The p150-Spir protein provides a link between c-Jun N-terminal kinase function and actin reorganization

AbstractThe Jun N-terminal kinase (JNK) is a downstream effector of Rac and Cdc42 GTPases involved in actin reorganization [1–3]. A role of the Drosophila JNK homologue, Basket (DJNK/Bsk), in the regulation of cell shape changes and actin reorganization arises from its function in the process of dorsal closure [4–6]. One potential mechanism for induction of cytoskeletal changes by JNK is via transcriptional activation of the decapentaplegic gene (dpp, a member of the TGFβ superfamily) [8]. A direct link between JNK signalling and actin organization has not yet been found, however. We have identified a novel DJNK-interacting protein, p150-Spir, that belongs to the Wiscott–Aldrich syndrome protein (WASP) homology domain 2 (WH2) family of proteins involved in actin reorganization [7,8]. It is a multidomain protein with a cluster of four WH2 domains, a modified FYVE zinc-finger motif [9], and a DEJL motif, a docking site for JNK [10], at its carboxy-terminal end. In mouse fibroblasts, p150-Spir colocalized with F-actin and its overexpression induced clustering of filamentous actin around the nucleus. When coexpressed with p150-Spir in NIH 3T3 cells, JNK translocated to and colocalizes with p150-Spir at discrete spots around the nucleus. Carboxy-terminal sequences of p150-Spir were phosphorylated by JNK both in vitro and in vivo. We conclude that p150-Spir is a downstream target of JNK function and provides a direct link between JNK and actin organization

The Spir actin organizers are involved in vesicle transport processes

AbstractThe p150-Spir protein, which was discovered as a phosphorylation target of the Jun N-terminal kinase, is an essential regulator of the polarization of the Drosophila oocyte [1, 2]. Spir proteins are highly conserved between species and belong to the family of Wiskott-Aldrich homology region 2 (WH2) proteins involved in actin organization. The C-terminal region of Spir encodes a zinc finger structure highly homologous to FYVE motifs [2]. A region with high homology between the Spir family proteins is located adjacent (N-terminal) to the modified FYVE domain and is designated as “Spir-box.” The Spir-box has sequence similarity to a region of rabphilin-3A, which mediates interaction with the small GTPase Rab3A [3]. Coexpression of p150-Spir and green fluorescent protein-tagged Rab GTPases in NIH 3T3 cells revealed that the Spir protein colocalized specifically with the Rab11 GTPase, which is localized at the trans-Golgi network (TGN), post-Golgi vesicles, and the recycling endosome [4]. The distinct Spir localization pattern was dependent on the integrity of the modified FYVE finger motif and the Spir-box. Overexpression of a mouse Spir-1 dominant interfering mutant strongly inhibited the transport of the vesicular stomatitis virus G (VSV G) protein to the plasma membrane. The viral protein was arrested in membrane structures, largely colocalizing with the TGN marker TGN46. Our findings that the Spir actin organizer is targeted to intracellular membrane structures by its modified FYVE zinc finger and is involved in vesicle transport processes provide a novel link between actin organization and intracellular transport

Protein kinase Cα activates RAF-1 by direct phosphorylation.

THE kinase Raf-1 can be activated by treatment of cells with mitogens and by the protein kinase C (PKC) activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) (reviewed in refs 1, 2). Activated Raf-1 triggers a protein kinase cascade by direct phosphorylation of MAP kinase kinase3-5, resulting in phosphorylation of ternary complex factor6 and Jun7,8 by MAP kinase. Here we investigate the molecular mechanism and biological consequences of PKCα-mediated Raf-1 activation in NIH3T3 fibroblasts. PKCα directly phosphorylates and activates Raf-1 both in vitro and in vivo. PKCα induces Raf-1 phosphorylation at several sites, including a serine residue at position 499. Mutation of serine at this position or at residue 259 does not abrogate Raf-1 stimulation by a combination of Ras plus the src tyrosine kinase Lck, but severely impedes Raf-1 activation by PKCα. Consistent with such a direct interaction is the observation that Raf-1 and PKCα cooperate in the transformation of NIH3T3 cells. The Ser499 phosphorylation site is necessary for this synergism

Activation of c-Jun NH 2-Terminal Kinase/Stress-activated Protein Kinase (JNK/SAPK) Is Critical for Hypoxia-induced Apoptosis of Human Malignant Melanoma 1

Mitogen-activated protein kinase (MAPK) signaling was examined in malignant melanoma cells exposed to hypoxia. Here we demonstrate that hypoxia induced a strong activation of the c-Jun NH 2-terminal kinase (JNK), also termed stress-activated protein kinase (SAPK), in the melanoma cell line 530 in vitro. Other members of the MAPK family, e.g., extracellular signalregulated kinase and p38, remained unaffected by the hypoxic stimulus. Activated JNK/SAPK could also be observed in the vicinity of hypoxic tumor areas in melanoma metastases as detected by immunohistochemistry. Functional analysis of JNK/SAPK activation in the melanoma cell line 530 revealed that activation of JNK/SAPK is involved i

Activation of c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) is critical for hypoxia-induced apoptosis of human malignant melanoma.

