kinases were involved in responses to sorbitol and lipopolysaccharide. Here we have extended that analysis to the possible MAPK kinase kinase kinases (MAP4Ks) in the JNK pathway. Fray, a putative Drosophila MAP4K, provided a major contribution to JNK activation by sorbitol. To explore the possible link to JNK in mammalian cells, we isolated and characterized OSR1 (oxidative stress-responsive 1), one of two human Fray homologs. OSR1 is a 58-kDa protein of 527 amino acids that is widely expressed in mammalian tissues and cell lines. Of potential regulators surveyed, endogenous OSR1 is activated only by osmotic stresses, notably sorbitol and to a lesser extent NaCl. However, OSR1 did not increase the activity of coexpressed JNK, nor did it activate three other MAPKs, p38, ERK2, and ERK5. A two-hybrid screen implicated another Ste20p family member, the p21-activated protein kinase PAK1, as an OSR1 target. OSR1 phosphorylated threonine 84 in the N-terminal regulatory domain of PAK1. Replacement of threonine 84 with glutamate reduced the activation of PAK1 by an active form of the small G protein Cdc42, suggesting that phosphorylation by OSR1 modulates the G protein sensitivity of PAK isoforms.     

Unbalanced activation of ERK1/2 and MEK1/2 in apigenin-induced HeLa cell death

Apigenin, a dietary bioflavonoid with anticarcinogenic properties, was highly cytotoxic for HeLa cells (incubated with 0.5% FBS). This effect was accompanied with a marked increase in ERK1/2 but not MEK1/2 phosphorylation. The cytotoxic effects of apigenin were attenuated by the stimulation of these cells with 10% FBS, which provoked an increase in the phosphorylation levels of MEK1/2 and ERK1/2. The steps in the ERK1/2 pathway relevant to the cytotoxic effects of apigenin, as well as the contribution of other signaling pathways, were investigated. The activation of the pathway by transfection with the constitutively active Ras mutant (RasV12) conferred protection to serum-starved HeLa cells against apigenin, whereas the constitutively active MEK(E) mutant did not. MEK inhibitors (PD098059 or U0126) blocked ERK1/2 phosphorylation induced by apigenin and conferred partial protection against this flavonoid. The effects of apigenin did not involve p38-MAPK or JNK1/2, and were not simply due to inhibition of PI3kinase or protein kinase CK2. These data suggest that the deregulation of the ERK1/2 pathway, due to the potentiation of ERK1/2 phosphorylation without increasing MEK1/2 phosphorylation, is involved in apigenin-induced HeLa cell death.     

Novel role for JNK as a stress-activated Bcl2 kinase

Interleukin (IL)-3-induced Bcl2 phosphorylation at Ser(70) may be required for its full and potent antiapoptotic activity. However, in the absence of IL-3, increased expression of Bcl2 can also prolong cell survival. To determine how Bcl2 may be functionally phosphorylated following IL-3 withdrawal, a stress-activated Bcl2 kinase (SAK) was sought. Results indicate that anisomycin, a potent activator of the stress kinase JNK/SAPK, can induce Bcl2 phosphorylation at Ser(70) and that JNK1 can be latently activated following IL-3 withdrawal to mediate Bcl2 phosphorylation. JNK1 directly phosphorylates Bcl2 in vitro, co-localizes with Bcl2, and collaborates with Bcl-2 to mediate prolonged cell survival in the absence of IL-3 or following various stress applications. Dominant-negative (DN)-JNK1 can block both anisomycin and latent IL-3 withdrawal-induced Bcl2 phosphorylation (>90%) and potently enhances cell death. Furthermore, low dose okadaic acid (OA), a potent protein phosphatase 1 and 2A inhibitor, can activate the mitogen-activated protein kinases JNK1 and ERK1/2, but not p38 kinase, to induce Bcl2 phosphorylation and prolong cell survival in factor-deprived cells. Since PD98059, a specific MEK inhibitor, can only partially inhibit OA-induced Bcl2 phosphorylation but completely blocks OA-induced Bcl2 phosphorylation in cells expressing DN-JNK1, this supports the conclusion that OA may stimulate Bcl2 phosphorylation via a mechanism involving both JNK1 and ERK1/2. Collectively, these findings indicate a novel role for JNK1 as a SAK and may explain, at least in part, how functional phosphorylation of Bc12 can occur in the absence of growth factor.     

ERK2 is required for FGF1-induced JNK1 phosphorylation in Xenopus oocyte expressing FGF receptor 1

A possible connection between the ERK2 and JNK1 MAP kinases transduction cascades was investigated in Xenopus oocytes expressing FGFR1 stimulated by FGF1. Injection of various inhibitors for the Shc/Grb2/Ras/Mos/MEK/ERK2 cascade blocked FGF1-induced germinal vesicle breakdown (GVBD), as well as ERK2 and JNK1 phosphorylation. JNK1 was found to be activated downstream of ERK2, since injection of an active ERK2 triggered JNK1 phosphorylation and inhibition of ERK2 either by a MEK inhibitor or the MKP3 phosphatase blocked JNK1 phosphorylation. These results demonstrated that in FGFR1 signalling JNK1 phosphorylation depends on ERK2.