Item does not contain fulltextMitogen-activated protein kinase (MAPK) signaling was examined in malignant melanoma cells exposed to hypoxia. Here we demonstrate that hypoxia induced a strong activation of the c-Jun NH2-terminal kinase (JNK), also termed stress-activated protein kinase (SAPK), in the melanoma cell line 530 in vitro. Other members of the MAPK family, e.g., extracellular signal-regulated kinase and p38, remained unaffected by the hypoxic stimulus. Activated JNK/SAPK could also be observed in the vicinity of hypoxic tumor areas in melanoma metastases as detected by immunohistochemistry. Functional analysis of JNK/SAPK activation in the melanoma cell line 530 revealed that activation of JNK/SAPK is involved in hypoxia-mediated tumor cell apoptosis. Both a dominant negative mutant of JNK/SAPK (SAPKbeta K-->R) and a dominant negative mutant of the immediate upstream activator of JNK/SAPK, SEK1 (SEK1 K-->R), inhibited hypoxia-induced apoptosis in transient transfection studies. In contrast, overexpression of the wild-type kinases had a slight proapoptotic effect. Inhibition of extracellular signal-regulated kinase and p38 pathways by the chemical inhibitors PD98058 and SB203580, respectively, had no effect on hypoxiainduced apoptosis. Under normoxic conditions, no influence on apoptosis regulation was observed after inhibition of all three MAPK pathways. In contrast to recent findings, JNK/SAPK activation did not correlate with Fas or Fas ligand (FasL) expression, suggesting that the Fas/FasL system is not involved in hypoxia-induced apoptosis in melanoma cells. Taken together, our data demonstrate that hypoxia-induced JNK/SAPK activation appears to play a critical role in apoptosis regulation of melanoma cells in vitro and in vivo, independent of the Fas/FasL system

Impaired binding of 14-3-3 to C-RAF in noonan syndrome suggests new approaches in diseases with increased ras signaling

The Ras-RAF-mitogen-activated protein kinase (Ras-RAF-MAPK) pathway is overactive in many cancers and in some developmental disorders. In one of those disorders, namely, Noonan syndrome, nine activating C-RAF mutations cluster around Ser(259), a regulatory site for inhibition by 14-3-3 proteins. We show that these mutations impair binding of 14-3-3 proteins to C-RAF and alter its subcellular localization by promoting Ras-mediated plasma membrane recruitment of C-RAF. By presenting biophysical binding data, the 14-3-3/C-RAFpS(259) crystal structure, and cellular analyses, we indicate a mechanistic link between a well-described human developmental disorder and the impairment of a 14-3-3/target protein interaction. As a broader implication of these findings, modulating the C-RAFSer(259)/14-3-3 protein-protein interaction with a stabilizing small molecule may yield a novel potential approach for treatment of diseases resulting from an overactive Ras-RAF-MAPK pathway

In Vitro and In Vivo Antitumor Activity of a Novel Semisynthetic Derivative of Cucurbitacin B

Lung cancer is the most deadly type of cancer in humans, with non-small-cell lung cancer (NSCLC) being the most frequent and aggressive type of lung cancer showing high resistance to radiation and chemotherapy. Despite the outstanding progress made in anti-tumor therapy, discovering effective anti-tumor drugs is still a challenging task. Here we describe a new semisynthetic derivative of cucurbitacin B (DACE) as a potent inhibitor of NSCLC cell proliferation. DACE arrested the cell cycle of lung epithelial cells at the G2/M phase and induced cell apoptosis by interfering with EGFR activation and its downstream signaling, including AKT, ERK, and STAT3. Consistent with our in vitro studies, intraperitoneal application of DACE significantly suppressed the growth of mouse NSCLC that arises from type II alveolar pneumocytes due to constitutive expression of a human oncogenic c-RAF kinase (c-RAF-1-BxB) transgene in these cells. Taken together, these findings suggest that DACE is a promising lead compound for the development of an anti-lung-cancer drug